RESUMEN
The non-receptor tyrosine kinase Src plays a key role in cell division, migration, adhesion, and survival. Src is overactivated in several cancers, where it transmits signals that promote cell survival, mitosis, and other important cancer hallmarks. Src is therefore a promising target in cancer therapy, but the underlying mechanisms are still uncertain. Here we show that Src is highly conserved across different species. Src expression increases during mitosis and is localized to the chromosomal passenger complex. Knockdown or inhibition of Src induces multipolar spindle formation, resulting in abnormal expression of the Aurora B and INCENP components of the chromosomal passenger complex. Molecular mechanism studies have found that Src interacts with and phosphorylates INCENP. This then leads to incorrect chromosome arrangement and segregation, resulting in cell division failure. Herein, Src and chromosomal passenger complex co-localize and Src inhibition impedes mitotic progression by inducing multipolar spindle formation. These findings provide novel insights into the molecular basis for using Src inhibitors to treat cancer.
Asunto(s)
Antineoplásicos , Genes src , Mitosis , Proteínas Proto-Oncogénicas pp60(c-src) , Humanos , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Citoesqueleto/metabolismo , Genes src/efectos de los fármacos , Mitosis/efectos de los fármacos , Huso Acromático/genética , Huso Acromático/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Antineoplásicos/farmacologíaRESUMEN
Psoralea corylifolia (P corylifolia) has been popularly applied in traditional Chinese medicine decoction for treating osteoporosis and promoting fracture healing since centuries ago. However, the bioactive natural components remain unknown. In this study, applying comprehensive two-dimensional cell membrane chromatographic/C18 column/time-of-flight mass spectrometry (2D CMC/C18 column/TOFMS) system, neobavaisoflavone (NBIF), for the first time, was identified for the bioaffinity with RAW 264.7 cells membranes from the extracts of P corylifolia. Here, we revealed that NBIF inhibited RANKL-mediated osteoclastogenesis in bone marrow monocytes (BMMCs) and RAW264.7 cells dose dependently at the early stage. Moreover, NBIF inhibited osteoclasts function demonstrated by actin ring formation assay and pit-formation assay. With regard to the underlying molecular mechanism, co-immunoprecipitation showed that both the interactions of RANK with TRAF6 and with c-Src were disrupted. In addition, NBIF inhibited the phosphorylation of P50, P65, IκB in NF-κB pathway, ERK, JNK, P38 in MAPKs pathway, AKT in Akt pathway, accompanied with a blockade of calcium oscillation and inactivation of nuclear translocation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In vivo, NBIF inhibited osteoclastogenesis, promoted osteogenesis and ameliorated bone loss in ovariectomized mice. In summary, P corylifolia-derived NBIF inhibited RANKL-mediated osteoclastogenesis by suppressing the recruitment of TRAF6 and c-Src to RANK, inactivating NF-κB, MAPKs, and Akt signalling pathways and inhibiting calcium oscillation and NFATc1 translocation. NBIF might serve as a promising candidate for the treatment of osteoclast-associated osteopenic diseases.
Asunto(s)
Genes src/efectos de los fármacos , Isoflavonas/farmacología , Osteogénesis/efectos de los fármacos , Ligando RANK/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Línea Celular , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células RAW 264.7RESUMEN
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.
Asunto(s)
Quimiocina CCL2/biosíntesis , Genes src/fisiología , Neoplasias Inflamatorias de la Mama/metabolismo , Interleucina-8/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias de la Mama Triple Negativas/metabolismo , Adulto , Anciano , Antineoplásicos/farmacología , Línea Celular Tumoral , Dasatinib/farmacología , Femenino , Genes src/efectos de los fármacos , Humanos , Neoplasias Inflamatorias de la Mama/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Persona de Mediana Edad , Proteolisis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/fisiologíaRESUMEN
Advanced systemic mastocytosis is a rare and still untreatable disease. Blocking antibodies against inhibitory receptors, also known as "immune checkpoints", have revolutionized anti-cancer treatment. Inhibitory receptors are expressed not only on normal immune cells, including mast cells but also on neoplastic cells. Whether activation of inhibitory receptors through monoclonal antibodies can lead to tumor growth inhibition remains mostly unknown. Here we show that the inhibitory receptor Siglec-7 is expressed by primary neoplastic mast cells in patients with systemic mastocytosis and by mast cell leukemia cell lines. Activation of Siglec-7 by anti-Siglec-7 monoclonal antibody caused phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP-1), reduced phosphorylation of KIT and induced growth inhibition in mast cell lines. In SCID-beige mice injected with either the human mast cell line HMC-1.1 and HMC-1.2 or with Siglec-7 transduced B cell lymphoma cells, anti-Siglec-7 monoclonal antibody reduced tumor growth by a mechanism involving Siglec-7 cytoplasmic domains in "preventive" and "treatment" settings. These data demonstrate that activation of Siglec-7 on mast cell lines can inhibit their growth in vitro and in vivo. This might pave the way to additional treatment strategies for mastocytosis.
Asunto(s)
Lectinas/agonistas , Leucemia de Mastocitos/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Diferenciación Mielomonocítica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Genes src/efectos de los fármacos , Humanos , Leucemia de Mastocitos/patología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/patología , Masculino , Mastocitosis/tratamiento farmacológico , Ratones , Ratones SCID , Persona de Mediana Edad , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 6/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Genes src/fisiología , Osteoclastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ligando RANK/farmacología , Especies Reactivas de Oxígeno/metabolismo , Saponinas/toxicidad , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Genes src/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Osteoclastos/efectos de los fármacos , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidoresRESUMEN
BACKGROUND: Sepsis is defined as end-organ dysfunction resulting from the host's inflammatory response to infection. One of the most common sepsis-injured organs is the kidneys, resulting in acute kidney injury (AKI) that contributes to the high morbidity and mortality, especially patients in the intensive care unit. Fisetin, a naturally occurring flavonoid, has been reported to protect against the rat of lipopolysaccharide (LPS)-induced acute lung injury. However, the effect of fisetin on septic AKI remains unknown. PURPOSE: The current study proposed to systematically investigate the renoprotective effects and the underlying mechanisms of fisetin in septic AKI mice. METHODS: The model of septic AKI was established on male C57BL/6â¯J mice by a single intraperitoneal injection of LPS (10â¯mg/kg). Fisetin was administrated by gavage at 100â¯mg/kg for 3 consecutive days before LPS injection and the mice were sacrificed at 16â¯h after LPS injection. The serum and kidney samples were evaluated for biochemical analysis, histopathological examinations as well as inflammation and apoptosis related gene/protein expression. RESULTS: Pretreatment with fisetin significantly alleviated the elevated levels of serum creatinine and blood urea nitrogen in LPS-treated mice. Consistently, LPS induced renal damage as implied by histopathological score and the increased injury markers NGAL and KIM-1, which was attenuated by fisetin. Meanwhile, LPS injection triggered proinflammatory cytokine production and inflammation related proteins in the kidneys. However, fisetin inhibited renal expression of IL-6, IL-1ß, TNF-α, HMGB1, iNOS and COX-2 to improve inflammatory response. Furthermore, fisetin effectively reduced the number of TUNEL positive apoptotic cells and suppressed apoptotic protein of Bcl-2, BAX and cleaved caspase-3 in the kidneys of LPS-induced septic AKI. Mechanistically, LPS stimulated the expression of TLR4 and the phosphorylation of NF-κB p65, MAPK (p38, ERK1/2 and JNK), Src and AKT in the injured kidneys, while fisetin notably suppressed the corresponding protein expression. CONCLUSION: Fisetin alleviated kidney inflammation and apoptosis to protect against LPS-induced septic AKI mice via inhibiting Src-mediated NF-κB p65 and MAPK signaling pathways.
Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Flavonoides/farmacología , Inflamación/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Lesión Renal Aguda/metabolismo , Animales , Flavonoles , Genes src/efectos de los fármacos , Inflamación/metabolismo , Riñón/efectos de los fármacos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Sepsis/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Ceramidas/farmacología , Genes src/fisiología , Interleucina-1beta/farmacología , Potenciación a Largo Plazo/fisiología , Receptores AMPA/biosíntesis , Animales , Células Cultivadas , Expresión Génica , Genes src/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Polimerizacion/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidoresRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Periploca forrestii Schltr. is a classical traditional Chinese medicine (TCM) called Heilonggu (HLG) in China. According to the theory of TCM, it possesses the efficacy of eliminating wind and removing dampness. In clinical practice, it is commonly used for the treatment of rheumatoid arthritis. The present work aimed to evaluate the anti-rheumatism activity of HLG ethanol extract and reveal the underlying molecular mechanism by employing an animal model of collagen-induced rheumatoid arthritis (CIA) in rats. MATERIALS AND METHODS: The CIA was induced in male Sprague-Dawley rats by intradermal injection of bovine collagen-II in complete Freund's adjuvant (CFA) at the base of tail. The rats received oral administration of HLG (200 and 400mg/kg) from day 1, with the treatment lasting for 28 days. A variety of indicators were measured for evaluation of anti-rheumatism effect, including paw swelling, arthritis scores, and histopathological changes. Furthermore, the serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2), as well as cyclooxygenase-2 (COX-2), nuclear factor NF-κB p65 and Src kinase in joint synovial tissues were detected to explore the possible mechanisms. RESULTS: The administration of HLG significantly restored type II collagen-induced arthritis in rats as evidenced by decrease in paw swelling and inflammatory factors in serum. Meanwhile, this treatment also notably reduced NF-κB p65 and COX-2 expression. Surprisingly, the activity of Src kinase was also inhibited demonstrated by downregulation of phosphorylated Src. CONCLUSION: Our results revealed that HLG possessed observable therapeutic action on collagen-induced arthritis by inhibiting the activation of Src and nuclear translocation of NF-κB in rats. HLG may serve as a potential candidate for the management of patients with RA.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Genes src/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Periploca , Extractos Vegetales/uso terapéutico , Tallos de la Planta/química , Transducción de Señal/efectos de los fármacos , Animales , Artritis Experimental/patología , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Articulaciones/metabolismo , Articulaciones/patología , Masculino , Periploca/química , Periploca/toxicidad , Extractos Vegetales/toxicidad , Tallos de la Planta/toxicidad , Ratas , Ratas Sprague-Dawley , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
UNLABELLED: Noninvasive, real-time, quantitative measurement of key biomarkers associated with cancer therapeutic interventions could provide a better understanding of cancer biology. We investigated in this study whether incorporating multiple molecular imaging approaches could be used to guide dasatinib anti-Src therapy and aid in the rational design of a combination therapy regimen. METHODS: Bioluminescence imaging, (18)F-FDG PET, integrin αvß3-targeted SPECT/CT, and vascular endothelial growth factor-targeted near-infrared fluorescence imaging were performed before and after dasatinib treatment in a tumor mouse model. RESULTS: There was no significant difference in the bioluminescence imaging signal or (18)F-FDG tumor uptake in dasatinib-treated tumors compared with the control tumors. However, the uptake of (99m)T-3PRGD2 (integrin αvß3-specific) and DyLight755-ranibizumab (vascular endothelial growth factor-specific) in the dasatinib-treated tumors was significantly lower than that in the control tumors. In vitro studies confirmed the antiangiogenic effects of dasatinib but indicated a lack of cytotoxicity. Dasatinib plus cytotoxic docetaxel elicited marked synergistic tumor growth inhibition in vivo. CONCLUSION: Visualization of post-Src inhibition tumor signatures through multiple imaging approaches facilitates sensitive and quantitative measurement of cancer biomarkers in vivo, thus aiding in the rational design of dasatinib combination therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Dasatinib/uso terapéutico , Genes src/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Quimioterapia Combinada , Fluorodesoxiglucosa F18/farmacocinética , Integrina alfaVbeta3/efectos de los fármacos , Luminiscencia , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Imagen Molecular , Ensayo de Unión Radioligante , Radiofármacos/farmacocinética , Ranibizumab/farmacocinética , Tomografía Computarizada de Emisión de Fotón Único , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
AIMS: Meso-dihydroguaiaretic acid (MDA) is known for its anti-inflammatory, anti-oxidant, anti-bacterial, and anti-tumor activity. However, the anti-breast cancer effect and the mechanism of MDA remain undefined. MAIN METHODS: In this study, we examined the anti-cancer activity and the mechanisms of action of MDA in breast cancer cell lines, 4T-1 and MCF-7 cells; and 4T-1 bearing mouse model. KEY FINDINGS: MDA showed cytotoxic effects on 4T-1 and MCF-7 cells in a dose-dependent manner. Moreover, MDA increased the amount of Annexin V-positive apoptotic bodies, phosphorylated JNK and p38 in 4T-1 cells. MDA also down-regulated cell-cycle dependent proteins, CDK-4 and cyclin D1; and induced cleaved caspase-3 in MDA-treated 4T-1 cells. We further verified that MDA-induced apoptosis is mediated by p38 and caspase-3 activation in 4T-1 cells. Next, we studied the effect of MDA treatment on cell migration and found that MDA significantly reduced cell migration. Moreover, MDA reduced EGFR and intergrin ß3 expression, and dephosphorylated Src in a dose-dependent manner in 4T-1 cells. Furthermore, we observed in vivo effect of MDA in 4T-1 cell inoculated mice. MDA (20mg/kg/day) significantly suppressed mammary tumor volume and activated caspase-3 in tumor tissues. SIGNIFICANCE: These results suggest novel targets of MDA in breast cancer in vitro and in vivo, making it a potential candidate as a chemotherapeutic drug.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Guayacol/análogos & derivados , Lignanos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Femenino , Genes src/efectos de los fármacos , Guayacol/farmacología , Humanos , Integrina beta3/biosíntesis , Integrina beta3/genética , Células MCF-7 , Neoplasias Mamarias Experimentales/tratamiento farmacológico , RatonesRESUMEN
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.
Asunto(s)
Fármacos del Sistema Nervioso Central/farmacología , Corteza Cerebral/efectos de los fármacos , Insulisina/metabolismo , Iridoides/farmacología , Neuronas/efectos de los fármacos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Células Cultivadas , Corteza Cerebral/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citosol/efectos de los fármacos , Citosol/enzimología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Factores de Transcripción Forkhead/metabolismo , Genes src/efectos de los fármacos , Genes src/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Células PC12 , PPAR gamma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
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.
Asunto(s)
Señalización del Calcio/fisiología , Sulfato de Dextran/toxicidad , Genes src/fisiología , MAP Quinasa Quinasa 7/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Uniones Estrechas/metabolismo , Animales , Células CACO-2 , Señalización del Calcio/efectos de los fármacos , Femenino , Genes src/efectos de los fármacos , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Uniones Estrechas/efectos de los fármacosRESUMEN
Germline mutations in the PTEN tumor-suppressor gene and germline variations in succinate dehydrogenase subunit D gene (SDHD-G12S, SDHD-H50R) are associated with a subset of Cowden syndrome and Cowden syndrome-like individuals (CS/CSL) and confer high risk of breast, thyroid and other cancers. However, very little is known about the underlying crosstalk between SDHD and PTEN in CS-associated thyroid cancer. Here, we show SDHD-G12S and SDHD-H50R lead to impaired PTEN function through alteration of its subcellular localization accompanied by resistance to apoptosis and induction of migration in both papillary and follicular thyroid carcinoma cell lines. Other studies have shown elevated proto-oncogene tyrosine kinase (SRC) activity in invasive thyroid cancer cells; so, we explore bosutinib, a specific inhibitor for SRC, to explore SRC as a mediator of SDH-PTEN crosstalk in this context. We show that SRC inhibition could rescue SDHD dysfunction-induced cellular phenotype and tumorigenesis only when wild-type PTEN is expressed, in thyroid cancer lines. Patient lymphoblast cells carrying either SDHD-G12S or SDHD-H50R also show increased nuclear PTEN and more oxidized PTEN after hydrogen peroxide treatment. Like in thyroid cells, bosutinib decreases oxidative PTEN in patient lymphoblast cells carrying SDHD variants, but not in patients carrying both SDHD variants and PTEN truncating mutations. In summary, our data suggest a novel mechanism whereby SDHD germline variants SDHD-G12S or SDHD-H50R induce thyroid tumorigenesis mediated by PTEN accumulation in the nucleus and may shed light on potential treatment with SRC inhibitors like bosutinib in PTEN-wild-type SDHD-variant/mutation positive CS/CSL patients and sporadic thyroid neoplasias.
Asunto(s)
Adenocarcinoma Folicular/metabolismo , Carcinoma/metabolismo , Núcleo Celular/metabolismo , Fosfohidrolasa PTEN/metabolismo , Succinato Deshidrogenasa/genética , Neoplasias de la Tiroides/metabolismo , Adenocarcinoma Folicular/genética , Compuestos de Anilina/farmacología , Apoptosis/efectos de los fármacos , Carcinoma/genética , Carcinoma Papilar , Línea Celular Tumoral , Genes src/efectos de los fármacos , Mutación de Línea Germinal , Síndrome de Hamartoma Múltiple/genética , Humanos , Nitrilos/farmacología , Oxidación-Reducción/efectos de los fármacos , Fosfohidrolasa PTEN/genética , Polimorfismo de Nucleótido Simple , Proto-Oncogenes Mas , Quinolinas/farmacología , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/genéticaRESUMEN
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.
Asunto(s)
Transformación Celular Neoplásica , Endometriosis/genética , Endometriosis/patología , Genes src/fisiología , Enfermedades del Ovario/genética , Enfermedades del Ovario/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Animales , Células Cultivadas , Endometriosis/tratamiento farmacológico , Células Epiteliales , Femenino , Genes src/efectos de los fármacos , Humanos , Ratones , Enfermedades del Ovario/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológicoRESUMEN
UNLABELLED: Hepatic stellate cell (HSC) activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR). Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 µM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 µM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine's inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway. CONCLUSIONS: Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III.
Asunto(s)
Acetaldehído/farmacología , Cafeína/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Receptor de Adenosina A2A/metabolismo , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Genes src/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed rapamycin-regulated targeted activation of pathways (RapRTAP), was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src-induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Genes src/efectos de los fármacos , Proteínas Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proteína Sustrato Asociada a CrK/genética , Proteína Sustrato Asociada a CrK/metabolismo , Citoplasma/enzimología , Citoplasma/ultraestructura , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Microscopía Fluorescente , Fenotipo , Seudópodos/efectos de los fármacos , Seudópodos/ultraestructura , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Genes src/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/farmacología , Inflamación/patología , ATPasas de Translocación de Protón Mitocondriales/farmacología , Factores de Acoplamiento de la Fosforilación Oxidativa/farmacología , Receptores CXCR4/biosíntesis , Transducción de Señal/efectos de los fármacos , Animales , Células Endoteliales/efectos de los fármacos , Genes src/efectos de los fármacos , Humanos , Hipoxia/patología , Inflamación/inducido químicamente , Ratones , Ratones TransgénicosRESUMEN
PURPOSE: We investigated the contribution of cytochrome P450 (CYP) 1B1 to hypertension and its pathogenesis by examining the effect of its selective inhibitor, 2,4,3',5'-tetramethoxystilbene (TMS), in spontaneously hypertensive rats (SHR). METHODS: Blood pressure (BP) was measured bi-weekly. Starting at 8 weeks, TMS (600 µg/kg, i.p.) or its vehicle was injected daily. At 14 weeks, samples were collected for measurement. RESULTS: TMS reversed increased BP in SHR (207 ± 7 vs. 129 ± 2 mmHg) without altering BP in Wistar-Kyoto rats. Increased CYP1B1 activity in SHR was inhibited by TMS (RLU: aorta, 5.4 ± 0.7 vs. 3.7 ± 0.7; heart, 6.0 ± 0.8 vs. 3.4 ± 0.4; kidney, 411 ± 45 vs. 246 ± 10). Increased vascular reactivity, cardiovascular hypertrophy, endothelial and renal dysfunction, cardiac and renal fibrosis in SHR were minimized by TMS. Increased production of reactive oxygen species and NADPH oxidase activity in SHR, were diminished by TMS. In SHR, TMS reduced increased plasma levels of nitrite/nitrate (46.4 ± 5.0 vs. 28.1 ± 4.1 µM), hydrogen-peroxide (36.0 ± 3.7 vs. 14.1 ± 3.8 µM), and thiobarbituric acid reactive substances (6.9 ± 1.0 vs. 3.4 ± 1.5 µM). Increased plasma levels of pro-inflammatory cytokines and catecholamines, and cardiac activity of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, c-Src tyrosine kinase, and protein kinase B in SHR were also inhibited by TMS. CONCLUSIONS: These data suggests that increased oxidative stress generated by CYP1B1 contributes to hypertension, increased cytokine production and sympathetic activity, and associated pathophysiological changes in SHR. CYP1B1 could be a novel target for developing drugs to treat hypertension and its pathogenesis.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Presión Sanguínea/fisiología , Hipertensión/metabolismo , Hipertensión/patología , Enfermedades Renales/metabolismo , Ratas Endogámicas SHR/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Presión Sanguínea/efectos de los fármacos , Sistema Cardiovascular/efectos de los fármacos , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patología , Catecolaminas/metabolismo , Citocromo P-450 CYP1B1 , Citocinas/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Fibrosis/metabolismo , Fibrosis/patología , Genes src/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Hipertrofia/metabolismo , Hipertrofia/patología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso/patología , NADPH Oxidasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Endogámicas SHR/fisiología , Ratas Endogámicas WKY , Especies Reactivas de Oxígeno/metabolismo , Estilbenos/farmacología , Superóxidos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
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.