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1.
Plant Mol Biol ; 102(4-5): 463-475, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31916083

RESUMO

The mechanism by which endogenous salicylic acid (SA) regulates leaf senescence remains elusive. Here we provide direct evidence that an enhancement of endogenous SA level, via chemical-induced upregulation of ISOCHORISMATE SYNTHASE 1 (ICS1), could significantly accelerate the senescence process of old leaves through mediation of the key SA signaling component NON EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1) in Arabidopsis. Importantly, by taking advantage of this chemically induced leaf senescence system, we identified a mitogen-activated protein kinase (MAPK) cascade MKK4/5-MPK1/2 that is required for the SA/NPR1-mediated leaf senescence. Both MKK4/5 and MPK1/2 exhibited SA-induced kinase activities, with MPK1/2 being the immediate targets of MKK4/5. Double mutants of mkk4 mkk5 and mpk1 mpk2 displayed delayed leaf senescence, while constitutive overexpression of the kinase genes led to premature leaf senescence. Such premature leaf senescence was suppressed when they were overexpressed in an SA synthesis defective mutant (sid2) or signaling detective mutant (npr1). We further showed that MPK1, but not MPK2, could directly phosphorylate NPR1. Meanwhile, MPK1 also mediated NPR1 monomerization. Notably, induction of disease resistance was significantly compromised in the single and double mutants of the kinase genes. Taken together, our data demonstrate that the MKK4/5-MPK1/2 cascade plays a critical role in modulating SA signaling through a complex regulatory network in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Sistema de Sinalização das MAP Quinases , Folhas de Planta/enzimologia , Ácido Salicílico/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Transdução de Sinais
2.
Gene ; 732: 144339, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31927008

RESUMO

OBJECTIVE: Previous studies have shown that follistatin-like protein 1 (FSTL1) is elevated in the synovial fluid of osteoarthritis and is associated with disease activity. The experiment was performed to stuy the effect and mechanism of FSTL1 on chondrocyte apoptosis in osteoarthritis. DESIGN: After the isolation of human normal and osteoarthritis (OA) chondrocytes, the expression of FSTL1 was detected by Q-PCR and western blot analyses. Chondrocytes were pre-transfected with FSTL1 overexpression plasmids then treated with SNP, and chondrocyte viability and apoptosis levels were detected by MTS and flow cytometry, respectively. Cartilage matrix gene expression was measured by Q-PCR and signal pathway-related proteins were assessed by western blot. RESULTS: The expression of FSTL1 in OA chondrocytes was markedly up-regulated compared with normal human chondrocytes (P < 0.05). The apoptosis rate of chondrocytes in the FSTL1 overexpression groups was highly elevated in the comparison with the negative control groups (P < 0.05). Additionally, FSTL1 potentiated protein abundances of MMP1, MMP3, MMP-9, and Bax as well as reduced Coll2a1 and Aggrecan and Bcl-2 expression. Furthermore, western blot results showed that the SAPK/JNK/Caspase3 signal pathway was significantly activated and the Ac-DEVD-FMK impaired FSTL1 induced chondrocyte apoptosis. CONCLUSION: FSTL1 promoted SNP-induced chondrocytes apoptosis by activating the SAPK/JNK/Caspase3 signal pathway.


Assuntos
Apoptose/fisiologia , Caspase 3/metabolismo , Condrócitos/citologia , Proteínas Relacionadas à Folistatina/fisiologia , MAP Quinase Quinase 4/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Óxido Nítrico/fisiologia , Transdução de Sinais/fisiologia , Idoso , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Proteínas Relacionadas à Folistatina/genética , Humanos , Pessoa de Meia-Idade , Osteoartrite/enzimologia , Osteoartrite/metabolismo , Osteoartrite/patologia , RNA Mensageiro/metabolismo
3.
J Clin Pathol ; 73(2): 116-119, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31506288

RESUMO

Beyond targeted therapy for patients with BRAF-mutated melanomas and immunotherapy in patients lacking BRAF mutations, anti-MEK therapy has been proposed in patients with advanced melanomas harbouring BRAF fusions. BRAF fusions diagnosis in patients with advanced melanomas is the subject of the present study. Using BRAF fluorescent in situ hybridisation (FISH), we searched for BRAF fusions in 74 samples of 66 patients with advanced BRAF/NRAS/KIT wild-type melanomas. We identified 2/66 (3%) patients with BRAF fusions in a brain metastasis of one patient and in a lymph node metastasis and in a cutaneous metastasis for the second patient with 90%-95% of tumour nuclei containing isolated 3'-BRAF FISH signals. As a result, we conclude that BRAF FISH in patients with advanced BRAF/NRAS/KIT wild-type melanomas is a valuable and easy-to-perform test to diagnose BRAF fusions and to identify patients who could benefit of anti-MEK targeted therapy.


Assuntos
Biomarcadores Tumorais/genética , GTP Fosfo-Hidrolases/genética , Fusão Gênica , Hibridização in Situ Fluorescente , Melanoma/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Feminino , Humanos , Masculino , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , Seleção de Pacientes , Medicina de Precisão , Valor Preditivo dos Testes , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/enzimologia
4.
Arch Oral Biol ; 110: 104605, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31751919

RESUMO

OBJECTIVE: The neuronal wiskott-aldrich syndrome protein (N-WASP) is a member of the wiskott-aldrich syndrome protein (WASP) family. N-WASP plays a vital role in promoting cell migration, receptor signaling and immune inflammatory responses. This study aimed to observe the changes in the expression of inflammatory factors and involving pathways after N-WASP knockdown in human gingival fibroblasts (HGFs). DESIGN: Gingival inflammatory condition of N-WASP knockout mice was evaluated by H&E staining. N-WASP in HGFs was knockdown by siRNA and the best knockdown efficiency was determined by qRT-PCR and immunofluorescence. The mRNA levels of interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), superoxide dismutase 2 (SOD2) and prostaglandin endoperoxide synthase 2 (PTGS2) were evaluated by qRT-PCR after N-WASP knockdown with or without mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors. The protein levels of IL-6, IL-8 and CCL2 were assessed by ELISA. Western blotting was used to detect the activation of NF-κB and MAPK signaling pathways. RESULTS: Gingival tissue from N-WASP knockout mice exhibited an inflammatory reaction. The expression of IL-6, IL-8, CCL2, SOD2 and PTGS2 was significantly upregulated after N-WASP knockdown in HGFs for 6, 24 and 48 h, except for the SOD2 at 6 h. N-WASP knockdown significantly activated the signaling pathways of NF-κB and MAPK. The inhibitors of p65, p38, ERK and JNK clearly decreased IL-6, IL-8, CCL2, SOD2 and PTGS2 expression after N-WASP knockdown. CONCLUSION: These data indicated that N-WASP deficiency in HGFs increases the production of inflammatory cytokine and is regulated via NF-κB and MAPK signaling pathways.


Assuntos
Citocinas , Fibroblastos , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Animais , Citocinas/metabolismo , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Gengiva/metabolismo , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
5.
Arch Oral Biol ; 110: 104602, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31734544

RESUMO

OBJECTIVE: Oxidative stress, which is defined as an imbalance between pro-oxidant and antioxidant systems, has been implicated in the development and/or progression of several inflammatory diseases, including periodontal disease. The reactive oxygen species (ROS) are the primary inducers of oxidative stress. In the induction of cytoprotective enzymes, the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling in antioxidant systems takes a main role. Notably, 10-oxo-trans-11-octadecenoic acid (KetoC), known as a bioactive metabolite generated by intestinal microorganisms, has been reported to have beneficial effects on several biological responses. Therefore, we investigated the antioxidant effect of KetoC on gingival epithelial cells (GECs) in this present study. METHODS: An SV40-T antigen-transformed human gingival epithelial cell line (Epi4) was used for experiments. The alteration of anti-oxidative stress related genes was analyzed by qPCR. The cellular ROS levels were evaluated by flow cytometry. To explore its molecular mechanisms, ARE promotor activity was analyzed by luciferase assay; the involvement of mitogen-activated protein kinase (MAPK) and G protein-coupled receptor 120 (GPR120) were evaluated by Western blotting and luciferase assay, respectively. RESULTS: KetoC significantly increased the expression of antioxidant-related genes in GECs. The level of ROS was significantly inhibited by the pretreatment of KetoC. Extracellular signal-regulated kinase (ERK) phosphorylation by KetoC promoted both the nuclear translocation of Nrf2 and its binding to the ARE in GECs. Further, GPR120 regulated the activation of KetoC induced-Nrf2-ARE signaling. CONCLUSION: KetoC exerts a protective function against the oxidative stress in GECs through GPR120-dependent ERK-Nrf2-ARE signaling.


Assuntos
Elementos de Resposta Antioxidante , Gengiva , Ácidos Linoleicos , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Transdução de Sinais , Antioxidantes , Células Epiteliais , Gengiva/citologia , Gengiva/metabolismo , Humanos , Ácidos Linoleicos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio
6.
Nat Commun ; 10(1): 5143, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723142

RESUMO

Molecular determinants governing the evolution of tumor subclones toward phylogenetic branches or fixation remain unknown. Using sequencing data, we model the propagation and selection of clones expressing distinct categories of BRAF mutations to estimate their evolutionary trajectories. We show that strongly activating BRAF mutations demonstrate hard sweep dynamics, whereas mutations with less pronounced activation of the BRAF signaling pathway confer soft sweeps or are subclonal. We use clonal reconstructions to estimate the strength of "driver" selection in individual tumors. Using tumors cells and human-derived murine xenografts, we show that tumor sweep dynamics can significantly affect responses to targeted inhibitors of BRAF/MEK or DNA damaging agents. Our study uncovers patterns of distinct BRAF clonal evolutionary dynamics and nominates therapeutic strategies based on the identity of the BRAF mutation and its clonal composition.


Assuntos
Evolução Clonal/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma de Pulmão/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Dano ao DNA , Dosagem de Genes , Loci Gênicos , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação/genética , Fenótipo , Inibidores de Proteínas Quinases/farmacologia
7.
BMC Bioinformatics ; 20(1): 615, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31779580

RESUMO

BACKGROUND: Bistability and ability to switch between two stable states is the hallmark of cellular responses. Cellular signaling pathways often contain bistable switches that regulate the transmission of the extracellular information to the nucleus where important biological functions are executed. RESULTS: In this work we show how the method of Gröebner bases can be used to detect bistability and output switchability. The method of Gröebner bases can be seen as a multivariate, non-linear generalization of the Gaussian elimination for linear systems which conveniently seperates the variables and drastically simplifies the simultaneous solution of polynomial equations. A necessary condition for fixed-point state bistability is for the Gröbner basis to have three distinct solutions for the state. A sufficient condition is provided by the eigenvalues of the local Jacobians. We also introduce the concept of output switchability which is defined as the ability of an output of a bistable system to switch between two different stable steady-state values. It is shown that bistability does not necessarily guarantee switchability of every state variable of the system. We further show that, for a bistable system, the necessary conditions for output switchability can be derived using the Gröebner basis. The theoretical results are incorporated into an analysis procedure and applied to several systems including the AKT (Protein kinase B), RAS (Rat Sarcoma) and MAPK (Mitogen-activated protein kinase) signal transduction pathways. Results demonstrate that the Gröebner bases can be conveniently used to analyze biological switches by simultaneously detecting bistability and output switchability. CONCLUSION: The Gröebner bases provides a novel methodology to analyze bistability. Results clarify the distinction between bistability and output switchability which is lacking in the literature. We have shown that theoretically, it is possible to have an output subspace of an n-dimensional bistable system where certain variables cannot switch. It is possible to construct such systems as we have done with two reaction networks.


Assuntos
Transdução de Sinais , Algoritmos , Cinética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
8.
Artif Cells Nanomed Biotechnol ; 47(1): 4030-4037, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31603005

RESUMO

Spinal cord injury (SCI) can lead to varying degrees of sensory and motor dysfunction. Salvianolic acid B (Sal-B) is the dominating bioactive constituent of Danshen, which has been reported to alleviate liver fibrosis and exert neuroprotective effects. But, the influence of Sal-B in SCI remains mysterious. The research planned to delve the protective function of Sal-B in hydrogen peroxide (H2O2)-caused PC-12 cell injury. H2O2-caused PC-12 cells injury model was built, CCK-8, Transwell and flow cytometry experiments were enforced to assess cell proliferation, migration and apoptosis. The microRNA (miR)-26a plasmid and the matching control were transfected into PC-12 cells, subsequently, the influence of miR-26a inhibition in H2O2-corrupted PC-12 cells was evaluated. The cell growth-correlated factors and PI3K/AKT and MEK/ERK pathways were assayed through western blot assay. Results corroborated that Sal-B eased H2O2-evoked injury in PC-12 cells. Ascended miR-26a was monitored in Sal-B and H2O2-exposed cells. MiR-26a inhibition annulled the protective action of Sal-B in H2O2-corrupted cells. The protective function of Sal-B was enabled through activating PI3K/AKT and MEK/ERK pathways. These findings delineated that Sal-B protected PC-12 cells against H2O2-caused injury through ascending miR-26a via initiating PI3K/AKT and MEK/ERK pathways. Highlights H2O2 causes PC-12 cell injury; Sal-B eases H2O2-caused PC-12 cell injury; Sal-B protects PC-12 cells against H2O2-caused injury via elevating miR-26a; Sal-B activates AKT and MEK/ERK pathways via modulating miR-26a.


Assuntos
Apoptose/efeitos dos fármacos , Benzofuranos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , MicroRNAs/genética , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Salvia miltiorrhiza/química , Transdução de Sinais/efeitos dos fármacos
9.
Res Vet Sci ; 126: 164-169, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31499425

RESUMO

Endometritis is one of the most common reproductive diseases caused by bacterial infection in the cow. Ferulic acid is a major effective component extracted from Ligusticum wallichii. Ferulic acid displays pharmacological effects such as anti-inflammation and antioxidation. The aim of the present study was to investigate the protective effects of ferulic acid on inflammation induced by lipopolysaccharide (LPS) in bovine endometrial epithelial cells (BEECs). BEECs were pretreated with ferulic acid followed by LPS treatment. QRT-PCR analysis showed the mRNA expression of LPS-induced proinflammatory cytokines (IL1B, IL6, TNFA, and IL8) was decreased with ferulic acid pretreatment. Western blot analysis showed that ferulic acid inhibited the degradation of IκB and phosphorylation of NF-κB p65. Ferulic acid suppressed the phosphorylation of MAPKs, including p38 and JNK. All of these results indicated that ferulic acid may be considered as a potential anti-inflammatory drug for curing endometritis.


Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Doenças dos Bovinos/tratamento farmacológico , Ácidos Cumáricos/administração & dosagem , Endometrite/veterinária , Inflamação/veterinária , Animais , Bovinos , Doenças dos Bovinos/induzido quimicamente , Endometrite/induzido quimicamente , Endometrite/tratamento farmacológico , Endométrio/efeitos dos fármacos , Endométrio/fisiopatologia , Células Epiteliais/efeitos dos fármacos , Feminino , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Plant Sci ; 287: 110190, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481213

RESUMO

Phosphatidic acid (PA) is a lipid secondary messenger involved in intracellular signaling in eukaryotes. It has been confirmed that PA mediates salt stress signaling by promoting activation of Mitogen-activated Protein Kinase 6 (MPK6) which phosphorylates Na+/H+ antiporter SOS1. However, the MPK6-upstream kinases and their relationship to PA remain unclear. Here, we found that, among the six tested Arabidopsis Mitogen-activated Protein Kinase Kinases (MKKs), PA specifically bound to MKK7 and MKK9 which phosphorylate MPK6, and promoted the activation of MKK7/MKK9. Based on phenotypic and physiological analyses, we found that MKK7 and MKK9 positively regulate Arabidopsis salt tolerance and are functionally redundant. NaCl treatment can induce significant increase in MKK7/MKK9 activities, and this depends, in part, on the Phospholipase Dα1 (PLDα1). MKK7 and MKK9 also mediate the NaCl-induced activation of MPK6. Furthermore, PA or NaCl treatment could induce translocation of MKK7/MKK9 to the plasma membrane, whereas this translocation disappeared in pldα1. These results indicate that PA binds to MKK7 and MKK9, increases their kinase activity and plasma membrane localization during Arabidopsis response to salt stress. Together with the PA-MPK6-SOS1 pathway identified previously, this mechanism may maximize the signal transduction efficiency, providing novel insights into the link between lipid signaling and MAPK cascade.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , MAP Quinase Quinase 7/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ácidos Fosfatídicos/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , MAP Quinase Quinase 7/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Estresse Salino , Tolerância ao Sal/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-31398390

RESUMO

Compound ammonium glycyrrhizin (CAG) protects hepatocytes from injury induced by lipopolysaccharide (LPS)/florfenicol (FFC) through a mitochondrial pathway. On this basis, the research was aimed to investigate whether CAG protects hepatocytes from injury induced by LPS/FFC through oxidative stress and the MAPK pathway. For liver injury induced by LPS/FFC, not only CAG can protect hepatocytes and prevent membrane permeability from being increased, but also the activities of ALT and AST were decreased significantly by CAG. Flow cytometry analysis indicated that the apoptosis rate (35.65 ±â€¯2.48%) of LPS/FFC group was significantly higher than that of the control group (8.60 ±â€¯0.32%). CAG (concentration of 0.01 µg/mL, 0.1 µg/mL, 1 µg/mL) significantly decreased the apoptosis rate (23.69 ±â€¯0.54%, 14.92 ±â€¯2.45% and 9.47 ±â€¯1.28%) for the liver injury induced by LPS/FFC. The activities of SOD and GSH were increased with the increased concentration of CAG, and the activity of MDA was decreased with the increased concentration of CAG. All the mRNA and proteins expression levels were increased by LPS/FFC-induced liver injury which associated with the MAPK pathway, and those of the CAG group were decreased with the increased concentration of CAG. And the change of caspase-3 activity was consistent with that of proteins and mRNA. It is suggested that LPS/FFC can induce liver injury through apoptosis and the CAG can protect hepatocytes from injury through the MAPK pathway and oxidative stress.


Assuntos
Compostos de Amônio/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Ácido Glicirrízico/farmacologia , Hepatócitos/metabolismo , Fígado/metabolismo , Animais , Apoptose/efeitos dos fármacos , Galinhas/metabolismo , Hepatócitos/patologia , Lipopolissacarídeos/toxicidade , Fígado/patologia , Masculino , Mitocôndrias/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Tianfenicol/análogos & derivados , Tianfenicol/toxicidade
12.
BMC Cancer ; 19(1): 764, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375085

RESUMO

BACKGROUND: MAPK/ERK kinases transmit signals from many growth factors/kinase receptors during normal cell growth/differentiation, and their dysregulation is a hallmark of diverse types of cancers. A plethora of drugs were developed to block this kinase pathway for clinical application. With the exception of a recently identified agent, EQW, most of these inhibitors target upstream factors but not ERK1/2; no activator of ERK1/2 is currently available. METHOD: A library of compounds isolated from medicinal plants of China was screened for anti-cancer activities. Three limonoid compounds, termed A1541-43, originally isolated from the plant Melia azedarach, exhibiting strong anti-leukemic activity. The anti-neoplastic activity and the biological target of these compounds were explored using various methods, including western blotting, flow cytometry, molecular docking and animal model for leukemia. RESULTS: Compounds A1541-43, exhibiting potent anti-leukemic activity, was shown to induce ERK1/2 phosphorylation. In contrast, the natural product Cedrelone, which shares structural similarities with A1541-43, functions as a potent inhibitor of ERK1/2. We provided evidence that A1541-43 and Cedrelone specifically target ERK1/2, but not the upstream MAPK/ERK pathway. Computational docking analysis predicts that compounds A1541-43 bind a region in ERK1/2 that is distinct from that to which Cedrelone and EQW bind. Interestingly, both A1541-43, which act as ERK1/2 agonists, and Cedrelone, which inhibit these kinases, exerted strong anti-proliferative activity against multiple leukemic cell lines, and induced robust apoptosis as well as erythroid and megakaryocytic differentiation in erythroleukemic cell lines. These compounds also suppressed tumor progression in a mouse model of erythroleukemia. CONCLUSIONS: This study identifies for the first time activators of ERK1/2 with therapeutic potential for the treatment of cancers driven by dysregulation of the MAPK/ERK pathway and possibly for other disorders.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Leucemia Eritroblástica Aguda/tratamento farmacológico , Limoninas/farmacologia , Limoninas/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melia azedarach/química , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células K562 , Leucemia Eritroblástica Aguda/mortalidade , Leucemia Eritroblástica Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Folhas de Planta/química , Transdução de Sinais/efeitos dos fármacos , Taxa de Sobrevida
13.
Folia Biol (Praha) ; 65(2): 70-87, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31464183

RESUMO

We investigated the detrimental effects of diabetes on myocardium of pregestational streptozotocin (STZ)-diabetic mother rats and their neonates via evaluations of oxidative redox, inflammatory and apoptotic pathways, also aiming to characterize whether calcitriol and/or pomegranate peel extract confer myocardial protection in hyperglycaemic dams and their foetuses via modulation of the Raf/MEK/ERK cascade. Sixty Sprague-Dawley female rats were randomized into five groups (N = 12): control, diabetic, diabetic treated with calcitriol and/or pomegranate peel extract (PPE), and mated with non-diabetic healthy males. After confirmation of pregnancy, treatments were kept until gestational day (E-18). Serum and cardiac tissues of mothers and foetuses were collected and processed for biochemical, histopathological, and molecular assessments. We observed that, compared to the control, diabetic mothers showed dramatically increased hyperglycaemia and hyperlipidaemia associated with decreased myocardial functions and disrupted maternal performance. Also, diabetic mothers and their neonates exhibited elevated levels of myocardial injury (troponin I, endothelin 1, creatine kinase-MB, lactate dehydrogenase), with increased pro-inflammatory cytokines (interleukin 1, interleukin 1ß, transforming growth factor ß) and oxidative redox. Concurrently, the MAPK pathway was significantly down-regulated with increased myocardial apoptotic activity. Furthermore, mRNA expression of angiogenic and fibrotic markers was significantly increased. Paradoxically, calcitriol and/or pomegranate peel extract alleviated these diabetic myocardial insults and normalized the aforementioned assayed parameters. Our findings hypothesized that calcitriol and/or pomegranate peel extract exerted cardioameliorative impacts due to their unique anti-oxidative and anti-inflammatory properties, and thus may be a promising treatment that directly targets the secondary myocardial complications of diabetes in dams and their offspring.


Assuntos
Apoptose , Calcitriol/uso terapêutico , Cardiomiopatias/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Sistema de Sinalização das MAP Quinases , Extratos Vegetais/uso terapêutico , /química , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Cardiomiopatias/complicações , Cardiomiopatias/patologia , Caspase 3/metabolismo , Citocinas/metabolismo , Dano ao DNA , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Feminino , Feto/patologia , Fibrose , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Miocárdio/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Reprodução , Espectroscopia de Infravermelho com Transformada de Fourier , Estreptozocina , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologia , Vitamina D/uso terapêutico , Quinases raf/metabolismo
14.
Artif Cells Nanomed Biotechnol ; 47(1): 3231-3238, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31373232

RESUMO

RAS mutations are frequent in non-small cell lung cancer (NSCLC). However, targeting RAS or the downstream/upstream effectors, such as tyrosine kinase inhibitors (TKIs), has been proved to be difficult. Here, we found that the stemness of KRAS-mutant NSCLC cells but not the KRAS-wild type NSCLC cells was promoted by TKIs treatment, as evident by the increase of ALDH1 activity, stemness marker expression and spheroid formation ability. Notably, SHP2 activation was found in KRAS-mutant NSCLC cells with TKIs treatment, as judged by the increase of tyrosine 542 phosphorylation (pSHP2 Y542), which activates the RAS/MEK/ERK pathway. On the contrary, inhibition of MEK was followed by a SHP2 activation in KRAS-mutant NSCLC cells. Additionally, inhibition of SHP2 attenuates the enhanced stemness of KRAS-mutant NSCLC cells induced by TKIs, characterized by decreasing ALDH1 activity, stemness marker expression and spheroid formation capacity, while had little effects on cell viability. Finally, we revealed that SHP2 inhibitor increased the sensitivity of TKIs and chemotherapy, which was potentiated by MEK inhibition. Our results suggest a possibility of using a combination of SHP2 inhibitor and TKIs for KRAS-mutant NSCLC treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/patologia , Mutação , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células-Tronco Neoplásicas/patologia
15.
Nat Commun ; 10(1): 2919, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266962

RESUMO

Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specific phosphorylation of ERK in response to transgenic KRASG12V in mouse intestinal organoids, while transgenic BRAFV600E activates ERK in all cells. Quantitative network modelling from perturbation data reveals that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of ERK in the intestine. Furthermore, we find that oncogenic KRAS, together with ß-Catenin, favours expansion of crypt cells with high ERK activity. Our experiments highlight key differences between oncogenic BRAF and KRAS in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.


Assuntos
Neoplasias do Colo/enzimologia , Mucosa Intestinal/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Especificidade da Espécie
16.
Dev Growth Differ ; 61(6): 365-377, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31270814

RESUMO

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Ectoderma/embriologia , Ectoderma/enzimologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/enzimologia , Sistema Nervoso/embriologia , Sistema Nervoso/enzimologia , Placa Neural/embriologia , Placa Neural/enzimologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transdução de Sinais
17.
Food Funct ; 10(7): 4231-4241, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31259337

RESUMO

The fruits of Lycium barbarum are considered medicinal foods with high nutritional value and bioactivity. In this study, we aimed to evaluate the effect of a crude L. barbarum polysaccharide (LBP) and two derived fractions, LBP-1 and LBP-2, on the lifespan of Drosophila melanogaster (fruit fly). The average lifespan of fruit flies was extended by supplementing their diet with either of the three LBP preparations. In vivo analysis of antioxidant activities detected increased superoxide dismutase (SOD) and catalase (CAT) activities and decreased malondialdehyde (MDA) levels. Dietary LBP supplements significantly reduced the mortality rate of fruit flies induced by paraquat and hydrogen peroxide. Importantly, the strongest anti-aging activity was exhibited by the LBP-2 fraction, containing arabinogalactan with a molecular weight of 9 × 104 Da. Further studies showed that the anti-aging activity of LBP was, at least in part, mediated by an age-related signaling pathway (MAPK, TOR, S6K) and the expression of longevity genes (Hep, MTH, and Rpn11).


Assuntos
Drosophila melanogaster/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Longevidade/efeitos dos fármacos , Animais , Antioxidantes/análise , Catalase/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Frutas/química , Regulação da Expressão Gênica , Peróxido de Hidrogênio/toxicidade , Malondialdeído/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Peso Molecular , Paraquat/toxicidade , Extratos Vegetais/farmacologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Superóxido Dismutase/metabolismo
18.
Emerg Microbes Infect ; 8(1): 934-945, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237474

RESUMO

Cytoskeletal rearrangement and acute cytotoxicity occur in Vibrio vulnificus-infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of V. vulnificus and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491-1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA11491-1971 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA11491-1971 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.


Assuntos
Toxinas Bacterianas/metabolismo , Citoesqueleto/metabolismo , Filaminas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Vibrioses/metabolismo , Vibrio vulnificus/metabolismo , Quinases Ativadas por p21/metabolismo , Motivos de Aminoácidos , Toxinas Bacterianas/toxicidade , Morte Celular , Citoesqueleto/genética , Filaminas/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Ligação Proteica , Vibrioses/genética , Vibrioses/microbiologia , Vibrioses/fisiopatologia , Vibrio vulnificus/química , Vibrio vulnificus/genética , Quinases Ativadas por p21/genética
19.
J Microbiol Biotechnol ; 29(8): 1248-1254, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216788

RESUMO

Identification of novel probiotic strains is of great interest in the field of functional foods. Specific strains of heat-killed bacteria have been reported to exert immunomodulatory effects. Herein, we investigated the immune-stimulatory function of heat-killed Lactobacillus plantarum KCTC 13314BP (LBP). Treatment with LBP significantly increased the production of TNF-α and IL-6 by macrophages. More importantly, LBP was able to enhance the phagocytic activity of macrophages against bacterial particles. Activation of p38, JNK, ERK, NF-κB, and STAT3 was involved in the immunomodulatory function of LBP. LBP treatment significantly increased production of TNF-α by bone marrow-derived macrophages and splenocytes, further confirming the immunostimulatory effect of LBP in primary immune cells. Interestingly, the immunomodulatory effects of LBP were much stronger than those of Lactobacillus rhamnosus GG, a well-known probiotic strain. These results indicate that LBP can be a promising immune-enhancing functional food agent.


Assuntos
Temperatura Alta , Fatores Imunológicos/farmacologia , Lactobacillus plantarum/imunologia , Fagócitos/imunologia , Fator de Transcrição STAT3/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Interleucina-6/metabolismo , Lactobacillus rhamnosus/imunologia , Macrófagos/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fagócitos/efeitos dos fármacos , Probióticos/farmacologia , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
20.
Molecules ; 24(11)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181779

RESUMO

Lung cancer is one of the most common malignancies and is an increasing cause of cancer-related deaths. In our previous study, a series of ferulic acid (FA) derivatives were designed and synthesized; they exhibited positive anti-cancer activities, especially for a compound labelled FXS-3. In this study, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, wherein it revealed the inhibitory effect of FXS-3 on the proliferation and metastasis of human lung cancer A549 cells. The further flow cytometry assay showed that FXS-3 induced apoptosis of A549 cells induced cell cycle arrest at the G0/G1 phase. The trans-well migration and Matrigel invasion assays revealed that FXS-3 inhibited the migration and invasion of A549 cells. By the western blotting analysis, FXS-3 increased the expression of B-cell lymphoma-2 (Bcl-2) associated X protein (Bax)/Bcl-2 ratio, inhibited matrix metalloproteinase (MMP)-2 and MMP-9, and regulated the extracellular signal-regulated kinase (ERK)/p38, c-Jun N-terminal kinase (JNK), protein kinase B (AKT)/mechanistic target of rapamycin (mTOR), as well as mitogen-activated protein kinase (MEK)/ERK signaling pathways. The subsequent A549 xenograft-bearing mouse model and tail vein injection of A549 cells induced pulmonary tumor metastasis model showed that FXS-3 significantly restrained the tumor growth and metastasis. In conclusion, FXS-3 might inhibit proliferation and metastasis of human lung cancer A549 cells by positively regulating JNK signaling pathway and negativly regulating ERK/p38, AKT/mTOR, and MEK/ERK signaling pathways, which provides important scientific basis for the development of anti-cancer drugs about FA derivatives.


Assuntos
Ácidos Cumáricos/farmacologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácidos Cumáricos/química , Humanos , Masculino , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Ensaios Antitumorais Modelo de Xenoenxerto
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