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1.
Tumour Biol ; 46(1): 1-11, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38728194

RESUMO

BACKGROUND: It is well established that most colorectal carcinomas arise from conventional adenomas through the adenoma-carcinoma sequence (ACS) model. mitogen-activated protein kinases (MAPKs) pathway has been reported as a crucial player in tumorigenesis. The MAPK signaling pathway is activated by different extracellular signals involving the "mitogen-activated/extracellular signal-regulated kinase 1 (MEK1)", and this induces the expression of genes involved in proliferation and cellular transformation. Diaphanous-related formin-3 (DIAPH3) acts as a potential metastasis regulator through inhibiting the cellular transition to amoeboid behavior in different cancer types. OBJECTIVE: The aim of the study was to investigate the pattern of immunohistochemical expression of MEK1 and DIAPH3 in colorectal adenoma (CRA) and corresponding colorectal carcinoma (CRC) specimens. METHODS: The immunohistochemical expression of DIAPH3 and MEK1 was examined in 43 cases of CRC and their associated adenomas using tissue microarray technique. RESULTS: MEK1 was overexpressed in 23 CRC cases (53.5%) and in 20 CRA cases (46.5%). DIAPH3 was overexpressed in 11 CRA cases (about 29%) which were significantly lower than CRC (22 cases; 58%) (P = 0.011). Both MEK1 and DIAPH3 overexpression were significantly correlated in CRC (P = 0.009) and CRA cases (P = 0.002). Tumors with MEK1 overexpression had a significantly higher tumor grade (P = 0.050) and perineural invasion (P = 0.017). CONCLUSIONS: Both MEK1 and DIAPH3 are overexpressed across colorectal ACS with strong correlation between them. This co- expression suggests a possible synergistic effect of MEK1 and DIAPH-3 in colorectal ACS. Further large-scale studies are required to investigate the potential functional aspects of MEK1 and DIAPH3 in ACS and their involvement in tumor initiation and the metastatic process.


Assuntos
Adenoma , Neoplasias Colorretais , Forminas , MAP Quinase Quinase 1 , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Forminas/genética , Forminas/metabolismo , Adenoma/patologia , Adenoma/genética , Adenoma/metabolismo , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Adulto , Imuno-Histoquímica , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Carcinoma/patologia , Carcinoma/genética , Carcinoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética
2.
Nat Commun ; 15(1): 3636, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710699

RESUMO

Polypharmacology drugs-compounds that inhibit multiple proteins-have many applications but are difficult to design. To address this challenge we have developed POLYGON, an approach to polypharmacology based on generative reinforcement learning. POLYGON embeds chemical space and iteratively samples it to generate new molecular structures; these are rewarded by the predicted ability to inhibit each of two protein targets and by drug-likeness and ease-of-synthesis. In binding data for >100,000 compounds, POLYGON correctly recognizes polypharmacology interactions with 82.5% accuracy. We subsequently generate de-novo compounds targeting ten pairs of proteins with documented co-dependency. Docking analysis indicates that top structures bind their two targets with low free energies and similar 3D orientations to canonical single-protein inhibitors. We synthesize 32 compounds targeting MEK1 and mTOR, with most yielding >50% reduction in each protein activity and in cell viability when dosed at 1-10 µM. These results support the potential of generative modeling for polypharmacology.


Assuntos
Simulação de Acoplamento Molecular , Humanos , Serina-Treonina Quinases TOR/metabolismo , Polifarmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 1/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Ligação Proteica , Descoberta de Drogas/métodos , Desenho de Fármacos , Sobrevivência Celular/efeitos dos fármacos
3.
Aging (Albany NY) ; 16(5): 4224-4235, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38431286

RESUMO

Alcoholic liver disease (ALD) serves as the leading cause of chronic liver diseases-related morbidity and mortality, which threatens the life of millions of patients in the world. However, the molecular mechanisms underlying ALD progression remain unclear. Here, we applied microarray analysis and experimental approaches to identify miRNAs and related regulatory signaling that associated with ALD. Microarray analysis identified that the expression of miR-99b was elevated in the ALD mouse model. The AML-12 cells were treated with EtOH and the expression of miR-99b was enhanced in the cells. The expression of miR-99b was positively correlated with ALT levels in the ALD mice. The microarray analysis identified the abnormally expressed mRNAs in ALD mice and the overlap analysis was performed with based on the differently expressed mRNAs and the transcriptional factors of miR-99b, in which STAT1 was identified. The elevated expression of STAT1 was validated in ALD mice. Meanwhile, the treatment of EtOH induced the expression of STAT1 in the AML-12 cells. The expression of STAT1 was positively correlated with ALT levels in the ALD mice. The positive correlation of STAT1 and miR-99b expression was identified in bioinformatics analysis and ALD mice. The expression of miR-99b and pri-miR-99b was promoted by the overexpression of STAT1 in AML-12 cells. ChIP analysis confirmed the enrichment of STAT1 on miR-99b promoter in AML-12 cells. Next, we found that the expression of mitogen-activated protein kinase kinase 1 (MAP2K1) was negatively associated with miR-99b. The expression of MAP2K1 was downregulated in ALD mice. Consistently, the expression of MAP2K1 was reduced by the treatment of EtOH in AML-12 cells. The expression of MAP2K1 was negative correlated with ALT levels in the ALD mice. We identified the binding site of MAP2K1 and miR-99b. Meanwhile, the treatment of miR-99b mimic repressed the luciferase activity of MAP2K1 in AML-12 cells. The expression of MAP2K1 was suppressed by miR-99b in the cells. We observed that the expression of MAP2K1 was inhibited by the overexpression of STAT1 in AML-12 cells. Meanwhile, the apoptosis of AML-12 cells was induced by the treatment of EtOH, while miR-99b mimic promoted but the overexpression of MAP2K1 attenuated the effect of EtOH in the cells. In conclusion, we identified the correlation and effect of STAT1, miR-99b, and MAP2K1 in ALD mouse model and hepatocyte. STAT1, miR-99b, and MAP2K1 may serve as potential therapeutic target of ALD.


Assuntos
Leucemia Mieloide Aguda , Hepatopatias Alcoólicas , MicroRNAs , Humanos , Animais , Camundongos , MAP Quinase Quinase 1/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Hepatócitos/metabolismo , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/metabolismo , Etanol , Leucemia Mieloide Aguda/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo
4.
Transplantation ; 108(5): 1127-1141, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38238904

RESUMO

BACKGROUND: Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. METHODS: We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. RESULTS: Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. CONCLUSIONS: These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.


Assuntos
Rejeição de Enxerto , Transplante de Coração , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Macrófagos , Camundongos Knockout , Animais , Transplante de Coração/efeitos adversos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/patologia , Rejeição de Enxerto/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , MAP Quinase Quinase 2/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 1/genética , Proteínas de Ligação a Hormônio da Tireoide , Camundongos Endogâmicos C57BL , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Masculino , Transdução de Sinais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Glicólise , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Modelos Animais de Doenças , Fenótipo , Aloenxertos
5.
J Vasc Res ; 60(4): 213-226, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37778342

RESUMO

INTRODUCTION: Cardiovascular disorders are characterized by vascular smooth muscle (VSM) transition from a contractile to proliferative state. Protease-activated receptor 2 (PAR2) involvement in this phenotypic conversion remains unclear. We hypothesized that PAR2 controls VSM cell proliferation in phenotype-dependent manner and through specific protein kinases. METHODS: Rat clonal low (PLo; P3-P6) and high passage (PHi; P10-P15) VSM cells were established as respective models of quiescent and proliferative cells, based on reduced PKG-1 and VASP. Western blotting determined expression of cytoskeletal/contractile proteins, PAR2, and select protein kinases. DNA synthesis and cell proliferation were measured 24-72 h following PAR2 agonism (SLIGRL; 100 nM-10 µm) with/without PKA (PKI; 10 µm), MEK1/2 (PD98059; 10 µm), and PI3K (LY294002; 1 µm) blockade. RESULTS: PKG-1, VASP, SM22α, calponin, cofilin, and PAR2 were reduced in PHi versus PLo cells. Following PAR2 agonism, DNA synthesis and cell proliferation increased in PLo cells but decreased in PHi cells. Western analyses showed reduced PKA, MEK1/2, and PI3K in PHi versus PLo cells, and kinase blockade revealed PAR2 controls VSM cell proliferation through PKA/MEK1/2. DISCUSSION: Findings highlight PAR2 and PAR2-driven PKA/MEK1/2 in control of VSM cell growth and provide evidence for continued investigation of PAR2 in VSM pathology.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Receptor PAR-2 , Ratos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , MAP Quinase Quinase 1/metabolismo , Músculo Liso Vascular/metabolismo , Proliferação de Células , Fosfatidilinositol 3-Quinases/metabolismo , DNA/metabolismo , Células Cultivadas
6.
Philos Trans R Soc Lond B Biol Sci ; 378(1890): 20220246, 2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37778380

RESUMO

Drug resistance is still a big challenge for cancer patients. We previously demonstrated that inhibiting peptidylarginine deiminase 2 (PADI2) enzyme activity with Cl-amine increases the efficacy of docetaxel (Doc) on tamoxifen-resistant breast cancer cells with PADI2 expression. However, it is not clear whether this effect applies to other tumour cells. Here, we collected four types of tumour cells with different PADIs expression and fully evaluated the inhibitory effect of the combination of PADIs inhibitor (BB-Cla) and Doc in vitro and in vivo on tumour cell growth. Results show that inhibiting PADIs combined with Doc additively inhibits tumour cell growth across the four tumour cells. PADI2-catalysed citrullination of MEK1 Arg 189 exists in the four tumour cells, and blocking the function of MEK1 Cit189 promotes the anti-tumour effect of Doc in these tumour cells. Further analysis shows that inhibiting MEK1 Cit189 decreases the expression of cancer cell stemness factors and helps prevent cancer cell stemness maintenance. Importantly, this combined treatment can partially restore the sensitivity of chemotherapy-resistant cells to docetaxel or cisplatin in tumour cells. Thus, our study provides an experimental basis for the combined therapeutic approaches using docetaxel- and PADIs inhibitors-based strategies in tumour treatment. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.


Assuntos
Antineoplásicos , Citrulinação , Docetaxel , Resistencia a Medicamentos Antineoplásicos , MAP Quinase Quinase 1 , Humanos , Docetaxel/farmacologia , Tamoxifeno , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Antineoplásicos/farmacologia
7.
J Vet Med Sci ; 85(9): 977-984, 2023 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-37495516

RESUMO

Melanoma is a highly aggressive and metastatic cancer occurring in both humans and dogs. Canine melanoma accounts for a significant proportion of neoplastic diseases in dogs, and despite standard treatments, overall survival rates remain low. Protein phosphatase 6 (PP6), an evolutionarily conserved serine/threonine protein phosphatase, regulates various biological processes. Additionally, the loss of PP6 function reportedly leads to the development of melanoma in humans. However, there are no reports regarding the role of PP6 in canine cancer cells. We, therefore, conducted a study investigating the role of PP6 in canine melanoma by using four canine melanoma cell lines: CMec1, CMM, KMeC and LMeC. PP6 knockdown increased phosphorylation levels of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) but not Akt. Furthermore, PP6 knockdown decreased sensitivity to trametinib, a MEK inhibitor, but did not alter sensitivity to Akt inhibitor. These findings suggest that PP6 may function as a tumor suppressor in canine melanoma and modulate the response to trametinib treatment. Understanding the role of PP6 in canine melanoma could lead to the development of more effective treatment strategies for this aggressive disease.


Assuntos
Doenças do Cão , Melanoma , Animais , Cães , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 1/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/farmacologia , Sistema de Sinalização das MAP Quinases , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Melanoma/tratamento farmacológico , Melanoma/veterinária , Linhagem Celular Tumoral , Doenças do Cão/tratamento farmacológico
8.
Cells ; 12(12)2023 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-37371121

RESUMO

Peptide Lv is a small endogenous secretory peptide that is proangiogenic through hyperpolarizing vascular endothelial cells (ECs) by enhancing the current densities of KCa3.1 channels. However, it is unclear how peptide Lv enhances these currents. One way to enhance the current densities of ion channels is to promote its trafficking and insertion into the plasma membrane. We hypothesized that peptide Lv-elicited KCa3.1 augmentation occurs through activating the mitogen-activated protein kinase kinase 1 (MEK1)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathways, which are known to mediate ion channel trafficking and membrane insertion in neurons. To test this hypothesis, we employed patch-clamp electrophysiological recordings and cell-surface biotinylation assays on ECs treated with peptide Lv and pharmaceutical inhibitors of ERK and Akt. Blocking ERK or Akt activation diminished peptide Lv-elicited EC hyperpolarization and increase in KCa3.1 current densities. Blocking PI3K or Akt activation decreased the level of plasma membrane-bound, but not the total amount of KCa3.1 protein in ECs. Therefore, the peptide Lv-elicited EC hyperpolarization and KCa3.1 augmentation occurred in part through channel trafficking and insertion mediated by MEK1-ERK and PI3K-Akt activation. These results demonstrate the molecular mechanisms of how peptide Lv promotes EC-mediated angiogenesis.


Assuntos
Células Endoteliais , MAP Quinases Reguladas por Sinal Extracelular , Proteínas Proto-Oncogênicas c-akt , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinase Quinase 1/metabolismo , Peptídeos , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
9.
Cell Biol Int ; 47(2): 480-491, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36273427

RESUMO

Cardiac hypertrophy caused by angiotensin II (Ang II) is essential for the pathological process of heart failure. The intermediate calcium-activated potassium channel (SK4) has been shown to be involved in the process of the inflammatory response, cell proliferation, and apoptosis. However, the role of SK4 in cardiac hypertrophy has not been elucidated. Cardiac hypertrophy in human-induced pluripotent stem cells-derived cardiomyocytes (HiPSC-CMs) was induced by Ang II. Cells were transfected with SK4 adenovirus or treated with SK4 inhibitor (TRAM-34). TUNEL staining was used to assess the levels of apoptosis. Real-time polymerase chain reaction and Western blot analysis were used to measure messenger RNA (mRNA) and protein levels, respectively. The present results showed that SK4 expression was upregulated in HiPSC-CMs stimulated by Ang II. The downregulation of SK4 by a specific inhibitor TRAM-34 markedly ameliorated cardiac hypertrophy (reflected by the mRNA levels of atrial natriuretic peptide, brain natriuretic peptide, and ß-myosin heavy chain) and apoptosis (reflected by the level of Caspase 3, Bax, and Bcl-2) induced by Ang II treatment. The action of SK4 in cardiac hypertrophy was mediated by Ras-Raf-mitogen-activated protein kinases 1/2 (MEK1/2)-extracellular-regulated protein kinases 1/2 (ERK1/2) and calcineurin (CN)-nuclear factors of activated T cells (NFAT) activation. Our studies demonstrated that inhibition of SK4 significantly alleviated cardiac hypertrophy induced by Ang II in hiPSC-CMs by targeting Ras-Raf-MEK1/2-ERK1/2 signaling and CN-NFAT signaling pathway. Our studies suggest that SK4 may serve as a potential therapeutic target that could delay hypertrophy.


Assuntos
Angiotensina II , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Fatores de Transcrição NFATC/metabolismo , Sistema de Sinalização das MAP Quinases , MAP Quinase Quinase 1/metabolismo , Transdução de Sinais , Cardiomegalia/metabolismo , RNA Mensageiro/metabolismo , Células-Tronco/metabolismo
10.
Biomed Res Int ; 2022: 7375661, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36203485

RESUMO

Purpose: Gastric cancer(GC)is one of the deadliest digestive tract tumors worldwide,existing studies suggest that dysregulated expression of microRNAs (miRNAs) plays an important role in the pathogenesis and progression of GC. This study aimed to investigate the expression, biological function, and downstream mechanism of miR-34c-5p in GC, provide new targets for gastric cancer diagnosis and treatment. Methods: The expression of miR-34c-5p in GC tissues and cell lines was examined by RT-qPCR. Cell wound healing, transwell and cell cloning assays were used to detect the effect of miR-34c-5p on the migration and invasion abilities, respectively, of GC cells. Western blot was performed to detect the expression of related proteins. Bioinformatics analysis was used to predict the binding of MAP2K1 to miR-34c-5p and the targeting relationship was confirmed by dual luciferase reporter assay. Results: The expression level of miR-34c-5p was significantly decreased in GC tissues and cell lines. miR-34c-5p overexpression inhibited migration, invasion, and colony formation of gastric cancer cells, the related protein E-cadherin expression was significantly increased and N-cadherin, vimentin, and PCNA expression were significantly decreased, while miR-34c-5p knockdown exerted the opposite effects. In addition, the targeting relationship between miR-34c-5p and MAP2K1 was predicted and confirmed, and further confirmed by rescue experiments that MAP2K1 alleviated the inhibitory effect of miR-34c-5p in GC. Conclusion: MiR-34c-5p is lowly expressed in GC, and it can target MAP2K1 to exert inhibitory effects on GC proliferation, invasion, and migration. These findings provide a promising biomarker and a potential therapeutic target for gastric cancer.


Assuntos
MicroRNAs , Neoplasias Gástricas , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 1/farmacologia , MAP Quinase Quinase 1/uso terapêutico , MicroRNAs/metabolismo , Processos Neoplásicos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Neoplasias Gástricas/patologia , Vimentina/metabolismo
11.
Cell Death Dis ; 13(8): 701, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35961969

RESUMO

Oral submucous fibrosis (OSF) is a chronic and insidious oral potentially malignant disorder associated with a 4-17% risk of oral squamous cell carcinoma (OSCC). Our previous study found that proteasomal activator 28 gamma (PA28γ) is frequently overexpressed in oral squamous cell carcinoma and negatively correlated with poor patient prognosis. However, the role of PA28γ in the occurrence and development of OSF remains unclear. Here, we screened PA28γ-related genes and investigated their function in OSF. We demonstrated that the expression of PA28γ was positively associated with MEK1 and gradually elevated from normal to progressive stages of OSF tissue. Arecoline, a pathogenic component of OSF, could upregulate the protein levels of PA28γ and phosphorylated MEK1 and contribute to epithelial to mesenchymal transition (EMT) in epithelial cells. Notably, PA28γ could interact with MEK1 and upregulate its phosphorylation level. Furthermore, arecoline upregulated BRAF, which can interact with PA28γ and upregulate its protein level. Additionally, BRAF, PA28γ, and MEK1 could form protein complexes and then enhance the MEK1/ERK signaling pathways. The concrete mechanism of the protein stability of PA28γ is that BRAF mediates its degradation by inhibiting its ubiquitination. These findings underscore the instrumental role of PA28γ in the BRAF/MEK1 pathway and enhanced EMT through MEK1/ERK activation in OSF.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Fibrose Oral Submucosa , Arecolina/farmacologia , Autoantígenos , Carcinoma de Células Escamosas/patologia , Transição Epitelial-Mesenquimal/genética , Humanos , MAP Quinase Quinase 1/metabolismo , Neoplasias Bucais/patologia , Fibrose Oral Submucosa/genética , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas B-raf , Carcinoma de Células Escamosas de Cabeça e Pescoço
12.
Cell Biochem Funct ; 40(5): 481-490, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35604288

RESUMO

MEK1 is an attractive target due to its role in selective extracellular-signal-regulated kinase phosphorylation, which plays a pivotal role in regulating cell proliferation. Another benefit of targeting the MEK protein is its unique hydrophobic pocket that can accommodate highly selective allosteric inhibitors. To date, various MEK1 inhibitors have reached clinical trials against several cancers, but they were discarded due to their severe toxicity and low efficacy. Thus, the development of allosteric inhibitors for MEK1 is the demand of the hour. In this in-silico study, molecular docking, long-term molecular dynamics (5 µs), and molecular mechanics Poisson-Boltzmann surface area analysis were undertaken to address the potential of quinolines as allosteric inhibitors. We selected four reference MEK1 inhibitors for the comparative analysis. The drug-likeness and toxicity of these molecules were also examined based on their ADMET and Toxicity Prediction by Komputer Assisted Technology profiles. The outcome of the analysis revealed that the quinolines (4m, 4o, 4s, and 4n) exhibited better stability and binding affinity while being nontoxic compared to reference inhibitors. We have reached the conclusion that these quinoline molecules could be checked by experimental studies to validate their use as allosteric inhibitors against MEK1.


Assuntos
Inibidores de Proteínas Quinases , Quinolinas , Sítio Alostérico , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia
13.
Cell Mol Life Sci ; 79(5): 281, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508574

RESUMO

MEK1 interactions with B-Raf and KSR1 are key steps in Ras/Raf/MEK/ERK signaling. Despite this, vital mechanistic details of how these execute signal transduction are still enigmatic. Among these is why, despite B-Raf and KSR1 kinase domains similarity, the B-Raf/MEK1 and KSR1/MEK1 complexes have distinct contributions to MEK1 activation, and broadly, what is KSR1's role. Our molecular dynamics simulations clarify these still unresolved ambiguities. Our results reveal that the proline-rich (P-rich) loop of MEK1 plays a decisive role in MEK1 activation loop (A-loop) phosphorylation. In the inactive B-Raf/MEK1 heterodimer, the collapsed A-loop of B-Raf interacts with the P-rich loop and A-loop of MEK1, minimizing MEK1 A-loop fluctuation and preventing it from phosphorylation. In the active B-Raf/MEK1 heterodimer, the P-rich loop moves in concert with the A-loop of B-Raf as it extends. This reduces the number of residues interacting with MEK1 A-loop, allowing increased A-loop fluctuation, and bringing Ser222 closer to ATP for phosphorylation. B-Raf αG-helix Arg662 promotes MEK1 activation by orienting Ser218 towards ATP. In KSR1/MEK1, the KSR1 αG-helix has Ala826 in place of B-Raf Arg662. This difference results in much fewer interactions between KSR1 αG-helix and MEK1 A-loop, thus a more flexible A-loop. We postulate that if KSR1 were to adopt an active configuration with an extended A-loop as seen in other protein kinases, then the MEK1 P-rich loop would extend in a similar manner, as seen in the active B-Raf/MEK1 heterodimer. This would result in highly flexible MEK1 A-loop, and KSR1 functioning as an active, B-Raf-like, kinase.


Assuntos
Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Trifosfato de Adenosina/metabolismo , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais
14.
Phytochemistry ; 198: 113154, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35245525

RESUMO

Three undescribed diterpenes including two ent-abietanes, euphomauritanol A, and euphomauritanol B, and one jatrophane, euphomauritanophane A, in addition to eight previously described metabolites were isolated from the MeOH-CH2Cl2 (1:1) extract of the Euphorbia mauritanica. The chemical structures of isolates were established based on the spectroscopic means including FT-IR, HRMS, 1D and 2D NMR. The absolute stereochemistry of the undescribed diterpenes was deduced by experimental and calculated TDDFT-electronic circular dichroism (ECD). The anti-proliferative effects of the isolated diterpenes were evaluated against B16-BL6, Hep G2, and Caco-2. The euphomauritanol A, euphomauritanol B, and euphomauritanophane A significantly inhibited the growth of murine melanoma B16-BL6 cell lines with IC50 10.28, 20.22, and 38.81 µM, respectively with no responses against the other cells. These activities were rationalized by molecular docking of the active compounds in BRAFV600E and MEK1 active sites. Moreover, the in-silico pharmacokinetics predictions by Swiss ADME revealed that the active compounds possessed favorable oral bioavailability and drug-likeness properties.


Assuntos
Diterpenos , Euphorbia , MAP Quinase Quinase 1 , Melanoma , Proteínas Proto-Oncogênicas B-raf , Animais , Células CACO-2 , Diterpenos/química , Diterpenos/farmacologia , Egito , Euphorbia/química , Células Hep G2 , Humanos , MAP Quinase Quinase 1/metabolismo , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Proto-Oncogênicas B-raf/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Lancet ; 399(10324): 541-553, 2022 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-35123694

RESUMO

BACKGROUND: Low-grade serous carcinoma of the ovary or peritoneum is characterised by MAPK pathway aberrations and its reduced sensitivity to chemotherapy relative to high-grade serous carcinoma. We compared the MEK inhibitor trametinib to physician's choice standard of care in patients with recurrent low-grade serous carcinoma. METHODS: This international, randomised, open-label, multicentre, phase 2/3 trial was done at 84 hospitals in the USA and UK. Eligible patients were aged 18 years or older with recurrent low-grade serous carcinoma and measurable disease, as defined by Response Evaluation Criteria In Solid Tumors version 1.1, had received at least one platinum-based regimen, but not all five standard-of-care drugs, and had received an unlimited number of previous regimens. Patients with serous borderline tumours or tumours containing low-grade serous and high-grade serous carcinoma were excluded. Eligible patients were randomly assigned (1:1) to receive either oral trametinib 2 mg once daily (trametinib group) or one of five standard-of-care treatment options (standard-of-care group): intravenous paclitaxel 80 mg/m2 by body surface area on days 1, 8, and 15 of every 28-day cycle; intravenous pegylated liposomal doxorubicin 40-50 mg/m2 by body surface area once every 4 weeks; intravenous topotecan 4 mg/m2 by body surface area on days 1, 8, and 15 of every 28-day cycle; oral letrozole 2·5 mg once daily; or oral tamoxifen 20 mg twice daily. Randomisation was stratified by geographical region (USA or UK), number of previous regimens (1, 2, or ≥3), performance status (0 or 1), and planned standard-of-care regimen. The primary endpoint was investigator-assessed progression-free survival while receiving randomised therapy, as assessed by imaging at baseline, once every 8 weeks for 15 months, and then once every 3 months thereafter, in the intention-to-treat population. Safety was assessed in patients who received at least one dose of study therapy. This trial is registered with ClinicalTrials.gov, NCT02101788, and is active but not recruiting. FINDINGS: Between Feb 27, 2014, and April 10, 2018, 260 patients were enrolled and randomly assigned to the trametinib group (n=130) or the standard-of-care group (n=130). At the primary analysis, there were 217 progression-free survival events (101 [78%] in the trametinib group and 116 [89%] in the standard-of-care group). Median progression-free survival in the trametinib group was 13·0 months (95% CI 9·9-15·0) compared with 7·2 months (5·6-9·9) in the standard-of-care group (hazard ratio 0·48 [95% CI 0·36-0·64]; p<0·0001). The most frequent grade 3 or 4 adverse events in the trametinib group were skin rash (17 [13%] of 128), anaemia (16 [13%]), hypertension (15 [12%]), diarrhoea (13 [10%]), nausea (12 [9%]), and fatigue (ten [8%]). The most frequent grade 3 or 4 adverse events in the standard-of-care group were abdominal pain (22 [17%]), nausea (14 [11%]), anaemia (12 [10%]), and vomiting (ten [8%]). There were no treatment-related deaths. INTERPRETATION: Trametinib represents a new standard-of-care option for patients with recurrent low-grade serous carcinoma. FUNDING: NRG Oncology, Cancer Research UK, Target Ovarian Cancer, and Novartis.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Epitelial do Ovário/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Administração Oral , Adulto , Idoso , Carcinoma Epitelial do Ovário/patologia , Feminino , Humanos , MAP Quinase Quinase 1/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia/patologia , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Intervalo Livre de Progressão , Padrão de Cuidado , Resultado do Tratamento , Reino Unido , Estados Unidos
16.
J Biol Chem ; 298(3): 101634, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35085550

RESUMO

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.


Assuntos
Sistema de Sinalização das MAP Quinases , MicroRNAs , Linfócitos T , Regiões 3' não Traduzidas , Animais , Ativação Linfocitária , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/imunologia , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , MicroRNAs/genética , MicroRNAs/imunologia , MicroRNAs/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Elk-1 do Domínio ets/imunologia , Proteínas Elk-1 do Domínio ets/metabolismo
17.
Cell Rep ; 38(2): 110223, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35021072

RESUMO

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development.


Assuntos
Ativação Linfocitária/fisiologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Alelos , Animais , Linfócitos B/metabolismo , Feminino , Humanos , Ativação Linfocitária/genética , MAP Quinase Quinase 1/fisiologia , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo
18.
Cell Mol Life Sci ; 79(1): 65, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013790

RESUMO

Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.


Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Fenamatos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células A549 , Adulto , Animais , COVID-19/metabolismo , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Citocinas/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos
19.
Cell Rep ; 37(13): 110155, 2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-34965411

RESUMO

During somatic reprogramming, cellular energy metabolism fundamentally switches from predominantly mitochondrial oxidative phosphorylation toward glycolysis. This metabolic reprogramming, also called the Warburg effect, is critical for the induction of pluripotency, but its molecular mechanisms remain poorly defined. Notably, SIRT2 is consistently downregulated during the reprogramming process and regulates glycolytic switch. Here, we report that downregulation of SIRT2 increases acetylation of mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) at Lys175, resulting in activation of extracellular signal-regulated kinases (ERKs) and subsequent activation of the pro-fission factor dynamin-related protein 1 (DRP1). In parallel, downregulation of SIRT2 hyperacetylates the serine/threonine protein kinase AKT1 at Lys20 in a non-canonical way, activating DRP1 and metabolic reprogramming. Together, our study identified two axes, SIRT2-MEK1-ERK-DRP1 and SIRT2-AKT1-DRP1, that critically link mitochondrial dynamics and oxidative phosphorylation to the somatic reprogramming process. These upstream signals, together with SIRT2's role in glycolytic switching, may underlie the Warburg effect observed in human somatic cell reprogramming.


Assuntos
Reprogramação Celular , Dinaminas/metabolismo , MAP Quinase Quinase 1/metabolismo , Dinâmica Mitocondrial , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirtuína 2/metabolismo , Acetilação , Dinaminas/genética , Metabolismo Energético , Fibroblastos/metabolismo , Glicólise , Humanos , MAP Quinase Quinase 1/genética , Mitocôndrias/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação Oxidativa , Proteínas Proto-Oncogênicas c-akt/genética , Sirtuína 2/genética
20.
Nat Struct Mol Biol ; 28(10): 847-857, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34625747

RESUMO

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.


Assuntos
Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Galectinas/química , Galectinas/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutagênese , Multimerização Proteica , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
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