RESUMEN
BACKGROUND: Amyloid-ß precursor protein (APP) is a highly conserved single transmembrane protein that has been linked to Alzheimer disease. Recently, the increased expression of APP in multiple types of cancers has been reported where it has significant correlation with the cancer cell proliferation. However, the function of APP in the pathogenesis of breast cancer has not previously been determined. In this study, we studied the pathological role of APP in breast cancer and revealed its potential mechanism. METHODS: The expression level of APP in multiple breast cancer cell lines was measured by Western blot analysis and the breast cancer tissue microarray was utilized to analyze the expression pattern of APP in human patient specimens. To interrogate the functional role of APP in cell growth and apoptosis, the effect of APP knockdown in MDA-MB-231 cells were analyzed. Specifically, multiple signal transduction pathways and functional alterations linked to cell survival and motility were examined in in vivo animal model as well as in vitro cell culture with the manipulation of APP expression. RESULTS: We found that the expression of APP is increased in mouse and human breast cancer cell lines, especially in the cell line possessing higher metastatic potential. Moreover, the analysis of human breast cancer tissues revealed a significant correlation between the level of APP and tumor development. Knockdown of APP (APP-kd) in breast cancer cells caused the retardation of cell growth in vitro and in vivo, with both the induction of p27(kip1) and caspase-3-mediated apoptosis. APP-kd cells also had higher sensitivity to treatment of chemotherapeutic agents, TRAIL and 5-FU. Such anti-tumorigenic effects shown in the APP-kd cells partially came from reduced pro-survival AKT activation in response to IGF-1, leading to activation of key signaling regulators for cell growth, survival, and pro-apoptotic events such as GSK3-ß and FOXO1. Notably, knock-down of APP in metastatic breast cancer cells limited cell migration and invasion ability upon stimulation of IGF-1. CONCLUSION: The present data strongly suggest that the increase of APP expression is causally linked to tumorigenicity as well as invasion of aggressive breast cancer and, therefore, the targeting of APP may be an effective therapy for breast cancer.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular , Precursor de Proteína beta-Amiloide/genética , Animales , Apoptosis/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Estadificación de Neoplasias , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring innovative and effective targeted therapies. The oncogene astrocyte-elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates insulin-like growth factor-binding protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. EXPERIMENTAL DESIGN: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarray and real-time PCR and ELISA in human HCC cell lines. Dual FISH was done to detect LOH at IGFBP7 locus. Stable IGFBP7-overexpressing clones were established in the background of AEG-1-overexpressing human HCC cells and were analyzed for in vitro proliferation and senescence and in vivo tumorigenesis and angiogenesis. RESULTS: IGFBP7 expression is significantly downregulated in human HCC samples and cell lines compared with normal liver and hepatocytes, respectively, and inversely correlates with the stages and grades of HCC. Genomic deletion of IGFBP7 was identified in 26% of patients with HCC. Forced overexpression of IGFBP7 in AEG-1-overexpressing HCC cells inhibited in vitro growth and induced senescence, and profoundly suppressed in vivo growth in nude mice that might be an end result of inhibition of angiogenesis by IGFBP7. CONCLUSION: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Accordingly, targeted overexpression of IGFBP7 might be a potential novel therapy for HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Genes Supresores de Tumor , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Neoplasias Hepáticas/genética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Pérdida de Heterocigocidad , Proteínas de la Membrana , Ratones , Ratones Desnudos , Estadificación de Neoplasias , Neovascularización Fisiológica/genética , Proteínas de Unión al ARN , Trasplante HeterólogoRESUMEN
Aggressive tumor growth, diffuse tissue invasion, and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogene that is overexpressed in several types of human cancers, including more than 90% of brain tumors. In addition, AEG-1 promotes gliomagenesis, particularly in the context of tumor growth and invasion, 2 primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from glioma patients indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.
Asunto(s)
Neoplasias Encefálicas/patología , Moléculas de Adhesión Celular/metabolismo , Glioma/patología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ácido Glutámico/metabolismo , Degeneración Nerviosa/patología , Oncogenes , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Proteína de Unión a CREB/metabolismo , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Transportador 2 de Aminoácidos Excitadores , Glioma/metabolismo , Humanos , Proteínas de la Membrana , Degeneración Nerviosa/metabolismo , Regiones Promotoras Genéticas , Proteínas de Unión al ARN , Ratas , Factor de Transcripción YY1/metabolismoRESUMEN
BACKGROUND & AIMS: Understanding the molecular pathogenesis of hepatocellular carcinoma (HCC) would facilitate development of targeted and effective therapies for this fatal disease. We recently demonstrated that the cellular transcription factor Late SV40 Factor (LSF) is overexpressed in more than 90% of human HCC cases, compared to the normal liver, and plays a seminal role in hepatocarcinogenesis. LSF transcriptionally upregulates osteopontin (OPN) that plays a significant role in mediating the oncogenic function of LSF. The present study aims at a better understanding of LSF function by analyzing the signaling pathway modulated by LSF. METHODS: Phospho-receptor tyrosine kinase (RTK) array was performed to identify which receptor tyrosine kinases are activated by LSF. Immunohistochemical analysis using tissue microarray was performed to establish correlation among LSF, OPN, and phospho-c-Met levels in HCC patients. Co-immunoprecipitation analysis was performed to check OPN-induced CD44 and c-Met interaction. Inhibition studies using chemicals and siRNAs were performed in vitro and in vivo using nude mice xenograft models to establish the importance of c-Met activation in mediating LSF function. RESULTS: Secreted OPN, induced by LSF, activates c-Met via a potential interaction between OPN and its cell surface receptor CD44. A significant correlation was observed among LSF, OPN, and activated c-Met levels in HCC patients. Chemical or genetic inhibition of c-Met resulted in profound abrogation of LSF-mediated tumorigenesis and metastasis in nude mice xenograft studies. CONCLUSIONS: The present findings elucidate a novel pathway of c-Met activation during hepatocarcinogenesis and support the rationale of using c-Met inhibitors as potential HCC therapeutics.
Asunto(s)
Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Osteopontina/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Ratones Desnudos , Modelos Biológicos , Trasplante de Neoplasias , Transducción de Señal , Trasplante HeterólogoRESUMEN
UNLABELLED: There is virtually no effective treatment for advanced hepatocellular carcinoma (HCC) and novel targets need to be identified to develop effective treatment. We recently documented that the oncogene Astrocyte elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis. Employing yeast two-hybrid assay and coimmunoprecipitation followed by mass spectrometry, we identified staphylococcal nuclease domain containing 1 (SND1), a nuclease in the RNA-induced silencing complex (RISC) facilitating RNAi-mediated gene silencing, as an AEG-1 interacting protein. Coimmunoprecipitation and colocalization studies confirmed that AEG-1 is also a component of RISC and both AEG-1 and SND1 are required for optimum RISC activity facilitating small interfering RNA (siRNA) and micro RNA (miRNA)-mediated silencing of luciferase reporter gene. In 109 human HCC samples SND1 was overexpressed in ≈74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to immortal normal hepatocytes. Increased RISC activity, conferred by AEG-1 or SND1, resulted in increased degradation of tumor suppressor messenger RNAs (mRNAs) that are target of oncomiRs. Inhibition of enzymatic activity of SND1 significantly inhibited proliferation of human HCC cells. As a corollary, stable overexpression of SND1 augmented and siRNA-mediated inhibition of SND1 abrogated growth of human HCC cells in vitro and in vivo, thus revealing a potential role of SND1 in hepatocarcinogenesis. CONCLUSION: We unravel a novel mechanism that overexpression of AEG-1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC.
Asunto(s)
Carcinoma Hepatocelular/etiología , Moléculas de Adhesión Celular/fisiología , Neoplasias Hepáticas/etiología , Proteínas Nucleares/fisiología , Complejo Silenciador Inducido por ARN/fisiología , Animales , Endonucleasas , Humanos , Proteínas de la Membrana , Ratones , Proteínas de Unión al ARN , Células Tumorales CultivadasRESUMEN
Since its initial identification and cloning in 2002, Astrocyte Elevated Gene-1 (AEG-1), also known as metadherin (MTDH), 3D3 and LYsine-RIch CEACAM1 co-isolated (LYRIC), has emerged as an important oncogene that is overexpressed in all cancers analyzed so far. Examination of a large cohort of patient samples representing diverse cancer indications has revealed progressive increase in AEG-1 expression with stages and grades of the disease and an inverse relationship between AEG-1 expression level and patient prognosis. AEG-1 functions as a bona fide oncogene by promoting transformation. In addition, it plays a significant role in invasion, metastasis, angiogenesis and chemoresistance, all important hallmarks of an aggressive cancer. AEG-1 is also implicated in diverse physiological and pathological processes, such as development, inflammation, neurodegeneration, migraine and Huntington's disease. AEG-1 is a highly basic protein with a transmembrane domain and multiple nuclear localization signals and it is present in the cell membrane, cytoplasm, nucleus, nucleolus and endoplasmic reticulum. In each location, AEG-1 interacts with specific proteins thereby modulating diverse intracellular processes the combination of which contributes to its pleiotrophic properties. The present review provides a snapshot of the current literature along with future perspectives on this unique molecule.
Asunto(s)
Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Neoplasias/genética , Oncogenes , Animales , Biomarcadores de Tumor , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana , Terapia Molecular Dirigida , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas de Unión al ARN , Transducción de Señal/fisiologíaRESUMEN
Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Animales , Astrocitos/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Transformación Celular Neoplásica , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Folículo Piloso/embriología , Folículo Piloso/metabolismo , Antígeno Ki-67/análisis , Antígeno Ki-67/genética , Hígado/embriología , Hígado/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas de Unión al ARN , Transducción de Señal , Piel/embriología , Piel/metabolismo , Médula Espinal/embriología , Médula Espinal/metabolismoRESUMEN
Our recent findings show that astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) samples, and AEG-1 plays a central role in regulating development and progression of HCC. In the present study, we elucidate a molecular mechanism of AEG-1-induced chemoresistance, an important characteristic of aggressive cancers. AEG-1 increases the expression of multidrug resistance gene 1 (MDR1) protein, resulting in increased efflux and decreased accumulation of doxorubicin, promoting doxorubicin resistance. Suppression of MDR1 by small interfering RNA or chemical reagents, or inhibition of AEG-1 or a combination of both genes, significantly increases in vitro sensitivity to doxorubicin. In nude mice xenograft studies, a lentivirus expressing AEG-1 short hairpin RNA, in combination with doxorubicin, profoundly inhibited growth of aggressive human HCC cells compared with either agent alone. We document that although AEG-1 does not affect MDR1 gene transcription, it facilitates association of MDR1 mRNA to polysomes, resulting in increased translation, and AEG-1 also inhibits ubiquitination and subsequent proteasome-mediated degradation of MDR1 protein. This study is the first documentation of a unique aspect of AEG-1 function (i.e., translational and posttranslational regulation of proteins). Inhibition of AEG-1 might provide a means of more effectively using chemotherapy to treat HCC, which displays inherent chemoresistance with aggressive pathology.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/enzimología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Lentivirus/genética , Proteínas de la Membrana , Ratones , Ratones Desnudos , Trasplante de Neoplasias , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARNRESUMEN
Hepatocellular carcinoma (HCC) is a highly aggressive cancer with no currently available effective treatment. Understanding of the molecular mechanism of HCC development and progression is imperative for developing novel, effective, and targeted therapies for this lethal disease. In this article, we document that the cellular transcription factor Late SV40 Factor (LSF) plays an important role in HCC pathogenesis. LSF protein was significantly overexpressed in human HCC cells compared to normal hepatocytes. In 109 HCC patients, LSF protein was overexpressed in >90% cases, compared to normal liver, and LSF expression level showed significant correlation with the stages and grades of the disease. Forced overexpression of LSF in less aggressive HCC cells resulted in highly aggressive, angiogenic, and multiorgan metastatic tumors in nude mice. Conversely, inhibition of LSF significantly abrogated growth and metastasis of highly aggressive HCC cells in nude mice. Microarray studies revealed that as a transcription factor, LSF modulated specific genes regulating invasion, angiogenesis, chemoresistance, and senescence. The expression of osteopontin (OPN), a gene regulating every step in tumor progression and metastasis, was robustly up-regulated by LSF. It was documented that LSF transcriptionally up-regulates OPN, and loss-of-function studies demonstrated that OPN plays an important role in mediating the oncogenic functions of LSF. Together, these data establish a regulatory role of LSF in cancer, particularly HCC pathogenesis, and validate LSF as a viable target for therapeutic intervention.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/metabolismo , Oncogenes , Factores de Transcripción/metabolismo , Animales , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proliferación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Osteopontina/genética , Osteopontina/metabolismo , Interferencia de ARN , Ratas , Análisis de Matrices Tisulares , Factores de Transcripción/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
Malignant gliomas including glioblastoma multiforme (GBM) and anaplastic astrocytomas are the most common primary brain tumors. Despite multimodal treatment including surgery, chemotherapy, and radiation, median survival for patients with GBMs is only 12 to 15 months. Identifying molecules critical for glioma progression is crucial for devising effective targeted therapy. In the present study, we investigated the potential contribution of astrocyte elevated gene-1 (AEG-1) in gliomagenesis and explored the possibility of AEG-1 as a therapeutic target for malignant glioma. We analyzed the expression levels of AEG-1 in 9 normal brain tissues and 98 brain tumor patient samples by Western blot analysis and immunohistochemistry. AEG-1 expression was significantly elevated in >90% of diverse human brain tumor samples including GBMs and astrocytic tumors, and also in human glioma cell lines compared with normal brain tissues and normal astrocytes. Knockdown of AEG-1 by small interfering RNA inhibited cell viability, cloning efficiency, and invasive ability of U87 human glioma cells and 9L rat gliosarcoma cells. We also found that matrix metalloproteases (MMP-2 and MMP-9) are involved in AEG-1-mediated invasion of glioma cells. In an orthotopic nude mouse brain tumor model using primary human GBM12 tumor cells, AEG-1 small interfering RNA significantly suppressed glioma cell growth in vivo. Taken together, these provocative results indicate that AEG-1 may play a crucial role in the pathogenesis of glioma and that AEG-1 could represent a viable potential target for malignant glioma therapy.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Glioma/metabolismo , Glioma/terapia , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Glioblastoma/enzimología , Glioblastoma/patología , Glioma/enzimología , Glioma/patología , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana , Ratones , Invasividad Neoplásica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Since its original cloning by subtraction hybridization in 2002, it is now evident that Astrocyte elevated gene-1 (AEG-1) is a key contributor to the carcinogenic process in diverse organs. AEG-1 protein expression is elevated in advanced stages of many cancers, which correlates with poor survival. In specific cancers, such as breast and liver cancer, the AEG-1 gene itself is amplified, further supporting a seminal role in tumorigenesis. Overexpression and inhibition studies both in in vitro and in in vivo models reveal the importance of AEG-1 in regulating multiple physiologically and pathologically relevant processes including proliferation, invasion, metastasis, and gene expression. AEG-1 is a single-pass transmembrane protein with multiple nuclear localization signals and no known domains or motifs. Although pertinent roles of AEG-1 in the carcinogenic process are established, its potential function (promotion of metastasis only versus functioning as a bona fide oncogene) as well as localization (cell surface versus nucleus) remain areas requiring further clarification. The present review critically evaluates what is currently known about AEG-1 and provides new perspectives relative to this intriguing molecule that may provide a rational target for intervening in the cancer phenotype.
Asunto(s)
Astrocitos/fisiología , Moléculas de Adhesión Celular/fisiología , Neoplasias/genética , Animales , Humanos , Proteínas de la Membrana , Proteínas de Unión al ARNRESUMEN
Astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) patients and plays a significant role in mediating aggressive progression of HCC. AEG-1 is known to augment invasion, metastasis, and angiogenesis, and we now demonstrate that AEG-1 directly contributes to another important hallmark of aggressive cancers, that is, resistance to chemotherapeutic drugs, such as 5-fluorouracil (5-FU). AEG-1 augments expression of the transcription factor LSF that regulates the expression of thymidylate synthase (TS), a target of 5-FU. In addition, AEG-1 enhances the expression of dihydropyrimidine dehydrogenase (DPYD) that catalyzes the initial and rate-limiting step in the catabolism of 5-FU. siRNA-mediated inhibition of AEG-1, LSF, or DPYD significantly increased the sensitivity of HCC cells to 5-FU in vitro and a lentivirus delivering AEG-1 siRNA in combination with 5-FU markedly inhibited growth of HCC cells xenotransplanted in athymic nude mice when compared to either agent alone. The present studies highlight 2 previously unidentified genes, AEG-1 and LSF, contributing to chemoresistance. Inhibition of AEG-1 might be exploited as a therapeutic strategy along with 5-FU-based combinatorial chemotherapy for HCC, a highly fatal cancer with currently very limited therapeutic options.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Moléculas de Adhesión Celular/genética , Proteínas de Unión al ADN/genética , Fluorouracilo/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Factores de Transcripción/genética , Animales , Carcinoma Hepatocelular/genética , Moléculas de Adhesión Celular/antagonistas & inhibidores , Línea Celular Tumoral , ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Dihidrouracilo Deshidrogenasa (NADP)/fisiología , Resistencia a Antineoplásicos/genética , Humanos , Antígeno Ki-67/análisis , Neoplasias Hepáticas/genética , Proteínas de la Membrana , Ratones , Proteínas de Unión al ARN , Timidilato Sintasa/genética , Factores de Transcripción/fisiologíaRESUMEN
Hepatocellular carcinoma (HCC) is a highly aggressive vascular cancer characterized by diverse etiology, activation of multiple signal transduction pathways, and various gene mutations. Here, we have determined a specific role for astrocyte elevated gene-1 (AEG1) in HCC pathogenesis. Expression of AEG1 was extremely low in human hepatocytes, but its levels were significantly increased in human HCC. Stable overexpression of AEG1 converted nontumorigenic human HCC cells into highly aggressive vascular tumors, and inhibition of AEG1 abrogated tumorigenesis by aggressive HCC cells in a xenograft model of nude mice. In human HCC, AEG1 overexpression was associated with elevated copy numbers. Microarray analysis revealed that AEG1 modulated the expression of genes associated with invasion, metastasis, chemoresistance, angiogenesis, and senescence. AEG1 also was found to activate Wnt/beta-catenin signaling via ERK42/44 activation and upregulated lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), the ultimate executor of the Wnt pathway, important for HCC progression. Inhibition studies further demonstrated that activation of Wnt signaling played a key role in mediating AEG1 function. AEG1 also activated the NF-kappaB pathway, which may play a role in the chronic inflammatory changes preceding HCC development. These data indicate that AEG1 plays a central role in regulating diverse aspects of HCC pathogenesis. Targeted inhibition of AEG1 might lead to the shutdown of key elemental characteristics of HCC and could lead to an effective therapeutic strategy for HCC.
Asunto(s)
Carcinoma Hepatocelular/patología , Moléculas de Adhesión Celular/genética , Neoplasias Hepáticas/patología , Animales , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Hepatocitos/fisiología , Humanos , Inflamación/genética , Neoplasias Hepáticas/genética , Factor de Unión 1 al Potenciador Linfoide/genética , Proteínas de la Membrana , Ratones , Ratones Desnudos , Estadificación de Neoplasias , ARN Mensajero/genética , ARN Neoplásico/genética , Proteínas de Unión al ARN , Valores de Referencia , Trasplante HeterólogoRESUMEN
We have shown that immunostimulated astrocytes produce excess nitric oxide (NO) and eventually peroxynitrite (ONOO(-)) that was closely associated with the glucose deprivation-potentiated death of astrocytes. The present study shows that activated p38 MAPK regulates ONOO(-) generation from lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-stimulated astrocytes. LPS+IFN-gamma-induced p38 MAPK activation and ONOO(-) generation were attenuated by SB203580 or SKF-86002, specific inhibitors of p38 MAPK. ONOO(-) generation was blocked by NADPH oxidase inhibitor, diphenyleneiodonium chloride, and nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester, suggesting both enzymes are involved in ONOO(-) generation. Inhibition of p38 MAPK suppressed LPS+IFN-gamma-induced NO production through down-regulating inducible form of NOS expression. It also suppressed LPS+IFN-gamma-induced NADPH oxidase activation and eventually, the inducible form of superoxide production. Transfection with dominant negative vector of p38 alpha reduced LPS+IFN-gamma-induced ONOO(-) generation through blocking both iNOS-derived NO production and NADPH oxidase-derived O2(-) production. Our results suggest that activated p38 MAPK may serve as a potential signaling molecule in ONOO(-) generation through dual regulatory mechanisms, involving iNOS induction and NADPH oxidase activation.
Asunto(s)
Astrocitos/enzimología , Encefalitis/enzimología , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ácido Peroxinitroso/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/inmunología , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Encefalitis/inmunología , Encefalitis/fisiopatología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Interferón gamma/inmunología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Óxido Nítrico/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
In spite of their pathophysiological importance in neuro-inflammatory diseases, little is known about the signal transduction pathways that lead to the induction of matrix metalloproteinases (MMPs) in the central nervous system. We reported previously that lipopolysaccharide (LPS) induced MMP-9 expression through ERK1/2 pathway in rat primary astrocytes (Glia 41:15-24, 2003). Here, we investigated the role of other MAPK pathways, including p38 and JNK/SAPK, on the regulation of MMP-9 expression in LPS-stimulated rat primary astrocytes. LPS activated both p38 and JNK in astrocytes. Treatment with a specific p38 MAPK inhibitor SB203580, but not JNK inhibitor SP600125, increased the LPS-stimulated MMP-9 expression in a concentration-dependent manner. Anti-inflammatory cytokines, including IFN-gamma and IL-4, activated p38 MAPK and decreased MMP-9 production in LPS-stimulated astrocytes. When p38 MAPK activation was blocked by SB203580, the inhibitory effects of these cytokines on MMP-9 induction were abolished. Finally, direct injection of SB203580 into the lateral ventricle of rat brain increased the LPS-induced MMP-9 activity in cerebral cortex. Altogether, these results suggest that p38 activation down-regulates the inflammatory stimulation-induced overexpression of MMP-9, both in primary astrocytes and in cerebral cortex. The elaborate interplay between ERK1/2 and p38 pathways provides a more sophisticated mechanism for regulating MMP-9 activity in neuroinflammatory diseases.
Asunto(s)
Astrocitos/enzimología , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 9 de la Matriz/genética , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Regulación hacia Abajo , Imidazoles/farmacología , Interferón gamma/farmacología , Interleucina-4/farmacología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Piridinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
We previously reported that in immunostimulated astrocytes, glucose deprivation induced cell death via the loss of ATP, reduced glutathione, and mitochondrial transmembrane potential. The cytotoxicity was due to reactive nitrogen and oxygen species and blocked by adenosine, a purine nucleoside, via the preservation of cellular ATP. Here, we investigated whether uridine, a pyrimidine nucleoside, could prevent the glucose deprivation-induced cytotoxicity in LPS+IFN-gamma-treated (immunostimulated) astrocytes. Glucose deprivation induced the death of immunostimulated cells, which was significantly reduced by uridine. Glucose deprivation rapidly decreased cellular ATP levels in immunostimulated astrocytes, which was also reversed by uridine. The inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine attenuated the protective effect of uridine. mRNA and protein expression for uridine phosphorylase, an enzyme catalyzing reversible phosphorolysis of uridine, were observed in rat brain as well as primary astrocytes. 5-(Phenylthio)acyclouridine (PTAU), a specific inhibitor of uridine phosphorylase, inhibited the protective effect of uridine. Additionally, the loss of mitochondrial transmembrane potential and reduced glutathione by glucose deprivation in immunostimulated cells was attenuated by uridine, which was also reversed by PTAU. These results provide the first evidence that uridine protects immunostimulated astrocytes against the glucose deprivation-induced death by preserving intracellular ATP through the action of uridine phosphorylase.
Asunto(s)
Astrocitos/fisiología , Muerte Celular/fisiología , Glucosa/metabolismo , Inmunización , Uridina Fosforilasa/metabolismo , Uridina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Células Cultivadas , Glutatión/metabolismo , Humanos , Interferón gamma/inmunología , Interferón gamma/farmacología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Potenciales de la Membrana , Fármacos Neuroprotectores/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Recently we have reported that glucose deprivation induces the potentiated death and loss of ATP in immunostimulated astroglia via the production of NO and eventually peroxynitrite. This study examined the role of the ERK1/2 signaling pathways in the glucose deprivation-induced death of immunostimulated astroglia. Immunostimulation with LPS+IFN-gamma induced the sustained activation of ERK1/2 for up to 48 h. Glucose deprivation caused the loss of ATP and consequently cell death in immunostimulated astroglia, which was significantly blocked by the treatment with the ERK kinase (MEK1) inhibitor, PD98059 (10-40 microM), to inhibit the ERK1/2 pathways. The systems for generating NO (iNOS) or superoxide (NADPH oxidase) were regulated by the ERK1/2 signaling pathways because the addition of PD98059 reduced the level of both. Interestingly, glucose deprivation caused an approximately two-fold increase in the level of peroxynitrite formation in immunostimulated astroglia, which was significantly reduced by the PD98059 treatment. This demonstrates that the ERK1/2 signaling pathways play an important role in glucose deprivation-induced death in immunostimulated astroglia by regulating the generation of NO, superoxide and their reaction product, peroxynitrite.
Asunto(s)
Astrocitos/enzimología , Isquemia Encefálica/metabolismo , Gliosis/metabolismo , Glucosa/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Animales , Astrocitos/efectos de los fármacos , Isquemia Encefálica/enzimología , Isquemia Encefálica/inmunología , Células Cultivadas , Técnicas de Cocultivo , Inhibidores Enzimáticos/farmacología , Gliosis/enzimología , Gliosis/inmunología , Glucosa/deficiencia , Inmunización , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , NADPH Oxidasas/biosíntesis , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Estrés Oxidativo/inmunología , Ácido Peroxinitroso/metabolismo , Ratas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Previously, we have shown that hydrogen peroxide (H2O2) and glucose deprivation (GD) induced ATP loss and cell death in astrocytes. Here, we reported that adenosine and related purine nucleos(t)ides recovered cellular ATP level and completely prevented the cell death in rat primary astrocytes co-treated with H2O2 and glucose deprivation. Time- and concentration-dependently, H2O2 induced cell death and ATP loss in glucose-deprived astrocytes. Adenosine or ATP prevented both astrocytic death and ATP loss caused by H2O2/GD in dose-dependent manner. Further, inhibition of adenosine deamination or transport with erythro-9-(-hydroxy-3-nonyl)adenosine or S-(4-nitrobenzyl)-6-thioinosine largely attenuated the protective effect of adenosine. Other purine nucleos(t)ides such as inosine, guanosine, ADP, AMP, ITP and GTP also showed similar protective effects. Adenosine or ATP also blocked the mitochondrial dysfunction and glutathione (GSH) depletion in H2O2-treated glucose-deprived astrocytes. The present results suggest that adenosine and related purine nucleos(t)ides may protect astrocytes from H2O2 and glucose deprivation induced the potentiated death by restoration of cellular ATP level.
Asunto(s)
Adenina/análogos & derivados , Adenosina/farmacología , Astrocitos/efectos de los fármacos , Glucosa/deficiencia , Peróxido de Hidrógeno/toxicidad , Purinas/farmacología , Teobromina/análogos & derivados , Tioinosina/análogos & derivados , Adenina/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/metabolismo , Bencimidazoles/metabolismo , Carbocianinas/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Técnicas In Vitro , L-Lactato Deshidrogenasa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Ratas , Ratas Sprague-Dawley , Teobromina/farmacología , Tioinosina/farmacología , Factores de Tiempo , Xantinas/farmacologíaRESUMEN
Activated astrocytes produce a large amount of bioactive molecules, including reactive oxygen and nitrogen species. Astrocytes are in general resistant to those reactive species. However, we previously reported that immunostimulated astrocytes became highly vulnerable to metabolic insults, such as glucose deprivation. In this study, we investigated whether H(2)O(2) production was associated with the increased vulnerability. Glucose deprivation for up to 8 hr did not change the intracellular level of H(2)O(2) in astrocytes. Treatment with lipopolysaccharide plus interferon-gamma for 48 hr evoked astroglial H(2)O(2) production; however, no apparent death or injury was observed in immunostimulated astrocytes. Glucose deprivation after 48 hr of immunostimulation markedly increased H(2)O(2) level, depleted adenosine triphosphate (ATP), and enhanced lactate dehydrogenase (LDH) release. The ATP depletion and LDH release were in part prevented by catalase, mannitol, and N-acetyl-L-cysteine. The enhanced level of H(2)O(2) in glucose-deprived immunostimulated astrocytes appeared to be secondary to the depletion of reduced glutathione. 4-(2-Aminoethyl)bebzenesulfonyl fluoride (AEBSF), an inhibitor of NADPH oxidase, reduced H(2)O(2) level and LDH release in glucose-deprived immunostimulated astrocytes. H(2)O(2), either endogenously produced or exogenously added, depolarized mitochondrial transmembrane potential in glucose-deprived astrocytes, leading to their ATP depletion and death. The present results strongly indicate that glucose deprivation causes deterioration of immunostimulated astrocytes by increasing the intracellular concentration of H(2)O(2).
Asunto(s)
Astrocitos/inmunología , Astrocitos/metabolismo , Muerte Celular/fisiología , Glucosa/deficiencia , Peróxido de Hidrógeno/metabolismo , Líquido Intracelular/metabolismo , Oxidantes/metabolismo , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Animales , Astrocitos/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Glucosa/metabolismo , Inmunización , Líquido Intracelular/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Lipopolisacáridos/farmacología , Corteza Prefrontal/citología , Corteza Prefrontal/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
In the present study, we investigated whether the activation of protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (Erk1/2) are involved in the induction of MMP-9 in lipopolysaccharide (LPS)-stimulated primary astrocytes. The expression of MMP-9 but not MMP-2 was increased by LPS. LPS treatment induced activation of Erk1/2 within 30 min, which was dose-dependently inhibited by PD98059, a specific inhibitor of the Erk kinase (MEK). In this condition, PD98059 blocked the increase in MMP-9 protein and mRNA level as well as gelatin-digesting activity. Inhibition of PKC activity blocked the LPS-induced activation of Erk1/2 as well as MMP-9 expression. In addition, activation of PKC by phorbol myristoyl acetate (PMA) activated Erk1/2 with concomitant increase in MMP-9 production. Moreover, treatment of PD98059 dose-dependently decreased the PMA-induced MMP-9 expression. The results from the present study suggest that induction of MMP-9 by LPS in rat primary astrocytes is mediated, at least in part, by the sequential activation of PKC and Erk1/2. The Erk1/2-mediated MMP-9 induction may provide insights into the regulation of MMP-9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation.