Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 143
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Theranostics ; 11(16): 8092-8111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335982

RESUMO

Active c-Src non-receptor tyrosine kinase localizes to the plasma membrane via N-terminal lipid modification. Membranous c-Src causes cancer initiation and progression. Even though transmembrane 4 L six family member 5 (TM4SF5), a tetraspan(in), can be involved in this mechanism, the molecular and structural influence of TM4SF5 on c-Src remains unknown. Methods: Here, we investigated molecular and structural details by which TM4SF5 regulated c-Src devoid of its N-terminus and how cell-penetrating peptides were able to interrupt c-Src activation via interference of c-Src-TM4SF5 interaction in hepatocellular carcinoma models. Results: The TM4SF5 C-terminus efficiently bound the c-Src SH1 kinase domain, efficiently to the inactively-closed form. The complex involved protein tyrosine phosphatase 1B able to dephosphorylate Tyr530. The c-Src SH1 domain alone, even in a closed form, bound TM4SF5 to cause c-Src Tyr419 and FAK Y861 phosphorylation. Homology modeling and molecular dynamics simulation studies predicted the directly interfacing residues, which were further validated by mutational studies. Cell penetration of TM4SF5 C-terminal peptides blocked the interaction of TM4SF5 with c-Src and prevented c-Src-dependent tumor initiation and progression in vivo. Conclusions: Collectively, these data demonstrate that binding of the TM4SF5 C-terminus to the kinase domain of inactive c-Src leads to its activation. Because this binding can be abolished by cell-penetrating peptides containing the TM4SF5 C-terminus, targeting this direct interaction may be an effective strategy for developing therapeutics that block the development and progression of hepatocellular carcinoma.


Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Tirosina Quinase CSK/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Genes src/genética , Genes src/fisiologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Tetraspaninas/genética , Tetraspaninas/metabolismo
2.
Exp Cell Res ; 399(1): 112439, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33359469

RESUMO

Yes-associated protein 1 (YAP1), a co-transcription activator, shuttles between the cytoplasm and the nucleus. Phosphorylation by large tumor suppressor kinases (LATS1/2) is the major determinant of YAP1 subcellular localization. Unphosphorylated YAP1 interacts with transcription factors in the nucleus and regulates gene transcription, while phosphorylated YAP1 is trapped in the cytoplasm and is degraded. We found that when U2OS and HeLa cells are exposed to 42 °C, YAP1 enters the nucleus within 30 min and returns to the cytoplasm at 4 h. SRC and HSP90 are involved in nuclear accumulation and return to the cytoplasm, respectively. Upon heat shock, LATS2 forms aggregates including protein phosphatase 1 and is dephosphorylated and inactivated. SRC activation is necessary for the formation of aggregates, while HSP90 is required for their dissociation. YAP1 is involved in heat shock-induced NF-κB signaling. Mechanistically, YAP1 is implicated in strengthening the interaction between RELA and DPF3, a component of SWI/SNF chromatin remodeling complex, in response to heat shock. Thus, YAP1 plays a role as a thermosensor.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Genes src/fisiologia , Resposta ao Choque Térmico/fisiologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Células HeLa , Resposta ao Choque Térmico/genética , Humanos , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico/genética , Transdução de Sinais/genética , Fator de Transcrição RelA/metabolismo , Células Tumorais Cultivadas
3.
Toxicol Appl Pharmacol ; 401: 115092, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32512068

RESUMO

Inflammatory breast cancer (IBC) is a highly metastatic and lethal breast cancer. As many as 25-30% of IBCs are triple negative (TN) and associated with low survival rates and poor prognosis. We found that the microenvironment of IBC is characterized by high infiltration of tumor associated macrophages (TAMs) and by over-expression of the cysteine protease cathepsin B (CTSB). TAMs in IBC secrete high levels of the cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared to non-IBC patients. Herein, we tested the roles of IL-8 and MCP-1/CCL2 in modulating proteolytic activity and invasiveness of TN-non-IBC as compared to TN-IBC and addressed the underlying molecular mechanism(s) for both cytokines. Quantitative real time PCR results showed that IL-8 and MCP-1/CCL2 were significantly overexpressed in tissues of TN-IBCs. IL-8 and MCP-1/CCL2 induced CTSB expression and activity of the p-Src and p-Erk1/2 signaling pathways relevant for invasion and metastasis in TN-non-IBC, HCC70 cells and TN-IBC, SUM149 cells. Dasatinib, an inhibitor of p-Src, and U0126, an inhibitor of p-Erk1/2, down-regulated invasion and expression of CTSB by HCC70 and SUM149 cells, a mechanism that is reversed by IL-8 and MCP-1/CCL2. Our study shows that targeting the cytokines IL-8 and MCP-1/CCL2 and associated signaling molecules may represent a promising therapeutic strategy in TN-IBC patients.


Assuntos
Quimiocina CCL2/biossíntese , Genes src/fisiologia , Neoplasias Inflamatórias Mamárias/metabolismo , Interleucina-8/biossíntese , Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dasatinibe/farmacologia , Feminino , Genes src/efeitos dos fármacos , Humanos , Neoplasias Inflamatórias Mamárias/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pessoa de Meia-Idade , Proteólise/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia
4.
Development ; 147(7)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32108024

RESUMO

Endothelial cell adhesion is implicated in blood vessel sprout formation, yet how adhesion controls angiogenesis, and whether it occurs via rapid remodeling of adherens junctions or focal adhesion assembly, or both, remains poorly understood. Furthermore, how endothelial cell adhesion is controlled in particular tissues and under different conditions remains unexplored. Here, we have identified an unexpected role for spatiotemporal c-Src activity in sprouting angiogenesis in the retina, which is in contrast to the dominant focus on the role of c-Src in the maintenance of vascular integrity. Thus, mice specifically deficient in endothelial c-Src displayed significantly reduced blood vessel sprouting and loss in actin-rich filopodial protrusions at the vascular front of the developing retina. In contrast to what has been observed during vascular leakage, endothelial cell-cell adhesion was unaffected by loss of c-Src. Instead, decreased angiogenic sprouting was due to loss of focal adhesion assembly and cell-matrix adhesion, resulting in loss of sprout stability. These results demonstrate that c-Src signaling at specified endothelial cell membrane compartments (adherens junctions or focal adhesions) control vascular processes in a tissue- and context-dependent manner.


Assuntos
Adesão Celular/genética , Células Endoteliais/fisiologia , Adesões Focais/genética , Genes src/fisiologia , Neovascularização Fisiológica/genética , Retina/embriologia , Animais , Células Cultivadas , Embrião de Mamíferos , Endotélio Vascular/embriologia , Endotélio Vascular/metabolismo , Feminino , Adesões Focais/metabolismo , Adesões Focais/fisiologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Knockout , Retina/metabolismo
5.
BMC Musculoskelet Disord ; 21(1): 112, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075617

RESUMO

BACKGROUND: Osteoporosis is a worldwide severe bone disease. This study aimed to evaluate the effect of polyphyllin VII on the genesis of osteoclasts from bone marrow macrophages (BMMs) and its potentiality as a therapeutic drug for osteoporosis. METHODS: BMMs were induced to differentiate into osteoclasts by RANKL and M-CSF. The cells were then treated with various concentrations of polyphyllin VII. Intracellular reactive oxygen species (ROS) measurement assay, resorption pit formation assay, tartrate-resistant acid phosphatase (TRAP) staining and TRAP activity assessment, cell viability assay, active GTPase pull-down assay, immunofluorescent staining, immunoblotting, and RT-PCR were performed. RESULTS: RANKL + M-CSF significantly increased TRAP activity, number of osteoclasts, number and area of lacunae, intracellular content of ROS, protein levels of Nox1, TRAF6, c-Src and p-PI3K, as well as the content of activated GTP-Rac1, which were significantly blocked by polyphyllin VII in a concentration-dependent manner. CONCLUSION: These findings suggested that polyphyllin VII inhibited differentiation of BMMs into osteoclasts through suppressing ROS synthesis, which was modulated by TRAF6-cSrc-PI3k signal transduction pathway including GTP-Rac1 and Nox1. Polyphyllin VII could be a therapeutic drug for osteoporosis.


Assuntos
Genes src/fisiologia , Osteoclastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligante RANK/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saponinas/toxicidade , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Genes src/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Osteoclastos/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores
6.
Ann N Y Acad Sci ; 1431(1): 72-84, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29984433

RESUMO

Although an association between cancer progression and matrix metalloproteinase (MMP) 2 and MPP9 expression has been known, the expression, nuclear localization, and physiologically controlled activation of these two MMPs have not been investigated in malignant mesothelioma cells. We examined the expression and intracellular localization of MMP2/9 in ZL55 malignant mesothelioma cells, as well as their regulation by ADP. Using real-time PCR, we showed that activation of the P2Y1 receptor by ADP increased the expression of MMP2/9 mRNAs; MMP2/9 collected from conditioned media also showed an increase in activity; and ADP induced the nuclear localization of MMP2/9. The effects of ADP on transcription of the MMPs were due to activation of c-Src, Akt, and NF-κB, while ERK1/2 phosphorylation was needed for the increase in enzymatic activity and the regulation of nuclear import. We also showed that the nuclear localization of MMP2/9 induced by ADP causes the cleavage and inactivation of poly-ADP-ribose polymerase-1. These findings may help to elucidate the mechanisms regulating MMP2/9 activation in ZL55 human epithelioid mesothelioma cells, and perhaps other cells. Therapeutic approaches that promote ADP accumulation in a tumor environment may constitute an effective means to induce anticancer activity.


Assuntos
Difosfato de Adenosina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Mesotelioma/metabolismo , Linhagem Celular Tumoral , Genes src/fisiologia , Humanos , Mesotelioma Maligno , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
7.
Oncogene ; 37(41): 5552-5568, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29899407

RESUMO

Neddylation is a cellular process that covalently conjugates substrate proteins with the small ubiquitin-like molecule NEDD8. As neddylation is required for fast turnover of proteins in proliferating cancer cells, the neddylation process is currently regarded as a potential target for cancer therapy. However, little is known about the role of neddylation in cancer invasion and metastasis. Unexpectedly, we here found that the neddylation blockade stimulates migration of lung cancer and glioblastoma cells. Mechanistically, C-CBL acts as the E3 ligase for neddylation of the proto-oncogene c-Src. After neddylation, c-Src is poly-ubiquitinated and degraded through the proteasome, which inhibits the PI3K-AKT pathway responsible for cell migration. In human lung cancer tissues, the downregulation of C-CBL was associated with c-Src/AKT, cancer metastasis, and poor survival in patients. Therefore, C-CBL is likely to play a tumor suppressive role by antagonizing a robust oncogenic signaling driven by c-Src. This study provides new insight about the role of neddylation in cancer metastasis. It also implies that the metastasis risk should be carefully evaluated before the clinical application of neddylation inhibitors as anticancer regimens.


Assuntos
Movimento Celular/fisiologia , Genes src/fisiologia , Glioblastoma/patologia , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Genes Supressores de Tumor , Glioblastoma/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Proteína NEDD8/metabolismo , Neoplasias , Pirimidinas/farmacologia
8.
J Neuroinflammation ; 15(1): 127, 2018 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712570

RESUMO

BACKGROUND: Brain inflammation including increases in inflammatory cytokines such as IL-1ß is widely believed to contribute to the pathophysiology of Alzheimer's disease. Although IL-1ß-induced impairments in long-term potentiation (LTP) in acute hippocampal slices and memory functions in vivo have been well documented, the neuron-specific molecular mechanisms of IL-1ß-mediated impairments of LTP and memory remain unclear. METHODS: This study uses an in vitro approach in primary hippocampal neurons to evaluate the effect of IL-1ß on chemical LTP (cLTP)-induced structural plasticity and signaling. RESULTS: We found that IL-1ß reduces both the surface expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 and the spine growth following cLTP. These effects of IL-1ß were mediated by impairing actin polymerization during cLTP, as IL-1ß decreased the cLTP-induced formation of F-actin, and the effect of IL-1ß on cLTP-induced surface expression of GluA1 can be mimicked by latrunculin, a toxin that disrupts dynamics of actin filaments, and can be prevented by jasplakinolide, a cell-permeable peptide that stabilizes F-actin. Moreover, live-cell imaging demonstrated that IL-1ß decreased the stability of the actin cytoskeleton in spines, which is required for LTP consolidation. We further examined the role of sphingolipid signaling in the IL-1ß-mediated impairment of spine plasticity and found that both the neutral sphingomyelinase inhibitor GW4869 and the inhibitor of Src kinase PP2 attenuated the IL-1ß-mediated suppression of cLTP-induced surface expression of GluA1 and actin polymerization. CONCLUSIONS: These findings support a mechanism by which IL-1ß, via the sphingomyelinase/ceramide/Src pathway, impairs structural spine remodeling essential for LTP consolidation and memory.


Assuntos
Actinas/metabolismo , Ceramidas/farmacologia , Genes src/fisiologia , Interleucina-1beta/farmacologia , Potenciação de Longa Duração/fisiologia , Receptores de AMPA/biossíntese , Animais , Células Cultivadas , Expressão Gênica , Genes src/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Polimerização/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/antagonistas & inibidores
9.
Mediators Inflamm ; 2017: 8123281, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28804222

RESUMO

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF-ß1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF-ß1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-ß1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α-SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF-ß1 on migration of A549 cells and suppressed TGF-ß1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-ß1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF-ß1 in upper airway chronic inflammatory disease such as CRS.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Genes src/fisiologia , Fator de Crescimento Transformador beta1/farmacologia , Células A549 , Movimento Celular/efeitos dos fármacos , Genes src/genética , Humanos , Pólipos Nasais/metabolismo , Técnicas de Cultura de Órgãos , Transdução de Sinais/efeitos dos fármacos
10.
J Endocrinol ; 233(2): 175-186, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28223314

RESUMO

Transgenic mice overexpressing growth hormone (GH) show increased hepatic protein content of the epidermal growth factor receptor (EGFR), which is broadly associated with cell proliferation and oncogenesis. However, chronically elevated levels of GH result in desensitization of STAT-mediated EGF signal and similar response of ERK1/2 and AKT signaling to EGF compared to normal mice. To ascertain the mechanisms involved in GH attenuation of EGF signaling and the consequences on cell cycle promotion, phosphorylation of signaling mediators was studied at different time points after EGF stimulation, and induction of proteins involved in cell cycle progression was assessed in normal and GH-overexpressing transgenic mice. Results from kinetic studies confirmed the absence of STAT3 and 5 activation and comparable levels of ERK1/2 phosphorylation upon EGF stimulation, which was associated with diminished or similar induction of c-MYC, c-FOS, c-JUN, CYCLIN D1 and CYCLIN E in transgenic compared to normal mice. Accordingly, kinetics of EGF-induced c-SRC and EGFR phosphorylation at activating residues demonstrated that activation of these proteins was lower in the transgenic mice with respect to normal animals. In turn, EGFR phosphorylation at serine 1046/1047, which is implicated in the negative regulation of the receptor, was increased in the liver of GH-overexpressing transgenic mice both in basal conditions and upon EGF stimulus. Increased basal phosphorylation and activation of the p38-mitogen-activated protein kinase might account for increased Ser 1046/1047 EGFR. Hyperphosphorylation of EGFR at serine residues would represent a compensatory mechanism triggered by chronically elevated levels of GH to mitigate the proliferative response induced by EGF.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica/fisiologia , Hormônio do Crescimento/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes src/genética , Genes src/fisiologia , Hormônio do Crescimento/genética , Humanos , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Mol Biol Cell ; 27(24): 3926-3936, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27733622

RESUMO

The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential.


Assuntos
Genes src/genética , Genes src/fisiologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Células COS , Proteína Tirosina Quinase CSK , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Cisteína/metabolismo , Proteínas de Fluorescência Verde , Lipoilação , Proteínas de Membrana , Membranas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Quinases da Família src/genética , Quinases da Família src/metabolismo
12.
CNS Neurol Disord Drug Targets ; 14(3): 370-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25544681

RESUMO

An increasing number of studies have demonstrated that insulin-degrading enzyme (IDE) plays an essential role in both the degradation and its activity of ß-amyloid (Aß). Therefore, the regulation of IDE expression and/or modification of IDE-dependent actions are two emerging strategies for the treatment of Alzheimer's disease (AD). We previously observed that geniposide, a novel agonist of glucagon-like peptide 1 receptor (GLP-1R), could attenuate Aß-induced neurotoxicity by regulating the expression of IDE in primary cortical neurons. However, the signal transduction mechanisms underlying this effect were not elucidated. The present study, therefore examined and explored the cell signaling transduction and molecular mechanisms by which geniposide induces the expression of IDE in primary cortical neurons. The current study revealed that LY294002 (an inhibitor for phosphatidyl inositol 3-kinase, PI3K), PP1 (inhibitor for c-Src), GW9662 (antagonist for peroxisome proliferator-activated receptor γ, PPARγ), H89 (an inhibitor for protein kinase A, PKA) and AG1478 (an antagonist for epidermal growth factor receptor, EGFR) prohibited the up-regulation of IDE induced by geniposide in primary cortical neurons. Further, geniposide also enhanced the phosphorylation of PPARγ and accelerated the release of phosphorylated FoxO1 (forkhead box O1) from nuclear fraction to the cytosol. Moreover, geniposide directly activated the activity of IDE promoter in PC12 cells, which confirmed the presence of the GLP-1 receptor. Taken together, our findings reveal for the first time the cell signaling transduction pathway of geniposide regulating the expression of IDE in neurons.


Assuntos
Fármacos do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Insulisina/metabolismo , Iridoides/farmacologia , Neurônios/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Células Cultivadas , Córtex Cerebral/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Genes src/efeitos dos fármacos , Genes src/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Células PC12 , PPAR gama/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos
13.
Mol Pharmacol ; 87(2): 150-61, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25391374

RESUMO

Resistance to the human epidermal growth factor receptor (HER2)-targeted antibody trastuzumab is a major clinical concern in the treatment of HER2-positive metastatic breast cancer. Increased expression or signaling from the insulin-like growth factor-1 receptor (IGF-1R) has been reported to be associated with trastuzumab resistance. However, the specific molecular and biologic mechanisms through which IGF-1R promotes resistance or disease progression remain poorly defined. In this study, we found that the major biologic effect promoted by IGF-1R was invasion, which was mediated by both Src-focal adhesion kinase (FAK) signaling and Forkhead box protein M1 (FoxM1). Cotargeting IGF-1R and HER2 using either IGF-1R antibodies or IGF-1R short hairpin RNA in combination with trastuzumab resulted in significant but modest growth inhibition. Reduced invasion was the most significant biologic effect achieved by cotargeting IGF-1R and HER2 in trastuzumab-resistant cells. Constitutively active Src blocked the anti-invasive effect of IGF-1R/HER2 cotargeted therapy. Furthermore, knockdown of FoxM1 blocked IGF-1-mediated invasion, and dual targeting of IGF-1R and HER2 reduced expression of FoxM1. Re-expression of FoxM1 restored the invasive potential of IGF-1R knockdown cells treated with trastuzumab. Overall, our results strongly indicate that therapeutic combinations that cotarget IGF-1R and HER2 may reduce the invasive potential of cancer cells that are resistant to trastuzumab through mechanisms that depend in part on Src and FoxM1.


Assuntos
Neoplasias da Mama/metabolismo , Quinase 1 de Adesão Focal/biossíntese , Fatores de Transcrição Forkhead/biossíntese , Regulação Neoplásica da Expressão Gênica , Receptor ErbB-2/biossíntese , Receptor IGF Tipo 1/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Proteína Forkhead Box M1 , Genes src/fisiologia , Humanos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Transdução de Sinais/fisiologia
14.
Biochem J ; 465(3): 503-15, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25377781

RESUMO

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and ß-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.


Assuntos
Sinalização do Cálcio/fisiologia , Sulfato de Dextrana/toxicidade , Genes src/fisiologia , MAP Quinase Quinase 7/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Junções Íntimas/metabolismo , Animais , Células CACO-2 , Sinalização do Cálcio/efeitos dos fármacos , Feminino , Genes src/efeitos dos fármacos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Junções Íntimas/efeitos dos fármacos
15.
PLoS One ; 9(11): e112636, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25390068

RESUMO

Bone morphogenic protein (BMP)-7 is a member of the transforming growth factor (TGF)-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvß3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvß3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.


Assuntos
Proteína Morfogenética Óssea 7/farmacologia , Neoplasias Ósseas/metabolismo , Movimento Celular/efeitos dos fármacos , Condrossarcoma/metabolismo , Genes src/fisiologia , Integrina alfaVbeta3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima/efeitos dos fármacos
16.
J Neuroimmune Pharmacol ; 9(5): 629-41, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24989845

RESUMO

Neuroinflammation plays critical roles in multiple sclerosis (MS). In addition to the part played by the lymphocytes, the underlying mechanisms could, in part, be also attributed to activation mediated by astrocytes. Macrophage inflammatory protein-1α (MIP-1α) has been implicated in a number of pathological conditions, specifically attributable to its potent chemottractant effects. Its modulation by IL-17, however, has received very little attention. In the present study, we demonstrated IL-17-mediated induction of MIP-1α in rat primary astroctyes through its binding to the cognate IL-17RA. Furthermore, this effect was mediated via the activation of Src, mitogen-activated protein kinases (MAPKs), PI3K/Akt and NF-kB pathways, culminating ultimately into increased expression of MIP-1α. Exposure of primary mouse astrocytes to IL-17 resulted in increased expression of glial fibrillary acidic protein and, this effect was abrogated in cells cultured in presence of the MIP-1α neutralizing antibody, thus underscoring its role in the activation of astrocytes. In vivo relevance of these findings was further corroborated in experimental autoimmune encephalomyelitis mice that demonstrated significantly increased activation of astrocytes with concomitant increased expression of MIP-1α in the corpus callosum compared with control group. Understanding the regulation of MIP-1α expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with multiple sclerosis.


Assuntos
Quimiocina CCL3/biossíntese , Genes src/fisiologia , Interleucina-17/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Esclerose Múltipla/metabolismo , NF-kappa B/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Interleucina-17/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/induzido quimicamente , Ratos , Ratos Sprague-Dawley
17.
Gynecol Oncol ; 135(1): 100-7, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24972189

RESUMO

BACKGROUND: Endometriosis is a common condition that is associated with an increased risk of developing ovarian carcinoma. Improved in vitro models of this disease are needed to better understand how endometriosis, a benign disease, can undergo neoplastic transformation, and for the development of novel treatment strategies to prevent this progression. METHODS: We describe the generation and in vitro characterization of novel TERT immortalized ovarian endometriosis epithelial cell lines (EEC16-TERT). RESULTS: Expression of TERT alone was sufficient to immortalize endometriosis epithelial cells. TERT immortalization induces an epithelial-to-mesenchymal transition and perturbation in the expression of genes involved in the development of ovarian cancer. EEC16-TERT was non-tumorigenic when xenografted into immunocompromised mice but grew in anchorage-independent growth assays in an epidermal growth factor and hydrocortisone dependent manner. Colony formation in agar was abolished by inhibition of Src, and the Src pathway was found to be activated in human endometriosis lesions. CONCLUSIONS: This new in vitro model system mimics endometriosis and the early stages of neoplastic transformation in the development of endometriosis associated ovarian cancer. We demonstrate the potential clinical relevance of this model by identifying Src activation as a novel pathway in endometriosis that could be targeted therapeutically, perhaps as a novel strategy to manage endometriosis clinically, or to prevent the development of endometriosis-associated ovarian cancer.


Assuntos
Transformação Celular Neoplásica , Endometriose/genética , Endometriose/patologia , Genes src/fisiologia , Doenças Ovarianas/genética , Doenças Ovarianas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Animais , Células Cultivadas , Endometriose/tratamento farmacológico , Células Epiteliais , Feminino , Genes src/efeitos dos fármacos , Humanos , Camundongos , Doenças Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico
18.
Am J Physiol Gastrointest Liver Physiol ; 306(11): G947-58, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24722904

RESUMO

The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and ß-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and ß-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism.


Assuntos
Ativação Enzimática/fisiologia , Genes src/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Junções Íntimas/fisiologia , Actinas/fisiologia , Junções Aderentes/fisiologia , Antracenos , Células CACO-2 , Humanos , Mecânica , Quinase de Cadeia Leve de Miosina/genética , Periodicidade , Fosforilação , Pirimidinas , Tirosina/análogos & derivados
19.
Hypertens Res ; 37(8): 708-15, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24646644

RESUMO

Vascular endothelial cells are exposed to an acidic pH, and CXC chemokine receptor type 4 (CXCR4) is a key protective molecule against acidosis. We investigated the effect of coupling factor 6 (CF6), a novel proton import activator, on CXCR4 signaling and its molecular mechanism. CF6 decreased CXCR4 expression in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. Pretreatment with small interfering RNA (siRNA) for hypoxia-inducible factor (HIF)-1α or PP1, a specific c-Src inhibitor, attenuated the CF6-induced decrease in CXCR4 without affecting CF6-induced intracellular acidosis. Chromatin immunoprecipitation revealed that CF6 enhanced the interaction between HIF-1α and the CXCR4 promoter at the hypoxia response element. CF6 also enhanced protein-protein interactions between phospho-c-Src and histone deacetylase 3 (HDAC3), but did not affect the binding of HDAC3 to the CXCR4 promoter at the hypoxia response element. Apoptotic cells, as measured by an Annexin-V-FITC Propidium Iodide Kit, were increased by CF6 in normoxia and hypoxia at 24 h; however, this increase was abolished by pretreatment with either siRNA for HIF-1α or the CXCR4 ligand. The coronary arteries and perivascular tissues obtained from CF6-overexpressing transgenic mice showed a lower expression of CXCR4 in the heart, increased wall thickness and infiltration of CD16-positive, CD206-positive or apoptotic cells. CF6 decreases CXCR4 expression through both HIF-1α- and c-Src-mediated mechanisms in vascular endothelial cells. Because CXCR4 has an important role in survival function, CF6 may have a role in the progression of arteriosclerosis via these complex mechanisms.


Assuntos
Apoptose/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Genes src/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/farmacologia , Inflamação/patologia , ATPases Mitocondriais Próton-Translocadoras/farmacologia , Fatores Acopladores da Fosforilação Oxidativa/farmacologia , Receptores CXCR4/biossíntese , Transdução de Sinais/efeitos dos fármacos , Animais , Células Endoteliais/efeitos dos fármacos , Genes src/efeitos dos fármacos , Humanos , Hipóxia/patologia , Inflamação/induzido quimicamente , Camundongos , Camundongos Transgênicos
20.
Mol Neurobiol ; 49(2): 658-72, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24018979

RESUMO

Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) were not completely understood. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 in RBA-1 cells and cells migration which were attenuated by pretreatment with the inhibitor of receptor tyrosine kinase (Genistein), c-Src (PP1), Jak2 (AG490), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), PKCs (Ro318220), PKCδ (Rottlerin), or NF-κB (Bay11-7082) and transfection with siRNA of c-Src, PDGFR, Akt, PKCδ, ATF2, p65, IKKα, or IKKß. In addition, thrombin-stimulated c-Src, Jak2, or PDGFR phosphorylation was inhibited by a thrombin inhibitor (PPACK), PP1, AG490, or AG1296. Thrombin further stimulated c-Src and PDGFR complex formation in RBA-1 cells. Thrombin also stimulated Akt and PKCδ phosphorylation and PKCδ translocation which were reduced by PPACK, PP1, AG490, AG1296, or LY294002. We further observed that thrombin markedly stimulated ATF2 or IκBα phosphorylation and NF-κB p65 translocation which were inhibited by Rottlerin or LY294002. Finally, thrombin stimulated in vivo binding of p65 to the MMP-9 promoter, which was reduced by pretreatment with Rottlerin or LY294002. These results concluded that in RBA-1 cells, thrombin activated a c-Src/Jak2/PDGFR/PI3K/Akt/PKCδ pathway, which in turn triggered ATF2 and NF-κB activation and ultimately induced MMP-9 expression associated with cell migration.


Assuntos
Astrócitos/metabolismo , Genes src/fisiologia , Janus Quinase 2/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Proteína Quinase C-delta/biossíntese , Receptores do Fator de Crescimento Derivado de Plaquetas/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Genes src/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Trombina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...