RESUMEN
The serine-threonine kinase Akt plays a fundamental role in cell survival, metabolism, proliferation, and migration. To keep these essential processes under control, Akt activity and stability must be tightly regulated; otherwise, life-threatening conditions might prevail. Although it is well understood that phosphorylation regulates Akt activity, much remains to be known about how its stability is maintained. Here, we characterize BAG5, a chaperone regulator, as a novel Akt-interactor and substrate that attenuates Akt stability together with Hsp70. BAG5 switches monoubiquitination to polyubiquitination of Akt and increases its degradation caused by Hsp90 inhibition and Hsp70 overexpression. Akt interacts with BAG5 at the linker region that joins the first and second BAG domains and phosphorylates the first BAG domain. The Akt-BAG5 complex is formed in serum-starved conditions and dissociates in response to HGF, coincident with BAG5 phosphorylation. BAG5 knockdown attenuated Akt degradation and facilitated its activation, whereas the opposite effect was caused by BAG5 overexpression. Altogether, our results indicate that Akt stability and signaling are dynamically regulated by BAG5, depending on growth factor availability.
Asunto(s)
Chaperonas Moleculares , Proteínas Proto-Oncogénicas c-akt , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ubiquitinación , Células HEK293 , Humanos , Animales , RatonesRESUMEN
Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.
Asunto(s)
Movimiento Celular , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Dominios Homólogos a Pleckstrina/genética , Seudópodos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Animales , Línea Celular , Humanos , Ratones , FosforilaciónRESUMEN
BACKGROUND: Histamine, through histamine H2 receptor (H2R), modulates different biological processes, involving the modulation of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Many evidences have demonstrated the existence and importance of the crossregulation between these two signaling pathways. The aim of the present work was to determine the molecular mechanisms leading to PI3K and ERK pathways modulation induced by the H2R agonist amthamine and to evaluate the possible interplay between them. METHODS: Phosphorylation levels of ERK and Akt were examined by Western blot in HEK293T cells expressing the human H2R, in the presence of H2R agonist and dominant negative mutants or pharmacological inhibitors of different proteins/pathways. Transcriptional activity assays were assessed to determine SRE activity. Amthamine-mediated cellular proliferation was investigated in MA-10A cells in the presence of PI3K inhibitor. RESULTS: H2R agonist inhibits PI3K/Akt/mTOR and stimulates Ras/MEK/ERK pathways. Moreover, PI3K/Akt/mTOR signaling inhibition is necessary to achieve H2R mediated ERK activation. In the presence of a constitutive active mutant of Akt, amthamine is not able to mediate ERK activation. This crosstalk affects classical ERK downstream targets such as Elk1 phosphorylation and the transcriptional activity of the SRE, classically associated to proliferation. We further demonstrate that amthamine-induced proliferation in Leydig MA-10 tumor cells, is enhanced by LY294002, a PI3K inhibitor. CONCLUSIONS: These results describe a crosstalk between PI3K/AKT/mTOR and Ras/MEK/ERK pathways induced by H2R stimulation with implications in cell proliferation. GENERAL SIGNIFICANCE: This work indicates that the modulation of PI3K/AKT/mTOR pathway by H2R in turn regulates Ras/MEK/ERK activation conditioning the proliferative capacity of the cells.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Histamina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Células HEK293 , Humanos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Tiazoles/farmacologíaRESUMEN
Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.
Asunto(s)
Proteínas ELAV/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitógenos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Regiones no Traducidas 3'/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Proteínas ELAV/genética , Proteína 1 Similar a ELAV , Células HEK293 , Células HeLa , Humanos , Ratones , Mutación , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas.
Asunto(s)
Extractos Celulares/farmacología , Movimiento Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Ganglios Linfáticos/inmunología , Melanoma , Receptores CCR7/genética , Animales , Línea Celular Tumoral , Células Dendríticas/citología , Humanos , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Masculino , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Receptores CCR7/inmunología , Receptores CCR7/metabolismoRESUMEN
G protein-coupled receptor signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather from complex interactions of multiple, branched signaling routes, i.e., signaling networks. In this work we present an exhaustive study of the cross-talk between H1 and H2 histamine receptors (H1R and H2R) in U937 cells and Chinese hamster ovary-transfected cells. By desensitization assays we demonstrated the existence of a crossdesensitization between both receptors independent of protein kinase A or C. H1R-agonist stimulation inhibited cell proliferation and induced apoptosis in U937 cells following treatment of 48 hours. H1R-induced antiproliferative and apoptotic response was inhibited by an H2R agonist suggesting that the cross-talk between both receptors modifies their function. Binding and confocal microscopy studies revealed cointernalization of both receptors upon treatment with the agonists. To evaluate potential heterodimerization of the receptors, sensitized emission fluorescence resonance energy transfer experiments were performed in human embryonic kidney 293T cells using H1R-cyan fluorescent protein and H2R-yellow fluorescent protein. To our knowledge these findings may represent the first demonstration of agonist-induced heterodimerization of the H1R and H2R. In addition, we also show that the inhibition of the internalization process did not prevent receptor crossdesensitization, which was mediated by G protein-coupled receptor kinase 2. Our study provides new insights into the complex signaling network mediated by histamine and further knowledge for the rational use of its ligands.
Asunto(s)
Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células CHO , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HEK293 , Histamina/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Humanos , Proteína Quinasa C/metabolismo , Transducción de Señal , Células U937RESUMEN
In C4-HD murine mammary carcinomas and in human breast cancer T47D cells, we showed that medroxyprogesterone acetate (MPA) induces a nuclear physical association between estrogen receptor alpha (ERa) and progesterone receptors (PR). The blockade of ERa inhibits cell proliferation mediated by progestins. We hypothesized that this nuclear association between ERa/PR is necessary to trigger progestin-induced cell proliferation and tumor growth. We demonstrated that fulvestrant (FUL, ICI182.780) induced complete regression of C4-HD tumors growing with progestins. MPA treatment induced an early increase in both CCND1 and MYC expression in T47D cells. The blockade of ERa prevented the MPA-dependent transcription of both genes. Specific binding of PR/ERa was observed at the same MPA-sensitive regions at the CCND1 and MYC gene promoters after chromatin immunoprecipitation (ChIP) analysis. ICI inhibited binding of ERa to both gene regulatory sequences while PR binding was unaffected. The nuclear colocalization between both receptors in T47D cells was confirmed by: confocal microscopy, Duolink assays and co-immunoprecipitation assays. In breast cancer samples we also observed a nuclear interaction between both steroid receptors. Our results indicate that the presence of ERa interacting with activated PR at the CCND1 and MYC promoters is required to trigger progestin-induced gene transcription and cell proliferation in breast cancer cells.
Asunto(s)
Carcinoma/patología , Estradiol/análogos & derivados , Receptor alfa de Estrógeno/fisiología , Neoplasias Mamarias Experimentales/patología , Receptores de Progesterona/fisiología , Animales , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/genética , Carcinoma/inducido químicamente , Carcinoma/tratamiento farmacológico , Proliferación Celular , Inmunoprecipitación de Cromatina , Ciclina D1/metabolismo , Estradiol/administración & dosificación , Receptor alfa de Estrógeno/efectos de los fármacos , Femenino , Fulvestrant , Genes myc , Humanos , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Acetato de Medroxiprogesterona/farmacología , Murinae , Progestinas/metabolismo , Receptores de Progesterona/efectos de los fármacos , Transcripción GenéticaRESUMEN
The expression of heme oxygenase-1 (HO-1) was shown to be increased in multiple tumors compared with their surrounding healthy tissues and was also observed to be up-regulated in oral squamous cell carcinomas (OSCC). However, conflicting results were obtained and little information is available regarding HO-1 significance in head and neck squamous cell carcinoma (HNSCC). Therefore, the aim of the present study was to perform a wide screening of HO-1 expression in a large collection of human primary HNSCCs and to correlate the results with clinical and pathological parameters. For this purpose, we investigated the expression of this protein by immunohistochemistry (IHC) in tissue microarrays (TMAs) of HNSCC and in an independent cohort of paraffin-embedded tumor specimens. HO-1 expression was further validated by real-time qPCR performed on selected laser capture-microdissected (LCM) oral tissue samples. Both the number of HO-1-positive samples and HO-1 immunoreactivity in the cancerous tissues were significantly higher than those in the non-tumor tissues. These results were confirmed at the mRNA level. Interestingly, HO-1 localization was observed in the nucleus, and the rate of nuclear HO-1 in HNSCC was higher than that in non-malignant tissues. Nuclear HO-1 was observed in HNSCC cell lines and increased even further following hemin treatment. Analysis of HO-1 expression and sub-cellular localization in a mouse model of squamous cell carcinoma (SCC) and in human HNSCC revealed that nuclear HO-1 increases with tumor progression. Taken together, these results demonstrate that HO-1 is up-regulated in HNSCC and that nuclear localization of HO-1 is associated with malignant progression in this tumor type.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Neoplasias de Cabeza y Cuello/enzimología , Hemo-Oxigenasa 1/metabolismo , Anciano , Animales , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Neoplasias de Cabeza y Cuello/diagnóstico , Neoplasias de Cabeza y Cuello/genética , Hemo-Oxigenasa 1/genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Adhesión en Parafina , Reacción en Cadena de la Polimerasa , Pronóstico , ARN Mensajero/metabolismo , Análisis de Matrices TisularesRESUMEN
Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth, but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) are required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear colocalization of activated ERα with PR. Treatment with the pure antiestrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies showed that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear colocalization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation.
Asunto(s)
Neoplasias de la Mama/patología , Ciclina D1/genética , Receptor alfa de Estrógeno/metabolismo , Genes myc , Neoplasias Mamarias Experimentales/patología , Receptores de Progesterona/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Procesos de Crecimiento Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Femenino , Fulvestrant , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Acetato de Medroxiprogesterona/farmacología , Ratones , Ratones Endogámicos BALB C , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Regiones Promotoras Genéticas , Receptores de Progesterona/genéticaRESUMEN
While changes in heme oxygenase (HO-1) in lung cancer have already been reported, conflicting results were obtained for enzyme expression in human lung cancer specimens. Therefore, the aim of this work was to study HO-1 expression in a large collection of human lung cancer samples. For this purpose, we analyzed the expression of HO-1 in an organized tissue microarray (TMA) and investigated its correlation with clinicopathological data. Ninety-six percent of tumor samples were positive for HO-1, and the expression of HO-1 was significantly higher in cancerous than in non-cancerous tissues. Importantly, HO-1 expression correlated with advanced stages and lymph node involvement. Additionally, quantitative RT-PCR in 18 pairs of human lung carcinomas and their adjacent non-malignant tissues was performed. Our results demonstrate that HO-1 protein is upregulated in epithelial malignant cells in NSCLC and its expression is associated with higher stages of the disease. Additionally, different subcellular localization is observed between tumor and adjacent non-malignant tissues.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Expresión Génica , Hemo-Oxigenasa 1/metabolismo , Neoplasias Pulmonares/enzimología , Animales , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/secundario , Línea Celular Tumoral , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Hemo-Oxigenasa 1/genética , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Ratones , Células 3T3 NIH , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Matrices Tisulares , Regulación hacia ArribaRESUMEN
SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 µM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Chaperonas de Histonas/metabolismo , Estrés Oxidativo , Transducción de Señal , Factores de Transcripción/metabolismo , Apoptosis , Western Blotting , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Supervivencia Celular , Citoplasma/efectos de los fármacos , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente , Glutatión/metabolismo , Células HEK293 , Chaperonas de Histonas/genética , Humanos , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , Factores de Transcripción/genética , Transfección , Regulación hacia Arriba , terc-Butilhidroperóxido/farmacologíaRESUMEN
BACKGROUND: Interleukin-21 (IL-21) controls the differentiation of T-helper Th17 cells and induces the production of IL-17 in this T-cell subtype. The aim of this study is to determine the relative expression of IL-21 in gingival tissues of chronic periodontitis patients and correlate/associate this expression with proinflammatory cytokines and clinical parameters of disease. METHODS: Samples of gingival biopsies were collected from chronic periodontitis patients (n = 10) and controls (n = 8). The mRNA expressions of IL-21, IL-1ß, IL-6, IL-17, IL-23, IL-10, and transforming growth factor-ß1 (TGF-ß1) were quantified using real-time reverse transcription-polymerase chain reaction. IL-21 levels were compared between chronic periodontitis and healthy gingival tissues and correlated with cytokine and clinical parameters of tissue destruction. RESULTS: A significant overexpression of IL-21, IL-1ß, IL-6, IL-17, and IL-23p19 was detected in periodontal disease-affected tissues compared to healthy gingival tissues. IL-10 and TGF-ß1 were, however, downregulated in periodontal lesions. IL-21 yielded significant positive correlations with probing depth, clinical attachment level, IL-1ß, and IL-6. In addition, IL-21 was negatively correlated with IL-10 and TGF-ß1. CONCLUSIONS: IL-21 was overexpressed in chronic periodontitis gingival tissues and correlated with clinical parameters of periodontal destruction and with proinflammatory cytokines. Therefore, IL-21 might play a role in the tissue destruction that characterizes chronic periodontal disease.
Asunto(s)
Periodontitis Crónica/inmunología , Citocinas/análisis , Mediadores de Inflamación/análisis , Interleucinas/análisis , Adulto , Índice de Placa Dental , Femenino , Encía/inmunología , Humanos , Interleucina-10/análisis , Interleucina-17/análisis , Interleucina-1beta/análisis , Interleucina-23/análisis , Subunidad p19 de la Interleucina-23/análisis , Interleucina-6/análisis , Masculino , Persona de Mediana Edad , Pérdida de la Inserción Periodontal/inmunología , Índice Periodontal , Bolsa Periodontal/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Factor de Crecimiento Transformador beta1/análisisRESUMEN
Fibroblast growth factor (FGF) receptor 2 (FGFR-2) polymorphisms have been associated with an increase in estrogen receptor and progesterone receptor (PR)-positive breast cancer risk; however, a clear mechanistic association between FGFR-2 and steroid hormone receptors remains elusive. In previous works, we have shown a cross talk between FGF2 and progestins in mouse mammary carcinomas. To investigate the mechanisms underlying these interactions and to validate our findings in a human setting, we have used T47D human breast cancer cells and human cancer tissue samples. We showed that medroxyprogesterone acetate (MPA) and FGF2 induced cell proliferation and activation of ERK, AKT, and STAT5 in T47D and in murine C4-HI cells. Nuclear interaction between PR, FGFR-2, and STAT5 after MPA and FGF2 treatment was also showed by confocal microscopy and immunoprecipitation. This effect was associated with increased transcription of PRE and/or GAS reporter genes, and of PR/STAT5-regulated genes and proteins. Two antiprogestins and the FGFR inhibitor PD173074, specifically blocked the effects induced by FGF2 or MPA respectively. The presence of PR/FGFR-2/STAT5 complexes bound to the PRE probe was corroborated by using NoShift transcription and chromatin immunoprecipitation of the MYC promoter. Additionally, we showed that T47D cells stably transfected with constitutively active FGFR-2 gave rise to invasive carcinomas when transplanted into NOD/SCID mice. Nuclear colocalization between PR and FGFR-2/STAT5 was also observed in human breast cancer tissues. This study represents the first demonstration of a nuclear interaction between FGFR-2 and STAT5, as PR coactivators at the DNA progesterone responsive elements, suggesting that FGFRs are valid therapeutic targets for human breast cancer treatment.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de Progesterona/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Acetato de Medroxiprogesterona/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Trasplante de NeoplasiasRESUMEN
In this article, we demonstrate the expression of functional progesterone binding sites at the cell membrane in murine mammary carcinomas that are stimulated by progestins and inhibited by antiprogestins. Using confocal immunofluorescence, ligand binding and cell compartment-specific western blots, we were able to identify the presence of the classical progesterone receptors. Medroxyprogesterone acetate (MPA) and RU-486 (1 × 10(-11) and 1 × 10(-8) M) behaved as agonists activating extracellular signal-regulated kinases (ERKs) and progestin-regulated proteins, except for Cyclin D1 and Tissue factor which failed to increase with 1 × 10(-8) M RU-486, an experimental condition that allows PR to bind DNA. These results predicted a full agonist effect at low concentrations of RU-486. Accordingly, at concentrations lower than 1 × 10(-11) M, RU-486 increased cell proliferation in vitro. This effect was abolished by incubation with the ERK kinase inhibitor PD 98059 or by OH-tamoxifen. In vivo, at a daily dose of 1.2 µg/kg body weight RU-486 increased tumor growth, whereas at 12 mg/kg induces tumor regression. Our results indicate that low concentrations of MPA and RU-486 induce similar agonistic non-genomic effects, whereas RU-486 at higher concentrations may inhibit cell proliferation by genomic-induced effects. This suggests that RU-486 should be therapeutically administered at doses high enough to guarantee its genomic inhibitory effect.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/metabolismo , Mifepristona/agonistas , Mifepristona/farmacología , Progestinas/agonistas , Progestinas/uso terapéutico , Receptores de Progesterona/metabolismo , Animales , Femenino , Acetato de Medroxiprogesterona/farmacología , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Trasplante de Neoplasias , Receptores de Progesterona/química , Esteroides/químicaRESUMEN
Over the past several years, we have been interested in understanding the mechanisms by which mammary carcinomas acquire hormone independence. We demonstrated that carcinoma associated fibroblasts participate in the ligand-independent activation of progesterone receptors inducing tumor growth. In this study, we used DNA microarrays to compare the gene expression profiles of tumors from the MPA mouse breast cancer model, one hormone-dependent (C4-HD) and one hormone-independent (C4-HI), using whole tumor samples or laser-captured purified stromal and epithelial cells obtained from the same tumors. The expression of selected genes was validated by immunohistochemistry and immunofluorescence assays. We identified 413 genes specifically expressed in tumor stroma. Eighty-five percent of these genes were upregulated, whereas the remaining 15% were downregulated in C4-HI relative to their expression in the C4-HD tumor stroma. Several matrix metallopeptidases were overexpressed in the C4-HI tumor microenvironment. On the other hand, 1100 genes were specifically expressed in the tumor parenchyma. Among them, the 29% were upregulated, whereas the remaining 71% were downregulated in C4-HI relative to C4-HD tumor epithelium. Steap, Pdgfc, Runx2, Cxcl9, and Sdf2 were among the genes with high expression in the C4-HI tumor parenchyma. Interestingly, Fgf2 was one of the few genes upregulated by MPA in C4-HD tumors, confirming its pivotal role in regulating tumor growth in this model. In conclusion, we demonstrate herein a gene expression profile that distinguishes both the epithelial and the stromal cells in mammary tumors with different hormone dependence, supporting the hypothesis that the tumor-associated stroma may contribute to hormone-independent tumor growth.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Carcinoma/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Mamarias Experimentales/genética , Acetato de Medroxiprogesterona/farmacología , Animales , Carcinoma/metabolismo , Carcinoma/patología , Análisis por Conglomerados , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Reproducibilidad de los Resultados , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Prostaglandina E/metabolismo , Western Blotting , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Proliferación Celular , Ciclooxigenasa 2/genética , Dinoprostona/genética , Femenino , Neoplasias de Cabeza y Cuello/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Receptores de Prostaglandina E/genéticaRESUMEN
Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/enzimología , Neoplasias de Cabeza y Cuello/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Progresión de la Enfermedad , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Humanos , Análisis por Micromatrices , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esfingolípidos/metabolismo , Regulación hacia ArribaRESUMEN
The Kaposi sarcoma-associated herpes virus-G protein-coupled receptor is a key molecule in the pathogenesis of Kaposi sarcoma, playing a central role in promoting vascular endothelial growth factor-driven angiogenesis and spindle cell proliferation. We studied the effects of 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)] and the analog TX527 on the proliferation of endothelial cells (SVECs) and SVECs transformed by the viral G protein-coupled receptor (SVEC-vGPCR). 1 alpha,25(OH)(2)D(3) and TX527 decreased SVEC-vGPCR and SVEC numbers, the response being time dependent and similar in both cell lines. Vitamin D receptor (VDR) levels increased on treatment with 10 nm 1 alpha,25(OH)(2)D(3) or 1 nm TX527 in a time-dependent manner (1.5-24 h) in SVECs and SVEC-vGPCR. Basal VDR levels were increased in SVEC-vGPCR. The antiproliferative effects were accompanied by reduction in cyclin D1 and accumulation of p27 in SVECs but not SVEC-vGPCR. Induction of VDR was blocked by transfection of short hairpin RNA against VDR in SVEC-vGPCR and the antiproliferative effects of 1 alpha,25(OH)(2)D(3) and TX527 were decreased, involving the VDR genomic pathway in the hormone and analog mechanism of action. In vivo experiments showed that 1 alpha,25(OH)(2)D(3) and TX527 decreased SVEC-vGPCR tumor progression when the tumor cells were implanted in nude mice. In conclusion, we have demonstrated that 1 alpha,25(OH)(2)D(3) and its TX527 analog have antiproliferative effects on the growth of endothelial cells transformed by the vGPCR in vitro and in vivo, the vitamin D receptor being part of the inhibitory mechanism of action.
Asunto(s)
Alquinos/farmacología , Proliferación Celular/efectos de los fármacos , Transformación Celular Viral/efectos de los fármacos , Colecalciferol/farmacología , Células Endoteliales/efectos de los fármacos , Vitamina D/análogos & derivados , Animales , Línea Celular Transformada , Transformación Celular Viral/genética , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células Endoteliales/patología , Femenino , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Receptores de Calcitriol/antagonistas & inhibidores , Receptores de Calcitriol/genética , Receptores de Quimiocina/genética , Receptores de Quimiocina/fisiología , Vitamina D/farmacologíaRESUMEN
Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases.
Asunto(s)
Simulación por Computador , Geraniltranstransferasa/metabolismo , Aminoácidos , Sitios de Unión , Catálisis , Hidrógeno/química , Terpenos/metabolismoRESUMEN
BACKGROUND: Based on in vitro studies, Rho guanine nucleotide exchange factors (RhoGEFs) are key regulators of mitogenic and transforming pathways. At least 1 family member, PDZ-RhoGEF, also integrates signaling between monomeric Rho G proteins and heterotrimeric G proteins through a so-called regulator of G-protein signaling (RGS) domain. Recently, the authors reported that 3 single-nucleotide polymorphisms (SNPs) in 2 members of the RGS family were associated with significant reductions in the risk of cancer. METHODS: For the current report, the authors studied the risk of lung cancer associated with a nonsynonymous SNP (rs868188; Ser1416Gly) in PDZ-RhoGEF in a large lung cancer case-control study of 2260 Caucasians and 369 Mexican Americans. RESULTS: Compared with individuals who had the wild-type genotype (AA), Mexican Americans with the variant genotypes (AG and GG) had a significantly reduced risk for lung cancer (odds ratio [OR], 0.57; 95% confidence interval [95%CI], 0.34-0.94). The protective effect appeared to be more evident in younger individuals (OR, 0.42; 95%CI, 0.20-0.91), men (OR, 0.36; 95%CI, 0.18-0.71), and ever smokers (OR, 0.50; 95%CI, 0.29-0.88). A joint effect was observed between Ser1416Gly and polymorphisms in 2 cell-cycle control genes: p53 (intron 3) and cyclin D1 (CCND1). Tallying the variant alleles of the 4 RGS gene SNPs, a gene-dosage effect was apparent. Compared with individuals who had < 3 variant alleles, patients with > or = 3 variant alleles had a 51% reduction in lung cancer risk (OR, 0.49; 95%CI, 0.28-0.88). CONCLUSIONS: To the authors' knowledge, this is the first epidemiological study to link PDZ-RhoGEF polymorphisms with cancer risk. The results suggest that there are interactions between RGS2, RGS6, and PDZ-RhoGEF and validate this family of proteins as key regulators of tumorigenesis.