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1.
Methods Mol Biol ; 2312: 237-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228294

RESUMO

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.


Assuntos
Engenharia Celular , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Biologia Sintética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Quinases raf/metabolismo
2.
Cancer Sci ; 112(10): 4187-4197, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34327762

RESUMO

Breast cancer is the most common cancer among women. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane protein that is highly expressed in many cancers, including breast cancer, has been shown to be a prognostic factor. We previously reported that GPNMB overexpression confers tumorigenic potential, as evidenced by invasive tumor growth in vivo, sphere formation, and cellular migration and invasion to non-tumorigenic mammary epithelial cells. In this study, we focused on the serine (S) residue in the intracellular domain of GPNMB (S530 in human isoform b and S546 in mouse), which is predicted to be a phosphorylation site. To investigate the roles of this serine residue, we made an antibody specific for S530-phosphorylated human GPNMB and a point mutant in which S530 is replaced by an alanine (A) residue, GPNMB(SA). Established GPNMB(SA) overexpressing cells showed a significant reduction in sphere formation in vitro and tumor growth in vivo as a result of decreased stemness-related gene expression compared to that in GPNMB(WT)-expressing cells. In addition, GPNMB(SA) impaired GPNMB-mediated cellular migration. Furthermore, we found that tyrosine kinase receptor signaling triggered by epidermal growth factor or fibroblast growth factor 2 induces the serine phosphorylation of GPNMB through activation of downstream oncoproteins RAS and RAF.


Assuntos
Glicoproteínas de Membrana/fisiologia , Serina/metabolismo , Animais , Especificidade de Anticorpos , Linhagem Celular Tumoral , Movimento Celular/genética , Fator de Crescimento Epidérmico/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Células MCF-7 , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Mutação Puntual , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Quinases raf/metabolismo , Proteínas ras/metabolismo
3.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299137

RESUMO

The KRAS mutation is one of the leading driver mutations in colorectal cancer (CRC), and it is usually associated with poor prognosis and drug resistance. Therapies targeting the epidermal growth factor receptor (EFGR) are widely used for end-stage CRC. However, patients with KRAS mutant genes cannot benefit from this therapy because of Ras signaling activation by KRAS mutant genes. Our previous study revealed the anti-proliferative effect of 4-acetyl-antroquinonol B (4-AAQB) on CRC cells, but whether the drug is effective in KRAS-mutant CRC remains unknown. We screened CRC cell lines harboring the KRAS mutation, namely G12A, G12C, G12V and G13D, with one wild type cell line as the control; SW1463 and Caco-2 cell lines were used for further experiments. Sulforhodamine B assays, together with the clonogenicity and invasion assay, revealed that KRAS-mutant SW1463 cells were resistant to cetuximab; however, 4-AAQB treatment effectively resensitized CRC cells to cetuximab through the reduction of colony formation, invasion, and tumorsphere generation and of oncogenic KRAS signaling cascade of CRC cells. Thus, inducing cells with 4-AAQB before cetuximab therapy could resensitize KRAS-mutant, but not wild-type, cells to cetuximab. Therefore, we hypothesized that 4-AAQB can inhibit KRAS. In silico analysis of the publicly available GEO (GSE66548) dataset of KRAS-mutated versus KRAS wild-type CRC patients confirmed that miR-193a-3p was significantly downregulated in the former compared with the latter patient population. Overexpression of miR-193a-3p considerably reduced the oncogenicity of both CRC cells. Furthermore, KRAS is a key target of miR-193a-3p. In vivo treatment with the combination of 4-AAQB and cetuximab significantly reduced the tumor burden of a xenograft mice model through the reduction of the expression of oncogenic markers (EGFR) and p-MEK, p-ERK, and c-RAF/p-c-RAF signaling, with the simultaneous induction of miR-193a-3p expression in the plasma. In summary, our findings provide strong evidence regarding the therapeutic effect of 4-AAQB on KRAS-mutant CRC cells. Furthermore, 4-AAQB effectively inhibits Ras singling in CRC cells, through which KRAS-mutant CRC can be resensitized to cetuximab.


Assuntos
Biomarcadores Tumorais/metabolismo , Cetuximab/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Ubiquinona/análogos & derivados , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Prognóstico , Células Tumorais Cultivadas , Ubiquinona/química , Ubiquinona/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
4.
Commun Biol ; 4(1): 688, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099862

RESUMO

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Associadas a Pancreatite/metabolismo , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/análise , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Proteínas Associadas a Pancreatite/análise , Transdução de Sinais , Células Tumorais Cultivadas , Quinases raf/metabolismo , Proteínas ras/metabolismo
5.
Mol Med Rep ; 23(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33880577

RESUMO

Esophageal squamous cell carcinoma (ESCC) is a major histological type of esophageal cancer, identified as a leading cause of tumor­associated death worldwide. In addition, long non­coding RNA (lncRNA) BRAF­activated non­coding RNA (BANCR) expression is increased in the plasma of patients with ESCC, which can be reversed by tumor resection. Thus, the aim of the present study was to investigate the underlying mechanism of BANCR in ESCC progression. The relative mRNA expression of BANCR was determined via reverse transcription­quantitative PCR. The cell behaviors of Eca­109 cells were detected using Cell Counting Kit­8, colony formation, wound healing and Transwell chamber assays. Finally, the expression levels of proteins involved in the Raf/MEK/ERK signaling pathway and cell metastasis were analyzed with western blotting. The results revealed that lncRNA BANCR was highly expressed in ESCC cells compared with in normal esophageal cells. BANCR overexpression enhanced proliferation, migration and invasion of ESCC cells, and BANCR silencing exerted opposite effects. Moreover, BANCR overexpression induced activation of the Raf/MEK/ERK signaling pathway in ESCC cells. Notably, U0126, a specific MEK inhibitor, decreased MEK and ERK expression, and blocked the promotive effects of BANCR overexpression on the proliferation, migration and invasion of ESCC cells. Overall, lncRNA BANCR facilitated the proliferation, migration and invasion of ESCC cells via the Raf/MEK/ERK signaling pathway. Thus, lncRNA BANCR may be a promising target for inhibiting ESCC growth and metastasis.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , RNA Longo não Codificante/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Progressão da Doença , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , RNA Longo não Codificante/genética , Quinases raf/metabolismo
6.
Genes (Basel) ; 12(4)2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-33920182

RESUMO

Oncogenic RAS (Rat sarcoma) mutations drive more than half of human cancers, and RAS inhibition is the holy grail of oncology. Thirty years of relentless efforts and harsh disappointments have taught us about the intricacies of oncogenic RAS signalling that allow us to now get a pharmacological grip on this elusive protein. The inhibition of effector pathways, such as the RAF-MEK-ERK pathway, has largely proven disappointing. Thus far, most of these efforts were aimed at blocking the activation of ERK. Here, we discuss RAF-dependent pathways that are regulated through RAF functions independent of catalytic activity and their potential role as targets to block oncogenic RAS signalling. We focus on the now well documented roles of RAF kinase-independent functions in apoptosis, cell cycle progression and cell migration.


Assuntos
Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Quinases raf/metabolismo , Proteínas ras/antagonistas & inibidores , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Quinases raf/genética
7.
Molecules ; 26(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923909

RESUMO

The binding free energy calculation of protein-ligand complexes is necessary for research into virus-host interactions and the relevant applications in drug discovery. However, many current computational methods of such calculations are either inefficient or inaccurate in practice. Utilizing implicit solvent models in the molecular mechanics generalized Born surface area (MM/GBSA) framework allows for efficient calculations without significant loss of accuracy. Here, GBNSR6, a new flavor of the generalized Born model, is employed in the MM/GBSA framework for measuring the binding affinity between SARS-CoV-2 spike protein and the human ACE2 receptor. A computational protocol is developed based on the widely studied Ras-Raf complex, which has similar binding free energy to SARS-CoV-2/ACE2. Two options for representing the dielectric boundary of the complexes are evaluated: one based on the standard Bondi radii and the other based on a newly developed set of atomic radii (OPT1), optimized specifically for protein-ligand binding. Predictions based on the two radii sets provide upper and lower bounds on the experimental references: -14.7(ΔGbindBondi)<-10.6(ΔGbindExp.)<-4.1(ΔGbindOPT1) kcal/mol. The consensus estimates of the two bounds show quantitative agreement with the experiment values. This work also presents a novel truncation method and computational strategies for efficient entropy calculations with normal mode analysis. Interestingly, it is observed that a significant decrease in the number of snapshots does not affect the accuracy of entropy calculation, while it does lower computation time appreciably. The proposed MM/GBSA protocol can be used to study the binding mechanism of new variants of SARS-CoV-2, as well as other relevant structures.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Algoritmos , Enzima de Conversão de Angiotensina 2/química , COVID-19/patologia , COVID-19/virologia , Entropia , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Quinases raf/química , Quinases raf/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo
8.
J Food Sci ; 86(6): 2753-2765, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33928646

RESUMO

25-Hydroxylprotopanaxadiol-3ß, 12ß, 20-triol (25-OH-PPD or AD-2) belongs to dammarane ginsenoside, and is commonly obtained from the acidic hydrolysate of total ginsensides of Panax ginseng. This study investigated the potential mechanism of AD-2 toward improving thioacetamide (TAA)-induced hepatic fibrosis in mice. Mice were divided into seven groups: control group, TAA model group, TAA + AD-2 (5, 10, and 20 mg/kg) groups, TAA + silymarin (100 mg/kg) group, and TAA + Fu Fang Biejia (FFBj; 300 mg/kg) group. All mice were treated to intraperitoneal TAA injection to establish a hepatic fibrosis model, and drugs were administered orally. The mechanism and related pathways underlying the AD-2-mediated action against hepatic fibrosis were explored by Western blotting and immunohistochemical staining. After AD-2 treatment, the expression levels of Lipin-1, SREBP1, and F4/80 significantly decreased, meanwhile the protein expressions levels of IL1ß, IL1R1, IL18, Bax, Bid, Bcl-2, and cFlips also decreased. Furthermore, AD-2 inhibited RAF and MEK pathways. The results demonstrate that AD-2 can alleviate hepatic fibrosis. The mechanism is likely related to the regulation of lipid accumulation, inflammatory response, apoptosis pathway, and Raf-MEK signaling pathways, which provide a basis for clinical research for the treatment of hepatic fibrosis. PRACTICAL APPLICATION: Ginsenoside is one of the main active ingredients of ginseng, and can alleviate the symptoms of various diseases, for example, hepatic fibrosis. This paper mainly used Western blotting to explore its possible mechanism of action. The goal was to provide a reference for the development of traditional Chinese medicines for hepatic fibrosis.


Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas/tratamento farmacológico , Ginsenosídeos/farmacologia , Inflamação/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Tioacetamida/toxicidade , Quinases raf/metabolismo , Animais , Doença Hepática Crônica Induzida por Substâncias e Drogas/etiologia , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Panax/química , Transdução de Sinais/efeitos dos fármacos
9.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33653954

RESUMO

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas p21(ras)/química , Quinases raf/química , Galectinas/química , Galectinas/genética , Galectinas/metabolismo , Humanos , Domínios Proteicos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/genética , Quinases raf/metabolismo
10.
Mol Med Rep ; 23(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649786

RESUMO

Spinal cord injury (SCI) remains a global challenge due to limited treatment strategies. Transcranial magnetic stimulation (TMS), bone marrow mesenchymal stem cell (BMSC) transplantation and downregulation of Raf/MEK/ERK signaling effectively improve SCI. The combination of BMSCs and TMS displays synergistic effects on vascular dementia. However, whether TMS displays a synergistic effect when combined with BMSC transplantation or Raf inhibitor (RafI) therapy for the treatment of SCI is not completely understood. The present study aimed to compare the therapeutic effect of monotherapy and combination therapy on SCI. In the present study, 8­week­old female Sprague Dawley rats were used to establish a model of SCI using the weight­drop method followed by treatment with monotherapy (TMS, BMSCs or RafI) or combination therapy (TMS+BMSCs or TMS+RafI). The effect of monotherapy and combination therapy on locomotor function, pathological alterations, neuronal apoptosis and expression of axonal regeneration­associated factors and Raf/MEK/ERK signaling­associated proteins in the spinal cord was analyzed by Basso, Beattie and Bresnahan (BBB) scoring, hematoxylin and eosin staining, TUNEL­neuronal nuclei (NeuN) staining and immunofluorescence or western blotting, respectively. The results demonstrated that compared with untreated SCI model rats, monotherapy significantly enhanced locomotor functional recovery, as evidenced by higher BBB scores, and slightly alleviated histopathological lesions of the spinal cord in SCI model rats. Furthermore, monotherapy markedly suppressed neuronal apoptosis and promoted axonal regeneration, as well as inhibiting astroglial activation in SCI model rats. The aforementioned results were demonstrated by significantly decreased numbers of apoptotic neurons, markedly decreased expression levels of glial fibrillary acidic protein (GFAP), significantly increased numbers of NeuN+ cells, markedly increased expression levels of growth­associated protein 43 (GAP­43) and significantly upregulated nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) expression levels in monotherapy groups (excluding the RafI monotherapy group) compared with untreated SCI model rats. In addition, monotherapy markedly suppressed activation of the Raf/MEK/ERK signaling pathway, as evidenced by significantly reduced p­Raf/Raf, p­MEK/MEK and p­ERK/ERK protein expression levels in monotherapy groups (excluding the BMSC monotherapy group) compared with untreated SCI model rats. Notably, combination therapy further alleviated SCI­induced spinal cord lesions and neuronal apoptosis, increased GAP­43, NGF and BDNF expression levels, downregulated GFAP expression levels and inhibited activation of the Raf/MEK/ERK signaling pathway in SCI model rats compared with the corresponding monotherapy groups. Therefore, it was hypothesized that compared with monotherapy, combination therapy displayed an improved therapeutic effect on SCI by further suppressing Raf/MEK/ERK signaling. The results of the present study provided an important basis for the clinical application of combination therapy.


Assuntos
Transplante de Medula Óssea/métodos , Indóis/farmacologia , Transplante de Células-Tronco Mesenquimais/métodos , Traumatismos da Medula Espinal/terapia , Sulfonamidas/farmacologia , Estimulação Magnética Transcraniana/métodos , Quinases raf/antagonistas & inibidores , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Terapia Combinada , Modelos Animais de Doenças , Feminino , Proteína GAP-43/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Atividade Motora/efeitos dos fármacos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Quinases raf/metabolismo
11.
Am J Chin Med ; 49(3): 753-765, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33641655

RESUMO

Lung cancer is the leading cause of cancer death in the world and classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). As tyrosine kinase inhibitors (TKIs), several triterpenoid saponins can target to epidermal growth factor receptor (EGFR), a widely used molecular therapeutic target, to exhibit remarkable anti-proliferative activities in cancer cells. As one of triterpenoid saponins, 20([Formula: see text])-ginsenoside Rg3 [20([Formula: see text])-Rg3] was confirmed to be an EGFR-TKI in this work. According to the quantitative real-time reverse transcription-PCR (qRT-PCR) and immunoblotting analysis, 20([Formula: see text])-Rg3 was certified to play a key role on EGFR/Ras/Raf/MEK/ERK signal pathway regulation. Our data demonstrated that 20([Formula: see text])-Rg3 might block the cell cycle at the G0/G1 phase by downregulating CDK2, Cyclin A2, and Cyclin E1. Molecular docking suggested that the combination of both hydrophobic and hydrogen-bonding interactions may help stabilizing the 20([Formula: see text])-Rg3-EGFR binding. Furthermore, their binding stability was assessed by molecular dynamics simulation. Taken together, these data provide the evidence that 20([Formula: see text])-Rg3 could prohibit A549 cell proliferation, probably by arresting the cell cycle at the G0/G1 phase via the EGFR/Ras/Raf/MEK/ERK pathway.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/genética , Ginsenosídeos/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Quinases raf/metabolismo , Proteínas ras/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ciclo Celular/genética , Receptores ErbB/metabolismo , Ginsenosídeos/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Terapia de Alvo Molecular , Fitoterapia
12.
J Med Chem ; 64(7): 3940-3955, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33780623

RESUMO

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.


Assuntos
Neoplasias/tratamento farmacológico , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinonas/uso terapêutico , Quinases raf/antagonistas & inibidores , Animais , Azetidinas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Cães , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos Nus , Estrutura Molecular , Mutação , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Piperidinas/uso terapêutico , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Quinazolinonas/química , Quinazolinonas/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/genética , Quinases raf/metabolismo
13.
J Ethnopharmacol ; 272: 113923, 2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-33617968

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Tanshinone-Ⅰ (TSNⅠ), a member of the mainly active components of Salvia miltiorrhiza Bunge (Dan Shen), which is widely used for the treatment for modern clinical diseases including cardiovascular and cerebrovascular diseases, has been reported to show the properties of anti-oxidation, anti-inflammation, neuroprotection and other pharmacological actions. However, whether TSNⅠ can improve neuron survival and neurological function against transient focal cerebral ischemia (tMCAO) in mice is still a blank field. AIM OF THE STUDY: This study aims to investigate the neuroprotective effects of TSNⅠ on ischemic stroke (IS) induced by tMCAO in mice and explore the potential mechanism of TSNⅠ against IS by combining network pharmacology approach and experimental verification. MATERIALS AND METHODS: In this study, the pivotal candidate targets of TSNⅠ against IS were screened by network pharmacology firstly. Enrichment analysis and molecular docking of those targets were performed to identify the possible mechanism of TSNⅠ against IS. Afterwards, experiments were carried out to further verify the mechanism of TSNⅠ against IS. The infarct volume and neurological deficit were evaluated by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Longa respectively. Immunohistochemistry was used to observe neuronal death in the hippocampus and cortical regions by detecting the change of NeuN. The predicting pathways of signaling-related proteins were assessed by Western blot in vitro and in vivo experiments. RESULTS: In vivo, TSNⅠ was found to dose-dependently decrease mice's cerebral infarct volume induced by tMCAO. In vitro, pretreatment with TSNⅠ could increase cell viability of HT-22 cell following oxygen-glucose deprivation (OGD/R). Moreover, the results showed that 125 candidate targets were identified, Protein kinase B (AKT) signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and mitogen-activated protein kinases 1 (MAPK1) and AKT1 could be bound to TSNⅠ more firmly by molecular docking analysis, which implies that TSNⅠ may play a role in neuroprotection through activating AKT and MAPK signaling pathways. Meanwhile, TSNⅠ was confirmed to significantly protect neurons from injury induced by IS through activating AKT and MAPK signaling pathways. CONCLUSION: In conclusion, our study clarifies that the mechanism of TSNⅠ against IS might be related to AKT and MAPK signaling pathways, which may provide the basic evidence for further development and utilization of TSNⅠ.


Assuntos
Abietanos/farmacologia , AVC Isquêmico/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Abietanos/uso terapêutico , Abietanos/toxicidade , Animais , Isquemia Encefálica/complicações , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipocampo/metabolismo , AVC Isquêmico/etiologia , AVC Isquêmico/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos ICR , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Fármacos Neuroprotetores/toxicidade , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Quinases raf/metabolismo
14.
Angew Chem Int Ed Engl ; 60(12): 6567-6572, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33427372

RESUMO

Cyclorasins 9A5 and 9A54 are 11-mer cyclic peptides that inhibit the Ras-Raf protein interaction. The peptides share a cell-penetrating peptide (CPP)-like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell-based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three-dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino-acid sidechains, Tle-2 and dVal-3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.


Assuntos
Peptídeos Cíclicos/farmacologia , Quinases raf/antagonistas & inibidores , Proteínas ras/antagonistas & inibidores , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/química , Conformação Proteica , Quinases raf/química , Quinases raf/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo
15.
Anticancer Res ; 41(1): 227-235, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33419817

RESUMO

BACKGROUND: 20(S)-Ginsenoside Rh2 (G-Rh2) has demonstrated therapeutic effects in many types of cancers. We aimed to investigate the potential anticancer activity and underlying mechanisms of G-Rh2 in oral cancer cells. MATERIALS AND METHODS: The antigrowth effect of G-Rh2 in oral cancer cells was stimulated by cell proliferation, soft agar colony formation, and migration and invasion assay. The cell cycle and apoptosis were detected by flow cytometry. The underlying mechanism of G-Rh2 in oral cancer cells was explored by immunoblotting. RESULTS: G-Rh2 significantly inhibited oral cancer cell growth by inducing apoptosis and cell cycle G0/G1-phase arrest. G-Rh2 inhibited oral cancer cell migration and invasion through regulation of epithelial-mesenchymal transition (EMT)-related proteins. G-Rh2 inhibited the Src/Raf/ERK signaling pathway in YD10B and Ca9-22 cells. CONCLUSION: G-Rh2 exerted anticancer activity in vitro by inhibiting the Src/Raf/ERK signaling pathway in oral cancer. G-Rh2 is a potential therapeutic drug for oral cancer treatment.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ginsenosídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases raf/metabolismo , Quinases da Família src/metabolismo , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Ginsenosídeos/química , Humanos , Neoplasias Bucais/metabolismo
17.
Clin Exp Hypertens ; 43(2): 142-150, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33070656

RESUMO

OBJECTIVE: To explore the effect of renal sympathetic denervation (RSD) on left ventricle hypertrophy and the Raf/MEK/ERK signaling pathway in spontaneously hypertensive rats (SHRs). METHODS: SHRs were divided into SHR, SHR + Sham, SHR + RSD and SHR + U0126 groups, with WKY rats as the baseline controls. The blood pressure of rats was observed, while myocardial fibrosis was evaluated through Masson staining. Thereafter, real-time quantitative polymerase chain reaction (qRT-PCR) was carried out to determine the levels of myocardial-hypertrophy-related markers, and Western blotting was used to measure the activity of the Raf/MEK/ERK signaling pathway. RESULTS: In comparison with the WKY group, significant increases were observed in the systolic pressure and diastolic pressure of rats from the other four groups at different time points after surgery. In addition, rats in these groups had obvious increases in LVMI, renal NE and IVSd and decreases in LVEDd, LVEF and LVFS. In addition, the CVF of myocardial tissues was increased, with the upregulation of ANP, BNP and ß-MHC and the downregulation of α-MHC. For the activity of the Raf/MEK/ERK signaling pathway, the levels of p-Raf/Raf, p-MEK/MEK and p-ERK1/2/ERK1/2 were all remarkably elevated (all P < .05). Further comparison with the SHR group showed that the above indexes in the rats were significantly improved in the RSD group and SHR + U0126 group (all P < .05). CONCLUSION: RSD may decrease blood pressure, mitigate hypertension-induced left ventricle hypertrophy and improve cardiac function efficiently in SHRs via the suppression of the Raf/MEK/ERK signaling pathway.


Assuntos
Hipertensão , Hipertrofia Ventricular Esquerda , Rim/inervação , Miocárdio , Simpatectomia/métodos , Animais , Biomarcadores/metabolismo , Fibrose/prevenção & controle , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Hipertensão/complicações , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertensão/cirurgia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/prevenção & controle , Sistema de Sinalização das MAP Quinases , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos Endogâmicos SHR , Quinases raf/metabolismo
18.
Environ Toxicol ; 36(5): 984-993, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33381906

RESUMO

Pesticide residues have become a healthy threaten of human beings. Among the pesticides, many of them have neurotoxicity. Extracellular Regulated Protein Kinases (ERK) pathway is an important signaling pathway that regulates a variety of downstream progress. In this work, peach (PRUNUS persica) and cherry (PRUNUS cerasus) were sampled from over 300 plantations in China and assessed for the residue risk. In mechanism studies, high-risk pesticide Avermectin showed a high activity inhibiting three neurotoxicity models, SH-SY5Y, PC-12 and SK-N-SH cells. At protein levels, ERK pathway proteins and their downstream proteins were obviously down-regulated. Moreover, the effects of low-dose Avermectin can be accumulated at protein levels in the low-dose long-term chronic toxicology detection.


Assuntos
Resíduos de Praguicidas , Quinases raf , China , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Ivermectina/análogos & derivados , MAP Quinase Quinase Quinases , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases raf/metabolismo
19.
Nat Commun ; 11(1): 6184, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273465

RESUMO

Environmental drought and high salinity impose osmotic stress, which inhibits plant growth and yield. Thus, understanding how plants respond to osmotic stress is critical to improve crop productivity. Plants have multiple signalling pathways in response to osmotic stress in which the phytohormone abscisic acid (ABA) plays important roles. However, since little is known concerning key early components, the global osmotic stress-signalling network remains to be elucidated. Here, we review recent advances in the identification of osmotic-stress activated Raf-like protein kinases as regulators of ABA-dependent and -independent signalling pathways and discuss the plant stress-responsive kinase network from an evolutionary perspective.


Assuntos
Pressão Osmótica , Plantas/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinases raf/metabolismo , Ativação Enzimática , Transdução de Sinais
20.
Molecules ; 25(21)2020 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-33171577

RESUMO

Coumarins, which occur naturally in the plant kingdom, are diverse class of secondary metabolites. With their antiproliferative, chemopreventive and antiangiogenetic properties, they can be used in the treatment of cancer. Their therapeutic potential depends on the type and location of the attachment of substituents to the ring. Therefore, the aim of our study was to investigate the effect of simple coumarins (osthole, umbelliferone, esculin, and 4-hydroxycoumarin) combined with sorafenib (specific inhibitor of Raf (Rapidly Accelerated Fibrosarcoma) kinase) in programmed death induction in human glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells lines. Osthole and umbelliferone were isolated from fruits: Mutellina purpurea L. and Heracleum leskowii L., respectively, while esculin and 4-hydroxycoumarin were purchased from Sigma Aldrich (St. Louis, MO, USA). Apoptosis, autophagy and necrosis were identified microscopically after straining with specific fluorochromes. The level of caspase 3, Beclin 1, PI3K (Phosphoinositide 3-kinase), and Raf kinases were estimated by immunoblotting. Transfection with specific siRNA (small interfering RNA) was used to block Bcl-2 (B-cell lymphoma 2), Raf, and PI3K expression. Cell migration was tested with the wound healing assay. The present study has shown that all the coumarins eliminated the MOGGCCM and T98G tumor cells mainly via apoptosis and, to a lesser extent, via autophagy. Osthole, which has an isoprenyl moiety, was shown to be the most effective compound. Sorafenib did not change the proapoptotic activity of this coumarin; however, it reduced the level of autophagy. At the molecular level, the induction of apoptosis was associated with a decrease in the expression of PI3K and Raf kinases, whereas an increase in the level of Beclin 1 was observed in the case of autophagy. Inhibition of the expression of this protein by specific siRNA eliminated autophagy. Moreover, the blocking of the expression of Bcl-2 and PI3K significantly increased the level of apoptosis. Osthole and sorafenib successfully inhibited the migration of the MOGGCCM and T98G cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cumarínicos/farmacologia , Glioblastoma/tratamento farmacológico , Magnoliopsida/química , Extratos Vegetais/farmacologia , Sorafenibe/farmacologia , 4-Hidroxicumarinas/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Esculina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Necrose/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/metabolismo , Umbeliferonas/farmacologia , Quinases raf/metabolismo
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