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1.
Anticancer Res ; 39(11): 5867-5877, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704811

RESUMO

BACKGROUND/AIM: The aim of this study was to examine clonal heterogeneity, to test the utility of liquid biopsy in monitoring disease progression and to evaluate the usefulness of ex vivo drug screening in a BRAF L597Q-mutated colorectal cancer (CRC) patient developing metastases during adjuvant therapy. MATERIALS AND METHODS: Next generation sequencing (NGS) and droplet digital PCR (ddPCR) were performed in samples from tumor tissues and liquid biopsies. Live cancer cells from a metastatic lesion were used in ex vivo drug sensitivity assays. RESULTS: We found evidence of continued dependence of MEK/MAPK pathway activation, but different activating mutations in primary tumor and metastases. Liquid biopsy based BRAF L597Q ddPCR testing was a sensitive personalized biomarker predicting the rise of clinically aggressive metastatic disease. Ex vivo drug sensitivity assays with BRAF L597Q mutated cells showed response to MEK/MAPK targeted therapies. CONCLUSION: The rare BRAF L597Q mutation may be associated with aggressive tumor behavior in CRC. Liquid biopsy can be used to capture clinically relevant tumor features.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/secundário , MAP Quinase Quinase 1/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Capecitabina/administração & dosagem , Evolução Clonal , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxaliplatina/administração & dosagem , Prognóstico
2.
Cancer Treat Rev ; 81: 101907, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31715422

RESUMO

The aberrant activation of RAS-derived mitogen-activated protein kinase (MAPK) signaling pathway plays a prominent role in tumorigenesis of an array of malignancies. The reasons are usually the upstream activated mutations including mitogen-activated protein kinase kinase 1/2 (MEK1/2). As oncogenic mutations, MEK1 mutations have been observed in a variety of malignancies including melanoma, histiocytic neoplasms, colorectal cancer and lung cancer. Presently, the use of trametinib, a highly selective MEK1/2 inhibitor, was limited to BRAF mutations, according to the approvals of FDA. Therefore, we consider that this is a question worth studying that whether malignancies with MEK1 mutations are sensitive to the treatment of trametinib. This review discussed the function of MEK1 mutations, retrieved the frequency and distribution of MEK1 mutations in various malignancies, and reviewed the basic experiments and clinical case reports on trametinib in the treatment of cell lines or patients with MEK1 mutations. Most studies have demonstrated that trametinib was effective to cells or tumor patients harboring MEK1 mutations, which suggest that the MEK1 mutations might be potential indications of trametinib therapy. In addition, it was also reported that resistance was observed in the treatment of trametinib, suggesting that different MEK1 mutations may have different response to trametinib, and further studies are necessary to distinguish that which MEK1 mutations are appropriate for the treatment with trametinib and which are not.


Assuntos
Antineoplásicos/uso terapêutico , MAP Quinase Quinase 1/genética , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/uso terapêutico , Pirimidinonas/uso terapêutico , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Animais , Antineoplásicos/farmacologia , Grupo com Ancestrais do Continente Asiático/genética , Histiocitose de Células de Langerhans/tratamento farmacológico , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Camundongos , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia
3.
Wei Sheng Yan Jiu ; 48(4): 611-620, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31601344

RESUMO

OBJECTIVE: To investigate the effect of chronic and acute swimming exercise intervention on the mitogen-activated extracellular signal-regulated kinase(MEK) and extracellular signal-regulated kinase 1(ERK1) phosphorylation level in adipose tissues of obesityinduced insulin resistance rats. METHODS: A total of 100 SD rats were randomly divided into control group(n=10) fed with normal diet and high-fat diet group(n=90) fed with high fat diet. After 8 weeks, one third rats(n=30) with upper weight in high-fat diet group were selected and randomly divided into high-fat diet sedentary group(n=10), chronic exercise group(n=10) and acute exercise group(n=10). Under another 8-week high-fat diet feeding, exercise intervention was performed according to the exercise procedure; control group was fed with normal diet for 8 weeks. After exercise intervention, visceral adipose tissues were separated and MEK and ERK1 phosphorylation level in adipose tissue was detected by Western blot method. RESULTS: Chronic exercise intervention significantly reduced body weight, visceral fat weight and visceral fat weight/body weight ratio(P<0. 01), and acute exercise intervention had no significant effect on body weight, visceral fat weight and visceral fat weight/body weight ratio. Both chronic and acute exercise intervention significantly increased body insulin sensitivity(P<0. 05), as well as significantly decreased MEK and ERK1 phosphorylation level in adipose tissues(P<0. 01). CONCLUSION: The improvement of obesity-induced insulin resistance by exercise might be related to inhibited phosphorylation of MEK and ERK1 in adipose tissues.


Assuntos
Tecido Adiposo/metabolismo , Resistência à Insulina/fisiologia , MAP Quinase Quinase 1/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Condicionamento Físico Animal , Natação , Animais , Dieta Hiperlipídica , Insulina , Fosforilação , Ratos , Ratos Sprague-Dawley
4.
Nat Genet ; 51(9): 1389-1398, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477929

RESUMO

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We used single-cell RNA sequencing to profile epithelial cells undergoing a spontaneous spatially determined EMT in the presence or absence of transforming growth factor-ß. Pseudospatial trajectory analysis identified continuous waves of gene regulation as opposed to discrete 'partial' stages of EMT. KRAS was connected to the exit from the epithelial state and the acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along the EMT. Inhibiting the KRAS effector MEK and its upstream activators EGFR and MET demonstrates that interruption of key signaling events reveals regulatory 'checkpoints' in the EMT continuum that mimic discrete stages, and reconciles opposing views of the program that controls EMT.


Assuntos
Transição Epitelial-Mesenquimal/genética , Redes Reguladoras de Genes , Testes Genéticos , Glândulas Mamárias Humanas/metabolismo , Análise de Célula Única/métodos , Sistemas CRISPR-Cas , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Glândulas Mamárias Humanas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
5.
J Enzyme Inhib Med Chem ; 34(1): 1544-1561, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31448648

RESUMO

In this paper, a series of novel 1H-dibenzo[a,c]carbazole derivatives of dehydroabietic acid bearing different N-(piperazin-1-yl)alkyl side chains were designed, synthesised and evaluated for their in vitro anticancer activities against three human hepatocarcinoma cell lines (SMMC-7721, HepG2 and Hep3B). Among them, compound 10g exhibited the most potent activity against three cancer cell lines with IC50 values of 1.39 ± 0.13, 0.51 ± 0.09 and 0.73 ± 0.08 µM, respectively. In the kinase inhibition assay, compound 10g could significantly inhibit MEK1 kinase activity with IC50 of 0.11 ± 0.02 µM, which was confirmed by western blot analysis and molecular docking study. In addition, compound 10g could elevate the intracellular ROS levels, decrease mitochondrial membrane potential, destroy the cell membrane integrity, and finally lead to the oncosis and apoptosis of HepG2 cells. Therefore, compound 10g could be a potent MEK inhibitor and a promising anticancer agent worthy of further investigations.


Assuntos
/farmacologia , Antineoplásicos/farmacologia , Carbazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , Piperazina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , /síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Carbazóis/síntese química , Carbazóis/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , MAP Quinase Quinase 1/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Piperazina/síntese química , Piperazina/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
6.
Cells ; 8(7)2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31323891

RESUMO

In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Gefitinibe/farmacologia , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Regulação para Cima
7.
Cells ; 8(7)2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31336670

RESUMO

The aim of this study was to compare the acute effects of thrombin and brain-derived neurotrophic factor (BDNF) on spontaneous miniature endplate potentials (MEPPs) and multiquantal evoked endplate potentials (EPPs) in mouse neuromuscular junctions (NMJs) of m. diaphragma and m. EDL. Intracellular microelectrode recordings of MEPPs and EPPs were used to evaluate the changes in acetylcholine (ACh) release in mature and newly-formed mouse NMJs. Thrombin (1 nM) increased the amplitude of MEPPs and EPPs by 25-30% in mature and newly-formed NMJs. This effect was due to an enhanced loading of synaptic vesicles with ACh and increase of ACh quantal size, since it was fully prevented by blocking of vesicular ACh transporter. It was also prevented by tropomyosin-related kinase B (TrkB) receptors inhibitor ANA12. Exogenous BDNF (1 nM) mimicked thrombin effect and increased the amplitude of MEPPs and EPPs by 25-30%. It required involvement of protein kinase A (PKA) and mitogen-activated protein kinase (MEK1/2)-mediated pathway, but not phospholipase C (PLC). Blocking A2A adenosine receptors by ZM241385 abolished the effect of BDNF, whereas additional stimulation of A2A receptors by CGS21680 increased MEPP amplitudes, which was prevented by MEK1/2 inhibitor U0126. At mature NMJs, BDNF enhanced MEPPs frequency by 30-40%. This effect was selectively prevented by inhibition of PLC, but not PKA or MEK1/2. It is suggested that interrelated effects of thrombin/BDNF in mature and newly-formed NMJs are realized via enhancement of vesicular ACh transport and quantal size increase. BDNF-induced potentiation of synaptic transmission involves the functional coupling between A2A receptor-dependent active PKA and neurotrophin-triggered MAPK pathway, as well as PLC-dependent increase in frequency of MEPPs.


Assuntos
Acetilcolina/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Trombina/farmacologia , Animais , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Tirosina Quinases/metabolismo , Receptor A2A de Adenosina/metabolismo , Transmissão Sináptica , Fosfolipases Tipo C/metabolismo
8.
Environ Toxicol Pharmacol ; 70: 103200, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31158732

RESUMO

OBJECTIVE: Plumbagin exerts effective anti-hepatocellular carcinoma (HCC) benefits, however, the detailed mechanisms behind these effects are not yet completely elucidated. The pharmacological targets and molecular mechanisms of plumbagin against HCC were revealed through conducting network pharmacology approach before experimentative verification. METHODS: The web-accessible databases of herbal ingredients' targets (HIT), Swiss-Target-Prediction and Super-Pred were used to predict the therapeutic targets of plumbagin, followed by combined with pathogenic targets of HCC from oncogenomic database of hepatocellular carcinoma (OncoDB.HCC) and Liverome databases to obtain the predominant targets of plumbagin-treating HCC. The database for annotation, visualization and integrated discovery (DAVID) was applied to output the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment by use of all predominant targets for computerized visualization. The validated data of human and cell culture were subjected to a group of medical imaging, biochemical tests and immunostaining, respectively. RESULTS: As revealed in bioinformatic data, 19 predominant targets of plumbagin-treating HCC were obtained, and 5 top targets of TP53, MAPK1, MAP2K1, RAF1 and CCND1 were the most important biomolecules in anti-HCC effects exerted by plumbagin. Other identifiable 102 GO items were showed, including 66 biological processes, and 12 cellular components, 24 molecular functions. And 67 KEGG pathways were mainly involved in neoplastic signaling. In human data, HCC sections showed increased expressions of hepatocellular TP53, MAPK1, accompanied with positive clinical imaging results for HCC. In plumbagin-treated HepG2 cells, reduced TP53, MAPK1 protein expressions were observed, accompanied with cell arrest and apoptosis. CONCLUSION: Collectively, the pharmacological targets and mechanisms of plumbagin-treating HCC were predicted and integrated through the method of network pharmacology, followed by some investigative validations. Interestingly, these 5 predominant biomolecules may be the potential targets for screening and treating HCC.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Naftoquinonas/farmacologia , Biologia Computacional , Ciclina D1/metabolismo , Feminino , Células Hep G2 , Humanos , MAP Quinase Quinase 1/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteína Supressora de Tumor p53/metabolismo
9.
J Enzyme Inhib Med Chem ; 34(1): 955-972, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31072147

RESUMO

In this article, a series of novel quinoline derivatives of ursolic acid (UA) bearing hydrazide, oxadiazole, or thiadiazole moieties were designed, synthesised, and screened for their in vitro antiproliferative activities against three cancer cell lines (MDA-MB-231, HeLa, and SMMC-7721). A number of compounds showed significant activity against at least one cell line. Among them, compound 4d exhibited the most potent activity against three cancer cell lines with IC50 values of 0.12 ± 0.01, 0.08 ± 0.01, and 0.34 ± 0.03 µM, respectively. In particular, compound 4d could induce the apoptosis of HeLa cells, arrest cell cycle at the G0/G1 phase, elevate intracellular reactive oxygen species level, and decrease mitochondrial membrane potential. In addition, compound 4d could significantly inhibit MEK1 kinase activity and impede Ras/Raf/MEK/ERK transduction pathway. Therefore, compound 4d may be a potential anticancer agent and a promising lead worthy of further investigation.


Assuntos
Antineoplásicos/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , Oxidiazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Tiadiazóis/farmacologia , Triterpenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , MAP Quinase Quinase 1/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Oxidiazóis/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinolinas/química , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Tiadiazóis/química , Triterpenos/química
10.
Br J Cancer ; 120(9): 941-951, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944457

RESUMO

BACKGROUND: Mitogen-activated protein kinases (MEK 1/2) are central components of the RAS signalling pathway and are attractive targets for cancer therapy. These agents continue to be investigated in KRAS mutant colon cancer but are met with significant resistance. Clinical investigations have demonstrated that these strategies are not well tolerated by patients. METHODS: We investigated a biomarker of response for MEK inhibition in KRAS mutant colon cancers by LC-MS/MS analysis. We tested the MEK inhibitor in PIK3CA wild(wt) and mutant(mt) colon cancer cells. In addition, we tested the combinational effects of MEK and TNKS inhibitor in vitro and in vivo. RESULTS: We identified ß-catenin, a key mediator of the WNT pathway, in response to MEK inhibitor. MEK inhibition led to a decrease in ß-catenin in PIK3CA wt colon cancer cells but not in mt. Tumour regression was promoted by combination of MEK inhibition and NVP-TNS656, which targets the WNT pathway. Furthermore, inhibition of MEK promoted tumour regression in colon cancer patient-derived xenograft models expressing PIK3CA wt. CONCLUSIONS: We propose that inhibition of the WNT pathway, particularly ß-catenin, may bypass resistance to MEK inhibition in human PIK3CA mt colon cancer. Therefore, we suggest that ß-catenin is a potential predictive marker of MEK inhibitor resistance.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , beta Catenina/metabolismo , Acetamidas/farmacologia , Animais , Biomarcadores Farmacológicos/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias do Colo/metabolismo , Farmacorresistência Viral , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinonas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/antagonistas & inibidores
11.
Mol Cancer ; 18(1): 80, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953514

RESUMO

BACKGROUND: Accumulating evidence shows that, the dysregulation of circular RNAs (circRNAs) is associated with the progression of multiple malignancies. But, the underlying mechanisms by which has_circ_0032627 (circDLST) contributed to gastric cancer (GC) remain undocumented. METHODS: The expression and cellular localization of circDLST and its association with clinicopathological characteristics and prognosis in patients with GC was analysed by using fluorescence in situ hybridization. Gain- and loss-of-function experiments as well as a subcutaneous xenograft tumor model and a liver metastasis model from orthotopic implantation of GC tissues were conducted to assess the role of circDLST in GC cells. CircDLST specific binding with miR-502-5p was confirmed by dual luciferase gene report, RNA immunoprecipitation (RIP) assays and RIP-miRNA expression profiling. qRT-PCR and Western blot analysis was used to detect the effects of circDLST on miR-502-5p-mediated NRAS/MEK1/ERK1/2 signaling in GC cells. RESULTS: The expression levels of circDLST were dramatically elevated in GC tissues as compared with the adjacent normal tissues, and acted as an independent prognostic factor of poor survival in patients with GC. Knockdown of circDLST inhibited the cell viability, colony formation, DNA synthesis, cell invasion and liver metastasis in vitro and in vivo, whereas overexpression of circDLST had the opposite effects. Furthermore, circDLST was co-localized with miR-502-5p in the cytoplasm of GC cells, and acted as a sponge of miR-502-3p in GC cells, which abrogated the tumor promoting effects of circDLST by inactivating the NRAS/MEK1/ERK1/2 signaling in GC cells. CONCLUSION: CircDLST promotes the tumorigenesis and metastasis of GC cells by sponging miR-502-5p to activate the NRAS/MEK1/ERK1/2 signaling.


Assuntos
Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , MicroRNAs/genética , RNA/genética , Neoplasias Gástricas/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Transplante de Neoplasias , Prognóstico , Neoplasias Gástricas/genética , Análise de Sobrevida , Regulação para Cima
12.
Cell Commun Signal ; 17(1): 31, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971268

RESUMO

BACKGROUND: p21-activated kinase 1 (PAK1) plays a fundamental role in promoting the development and progression of several cancers and is a potential therapeutic target. However, the biological function and underlying mechanism of PAK1 in esophageal squamous cell carcinoma (ESCC) remain unclear. METHODS: The expression of PAK1 was detected in both ESCC cell lines and clinical samples. Cell growth was measured by MTT, focus formation and soft agar assays. Cell migration and invasion were detected by wound healing and transwell assays. Animal models of subcutaneous tumourigenicity and tail vein metastasis were performed to determine the inhibitory effect of pharmacological inhibitor IPA-3 on tumor growth and metastasis of ESCC cells. RESULTS: We found that PAK1 was frequently overexpressed in ESCC. Ectopic expression of PAK1 promoted cellular growth, colony formation and anchorage-independent growth. Overexpressing PAK1 also enhanced migration, invasion and the expression of MMP-2 and MMP-9 in ESCC cells. In contrast, silencing PAK1 by lentiviral knockdown or a specific inhibitor IPA-3 resulted in a contrary effect. Subsequent investigations revealed that Raf1/MEK1/ERK signaling pathway was involved in PAK1-mediated effect. Enhanced expression of Raf1 attenuated the inhibitory functions of PAK1 shRNA. Whereas blocking of Raf1 by shRNA or specific inhibition of MEK1 by U0126 antagonized the oncogenetic effect of PAK1 on ESCC cells. More importantly, Pharmacological inhibition of PAK1 by IPA-3 significantly suppressed tumor growth and lung metastasis of ESCC cells in vivo. CONCLUSIONS: These data support that PAK1 is an ideal target for the development of potential therapeutic drugs for ESCC patients even with metastasis.


Assuntos
Dissulfetos/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Naftóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Dissulfetos/uso terapêutico , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/secundário , Humanos , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Naftóis/uso terapêutico , Metástase Neoplásica , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Interferente Pequeno/metabolismo
13.
Nat Commun ; 10(1): 1897, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015486

RESUMO

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Cromatina/química , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Animais , Butadienos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nitrilos/farmacologia , Piperazinas/farmacologia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
14.
Can J Cardiol ; 35(4): 490-500, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30935640

RESUMO

BACKGROUND: The IκB kinase (IKK) complex has been found to have critical functions in cancer and the immune system. In particular, IKKα, which is a member of the IKK complex, has been shown to influence the inflammatory response and malignant diseases. However, the role of IKKα in macrophages after myocardial infarction (MI) remains largely unknown. METHODS: Sham or MI operations were performed on macrophage-specific IKKɑ knockout (mIKKɑ-/-) mice and IKKɑflox/flox littermates. We ligated the left anterior descending coronary artery of the MI group and observed the results at 3, 7, and 30 days after MI. RESULTS: We discovered more severe cardiac dysfunction with reduced angiogenesis, fibrosis, and collagen deposition in mIKKɑ-/- than in IKKɑflox/flox. In addition, we also observed that macrophages in mIKKɑ-/- were easier to polarize to the M1 phenotype and expressed more proinflammatory factors than IKKɑflox/flox. Mechanistically, IKKα deficiency in macrophages inhibited the alternative nuclear factor-κB/RelB pathway and enhanced the MEK1/2/ERK1/2 pathway. CONCLUSIONS: Overall, our data identified IKKɑ in the heart as a novel mediator that protected the heart from a severe inflammatory response and attenuated ventricular remodelling after MI by negatively regulating macrophage polarization to the M1 phenotype. Therefore, IKKα may serve as a potential therapeutic target for treatment after MI.


Assuntos
Quinase I-kappa B/metabolismo , Macrófagos/metabolismo , Infarto do Miocárdio/fisiopatologia , Remodelação Ventricular/fisiologia , Animais , Colágeno/metabolismo , Fibrose , Quinase I-kappa B/genética , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/imunologia , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Neovascularização Patológica/metabolismo
15.
Food Chem Toxicol ; 126: 303-312, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30840849

RESUMO

With the introduction of arsenic trioxide and all-trans retinoic acid, the prognosis of acute promyelocytic leukemia has greatly improved. However, all-trans retinoic acid resistance is still unresolved in acute promyelocytic leukemia relapsed patients. In this study, the clinical achievable concentration of 7-hydroxystaurosporine synergized with all-trans retinoic acid to induce terminal differentiation in all-trans retinoic acid resistant acute promyelocytic leukemia cell lines. Though 7-hydroxystaurosporine is a PKC inhibitor, PKC might not be involved in the combination-induced differentiation since other PKC selective inhibitors, Gö 6976 and rottlerin failed to cooperate with all-trans retinoic acid to trigger differentiation. The combination significantly enhanced the protein level of CCAAT/enhancer binding protein ß and/or PU.1 as well as activated MEK/ERK. U0126 (MEK specific inhibitor) not only suppressed the combination-induced differentiation but also restored the protein level of CCAAT/enhancer binding protein ß and/or PU.1. However, RAF-1 inhibitor had no inhibitory effect on MEK activation and the combination-induced differentiation. Therefore, the combination overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of CCAAT/enhancer binding protein ß and/or PU.1. These findings may provide a preclinical rationale for the potential role of this combination in the treatment of all-trans retinoic acid resistant acute promyelocytic leukemia patients.


Assuntos
Antineoplásicos/farmacologia , Leucemia Promielocítica Aguda/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Estaurosporina/análogos & derivados , Tretinoína/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Proto-Oncogênicas c-raf/genética , Estaurosporina/farmacologia
16.
Biochem Biophys Res Commun ; 512(1): 125-130, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30876692

RESUMO

Oxidative stress injury is one of the main mechanisms of ischemia-reperfusion (I/R) injury. The extracellular signal-regulated kinase (ERK1/2) pathway plays an important role in cardioprotective during acute myocardial infarction. In this study, we used constitutively active MEK1 gene (CaMEK) transfection strategy to investigate whether CaMEK provides a protective effect against apoptosis and autophagy induced by Hydrogen peroxide (H2O2) in neonatal rat cardiac ventricular cardiomyocytes (NCMs) and the underlying mechanisms. As a result, CaMEK attenuated H2O2-induced apoptosis and cytotoxicity in NCMs, evidenced by decreased apoptotic cells and the ratio of Bax/Bcl-2, increased the mitochondrial membrane potential (Δψm) and cell vitality and reduced the level of lactate dehydrogenase (LDH). Further studies revealed that CaMEK attenuated H2O2-induced autophagy, evidenced by the decreased LC3-Ⅱ/LC3-Ⅰratio and SQSTM1/p62 (p62) degradation. Furthermore, we demonstrated that CaMEK phosphorylated the ERK1/2 pathway-related proteins, ERK1/2, p70S6K and GSK3ß, in NCMs with H2O2 stimulation. In contrast, these effects could be reversed by co-treatment with the ERK1/2 inhibitor, PD98059. These results suggest that CaMEK plays an important role in protecting cardiomyocytes against H2O2-induced injury and autophagy in NCMs via ERK1/2 pathway. Therefore, transfection of CaMEK may provide a hopeful therapeutic strategy for I/R.


Assuntos
MAP Quinase Quinase 1/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cardiotônicos/metabolismo , Células Cultivadas , Feminino , Peróxido de Hidrogênio/toxicidade , MAP Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
17.
J Biol Chem ; 294(21): 8664-8673, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30858179

RESUMO

Most cancer cells are dependent on a network of deregulated signaling pathways for survival and are insensitive, or rapidly evolve resistance, to selective inhibitors aimed at a single target. For these reasons, drugs that target more than one protein (polypharmacology) can be clinically advantageous. The discovery of useful polypharmacology remains serendipitous and is challenging to characterize and validate. In this study, we developed a non-genetic strategy for the identification of pathways that drive cancer cell proliferation and represent exploitable signaling vulnerabilities. Our approach is based on using a multitargeted kinase inhibitor, SM1-71, as a tool compound to identify combinations of targets whose simultaneous inhibition elicits a potent cytotoxic effect. As a proof of concept, we applied this approach to a KRAS-dependent non-small cell lung cancer (NSCLC) cell line, H23-KRASG12C Using a combination of phenotypic screens, signaling analyses, and kinase inhibitors, we found that dual inhibition of MEK1/2 and insulin-like growth factor 1 receptor (IGF1R)/insulin receptor (INSR) is critical for blocking proliferation in cells. Our work supports the value of multitargeted tool compounds with well-validated polypharmacology and target space as tools to discover kinase dependences in cancer. We propose that the strategy described here is complementary to existing genetics-based approaches, generalizable to other systems, and enabling for future mechanistic and translational studies of polypharmacology in the context of signaling vulnerabilities in cancers.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/epidemiologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Receptores de Somatomedina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Antígenos CD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Células HCT116 , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo
18.
Oncogene ; 38(25): 5076-5090, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30905967

RESUMO

Genomic alterations in cancer cells result in vulnerabilities that clinicians can exploit using molecularly targeted drugs, guided by knowledge of the tumour genotype. However, the selective activity of these drugs exerts an evolutionary pressure on cancers that can result in the outgrowth of resistant clones. Use of rational drug combinations can overcome resistance to targeted drugs, but resistance may eventually develop to combinatorial therapies. We selected MAPK- and PI3K-pathway inhibition in colorectal cancer as a model system to dissect out mechanisms of resistance. We focused on these signalling pathways because they are frequently activated in colorectal tumours, have well-characterised mutations and are clinically relevant. By treating a panel of 47 human colorectal cancer cell lines with a combination of MEK- and PI3K-inhibitors, we observe a synergistic inhibition of growth in almost all cell lines. Cells with KRAS mutations are less sensitive to PI3K inhibition, but are particularly sensitive to the combined treatment. Colorectal cancer cell lines with inherent or acquired resistance to monotherapy do not show a synergistic response to the combination treatment. Cells that acquire resistance to an MEK-PI3K inhibitor combination treatment still respond to an ERK-PI3K inhibitor regimen, but subsequently also acquire resistance to this combination treatment. Importantly, the mechanisms of resistance to MEK and PI3K inhibitors observed, MEK1/2 mutation or loss of PTEN, are similar to those detected in the clinic. ERK inhibitors may have clinical utility in overcoming resistance to MEK inhibitor regimes; however, we find a recurrent active site mutation of ERK2 that drives resistance to ERK inhibitors in mono- or combined regimens, suggesting that resistance will remain a hurdle. Importantly, we find that the addition of low concentrations of the BCL2-family inhibitor navitoclax to the MEK-PI3K inhibitor regimen improves the synergistic interaction and blocks the acquisition of resistance.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Terapia de Alvo Molecular , Compostos de Anilina/administração & dosagem , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Transdução de Sinais/genética , Sulfonamidas/administração & dosagem , Células Tumorais Cultivadas
19.
Future Oncol ; 15(9): 967-977, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30638071

RESUMO

Approximately 50% of cutaneous melanomas harbor activating mutations of the BRAF-oncogene, making BRAF inhibitors (BRAFi) the standard treatment for this disease. However, disease responses are limited in duration mainly due to acquired resistance. Dual MAPK pathway inhibition with addition of a MEK inhibitor (MEKi) to a BRAFi improved the efficacy and tolerability compared with BRAFi alone. Cobimetinib (Cotellic®) is an orally bioavailable, potent and selective MEKi, which significantly improved response rates when combined with BRAFi vemurafenib (median overall survival: 22.3 months). The toxicity profile of cobimetinib is manageable and treatment discontinuation due to adverse events is uncommon. Present efforts are addressed to overcome resistance and improve long-term outcomes: based on the evidence of the immunomodulatory properties of BRAFi and MEKi, current clinical trials of combined targeted and immunotherapy are investigating the role of cobimetinib in the context of combination or as sequential treatments.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azetidinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Piperidinas/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Administração Oral , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azetidinas/uso terapêutico , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Melanoma/genética , Melanoma/mortalidade , Melanoma/patologia , Piperidinas/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Análise de Sobrevida , Fatores de Tempo , Resultado do Tratamento
20.
Curr Genet ; 65(3): 631-641, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30671596

RESUMO

Recombination, along with sister chromatid cohesion, is used during meiosis to physically connect homologous chromosomes so that they can be segregated properly at the first meiotic division. Recombination is initiated by the introduction of programmed double strand breaks (DSBs) into the genome, a subset of which is processed into crossovers. In budding yeast, the regulation of meiotic DSB repair is controlled by a meiosis-specific kinase called Mek1. Mek1 kinase activity promotes recombination between homologs, rather than sister chromatids, as well as the processing of recombination intermediates along a pathway that results in synapsis of homologous chromosomes and the distribution of crossovers throughout the genome. In addition, Mek1 kinase activity provides a readout for the number of DSBs in the cell as part of the meiotic recombination checkpoint. This checkpoint delays entry into the first meiotic division until DSBs have been repaired by inhibiting the activity of the meiosis-specific transcription factor Ndt80, a site-specific DNA binding protein that activates transcription of over 300 target genes. Recent work has shown that Mek1 binds to Ndt80 and phosphorylates it on multiple sites, including the DNA binding domain, thereby preventing Ndt80 from activating transcription. As DSBs are repaired, Mek1 is removed from chromosomes and its activity decreases. Loss of the inhibitory Mek1 phosphates and phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, promote Ndt80 activity such that Ndt80 transcribes its own gene in a positive feedback loop, as well as genes required for the completion of recombination and entry into the meiotic divisions. Mek1 is therefore the key regulator of meiotic recombination in yeast.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , MAP Quinase Quinase 1/metabolismo , Meiose , Saccharomycetales/genética , Segregação de Cromossomos , Saccharomycetales/fisiologia
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