RESUMEN
P-glycoprotein (P-gp), traditionally linked to cancer poor prognosis and multidrug resistance, is undetectable in normal gastric mucosa and overexpressed in gastric cancer (GC). We propose that P-gp may be involved in Helicobacter pylori (Hp)-related gastric carcinogenesis by inhibiting apoptosis. Aim of the study was to evaluate the expression of P-gp in fetal stomach and in Hp-related gastric carcinogenesis, the epigenetic control of the multi-drug resistance-1 (MDR1) gene, the localization and interaction between P-gp and Bcl-x(L) and the effect of the selective silencing of P-gp on cell survival. P-gp and Bcl-xl expression was evaluated by immunohistochemistry on 28 spontaneously abortive human fetuses, 66 Hp-negative subjects, 138 Hp-positive chronic gastritis (CG) of whom 28 with intestinal metaplasia (IM) and 45 intestinal type GCs. P-gp/Bcl-x(L) colocalization was investigated by confocal immunofluorescence microscopy and protein-protein interaction by co-immunoprecipitation, in basal conditions and after stress-induced apoptosis, in GC cell lines AGS and MKN-28 and hepatocellular carcinoma cell line Hep-G2. The role of P-gp in controlling apoptosis was evaluated by knocking down its expression with a specific small interfering RNAs in stressed AGS and MKN-28 cell lines. P-gp is expressed in the gastric mucosa of all human fetuses while, it is undetectable in adult normal mucosa and re-expressed in 30/110 Hp-positive non-IM-CG, 28/28 IM-CG and 40/45 GCs. P-gp expression directly correlates with that of Bcl-x(L) and with the promoter hypomethylation of the MDR1 gene. In GC cell lines, P-gp is localized on the plasma membrane and mitochondria where it colocalizes with Bcl-x(L). Co-immunoprecipitation confirms the physical interaction between P-gp and Bcl-x(L) in AGS, MKN-28 and Hep-G2, at both basal level and after stress-induced apoptosis. The selective silencing of P-gp sensitizes GC cells to stress-induced apoptosis. P-gp behaves as an oncofetal protein that, by cross-talking with Bcl-x(L), acts as an anti-apoptotic agent in Hp-related gastric carcinogenesis.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Biomarcadores de Tumor/metabolismo , Supervivencia Celular , Neoplasias Gástricas/metabolismo , Proteína bcl-X/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Feto Abortado , Adulto , Anciano , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Apoptosis , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Metilación de ADN , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Gastritis/metabolismo , Gastritis/patología , Gastritis/terapia , Silenciador del Gen/efectos de los fármacos , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Humanos , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Masculino , Metaplasia/metabolismo , Metaplasia/patología , Microscopía Confocal/métodos , Persona de Mediana Edad , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , Estómago/citología , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/terapia , Proteína bcl-X/inmunologíaRESUMEN
In this study, selenocystine, a nutritionally available selenoamino acid, was identified for the first time as a novel agent with anti proliferative activity on human keloids. The 20 µM concentration after 48 h treatment used here was the most effective to reduce keloid fibroblast growth. We analyzed the gene expression profile of selenocystine treatment response in keloid fibroblasts by the microarray system to characterize the effects of selenocystine on human keloids. The major alterations in keloid fibroblasts following selenocystine exposure included up-regulation of the genes encoding cell death and transcription factors. Prominent down-regulation of genes involved in development, cell adhesion and cytoskeleton, as well as extra cellular matrix genes, usually strongly up-regulated in keloids, resulted following selenocystine exposure. The range of the down-regulated genes and the degree of the decreased expression appeared to be correlated with the degree of the morphological alterations in selenocystine treated keloids.
Asunto(s)
Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Queloide/metabolismo , Selenocisteína/farmacología , Células Cultivadas , Humanos , Queloide/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PTPD1, a cytosolic non-receptor protein-tyrosine phosphatase, stimulates the Src-EGF transduction pathway. Localization of PTPD1 at actin cytoskeleton and adhesion sites is required for cell scattering and migration. Here, we show that during EGF stimulation, PTPD1 is rapidly recruited to endocytic vesicles containing the EGF receptor. Endosomal localization of PTPD1 is mediated by interaction with KIF16B, an endosomal kinesin that modulates receptor recycling at the plasma membrane. Silencing of PTPD1 promotes degradation of EGF receptor and inhibits downstream ERK signaling. We also found that PTPD1 is markedly increased in bladder cancer tissue samples. PTPD1 levels positively correlated with the grading and invasiveness potential of these tumors. Transgenic expression of an inactive PTPD1 mutant or genetic knockdown of the endogenous PTPD1 severely inhibited both growth and motility of human bladder cancer cells. These findings identify PTPD1 as a novel component of the endocytic machinery that impacts on EGF receptor stability and on growth and motility of bladder cancer cells.
Asunto(s)
Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Actinas/química , Biomarcadores de Tumor , Línea Celular Tumoral , Movimiento Celular , Citoesqueleto/metabolismo , Silenciador del Gen , Células HEK293 , Humanos , Cinesinas/química , Invasividad Neoplásica , Transducción de SeñalRESUMEN
PED (phosphoprotein enriched in diabetes) is a 15 kDa protein involved in many cellular pathways and human diseases including type II diabetes and cancer. We recently reported that PED is overexpressed in human cancers and mediates resistance to induced apoptosis. To better understand its role in cancer, we investigated on PED interactome in non-small cell lung cancer (NSCLC). By the Tandem Affinity Purification (TAP), we identified and characterized among others, Rac1, a member of mammalian Rho GTPase protein family, as PED-interacting protein. In this study we show that PED coadiuvates Rac1 activation by regulating AKT mediated Rac1-Ser(71) phosphorylation. Furthermore, we show that the expression of a constitutively active Rac, affected PED-Ser(104) phosphorylation, which is important for PED-regulated ERK 1/2 nuclear localization. Through specific Rac1-siRNA or its pharmacological inhibition, we demonstrate that PED augments migration and invasion in a Rac1-dependent manner in NSCLC. In conclusion, we show for the first time that PED and Rac1 interact and that this interaction modulates cell migration/invasion processes in cancer cells through ERK1/2 pathway.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas Reguladoras de la Apoptosis , Línea Celular , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Sistema de Señalización de MAP Quinasas/fisiología , Invasividad Neoplásica , Fosfoproteínas/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
Keloids are benign skin tumors and are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are characterized by formation of excess scar tissue beyond the boundaries of the wound. Keloids are often confused with hypertrophic scars because of an apparent lack of morphologic differences. The molecular distinction between scars and keloid is still controversial and, until today, there is no appropriate treatment yet for keloid disease. In this study, we have found, for the first time, p53 mutations in both hypertrophic scar and keloids fibroblasts from cultured cells to various extents. Since p53 plays a central role in the DNA damage response by inducing cell cycle arrest and/or apoptotic cell death, we also set up time course experiments making cell cultures at different times to investigate the phenomenon of apoptosis and its involvement in the process of pathological scarring in both hypertrophic scars and keloids. The extent of apoptosis in this study was investigated by DNA fragmentation and MTT assays, propidium iodide staining, p53 expression, and subcellular distribution. Moreover, the correlation of apoptosis and ROS levels in keloid and hypertrophic scars fibroblasts was assessed. Understanding the molecular mechanisms that determine the regulation of apoptosis during wound healing might allow us to therapeutically modulate these pathways so that apoptotic cell death is reactivated in dysregulated and hypertrophic cells.
Asunto(s)
Apoptosis , Cicatriz Hipertrófica/metabolismo , Fibroblastos/metabolismo , Queloide/metabolismo , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Humanos , Masculino , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Cicatrización de HeridasRESUMEN
Keloids are benign skin tumors that develop in individuals who have a positive family history of keloid disorders. Keloids are characterized by a deregulated woundhealing process, atypical fibroblasts with extreme deposition of extracellular matrix components, particularly collagen, increased cell proliferation and associated failure of apoptosis. Recently ingenolmebutate has been used as a novel agent with antiproliferative activity on human keloids as an alternative treatment option in patients, once conventional therapies have failed. We hypothesized that microRNAs (miR/miRNA) may be involved in the balance between lesion formation and repair. A comprehensive understanding of the molecular mechanism underlying the Ingenolmebutate response in keloid fibroblast following Ingenolmebutate exposure has been established previously. Therefore, the present study analyzed changes in miRNAs and apoptotic gene regulation in Ingenolmebutate treated keloid fibroblast, by reverse transcriptionquantitative polymerase chain reaction and a DNA fragmentation assay. The range of upregulated miRNAs and downregulated genes encoding cell death appeared to be associated with the degree of the morphological alterations in Ingenolmebutate treated keloids. In particular, the upregulation of miR34a was detected in keloid fibroblasts during and following Ingenolmebutate exposure. Keloid fibroblasts that overexpressed miR34a showed differential expression of genes involved in the apoptotic signaling pathway such as p53. In conclusion, the Ingenolmebutate treatment used here was effective in reducing keloid fibroblast growth in cell culture experiments and the expression of particular miRNAs modulated the proapoptotic gene expression following Ingenol-mebutate treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Diterpenos/farmacología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Queloide/tratamiento farmacológico , MicroARNs/genética , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Queloide/genética , Queloide/patologíaRESUMEN
The primary cilium emanates from the cell surface of growth-arrested cells and plays a central role in vertebrate development and tissue homeostasis. The mechanisms that control ciliogenesis have been extensively explored. However, the intersection between GPCR signaling and the ubiquitin pathway in the control of cilium stability are unknown. Here we observe that cAMP elevation promotes cilia resorption. At centriolar satellites, we identify a multimeric complex nucleated by PCM1 that includes two kinases, NEK10 and PKA, and the E3 ubiquitin ligase CHIP. We show that NEK10 is essential for ciliogenesis in mammals and for the development of medaka fish. PKA phosphorylation primes NEK10 for CHIP-mediated ubiquitination and proteolysis resulting in cilia resorption. Disarrangement of this control mechanism occurs in proliferative and genetic disorders. These findings unveil a pericentriolar kinase signalosome that efficiently links the cAMP cascade with the ubiquitin-proteasome system, thereby controlling essential aspects of ciliogenesis.
Asunto(s)
Cilios/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Animales , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Centriolos/metabolismo , Células HEK293 , Humanos , Hipogonadismo/genética , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Quinasas Relacionadas con NIMA/fisiología , Oryzias/embriología , Fosforilación , Proteolisis , Ataxias Espinocerebelosas/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45 S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45 S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45 S rDNA gene promoters.