Depósito Académico
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Results
- Toward the discovery of new biomarkers of hepatocellular carcinoma by proteomics(John Wiley and Sons, 2007) Corrales, F.J. (Fernando José); Fernandez-Irigoyen, J. (Joaquín); Prieto, J. (Jesús); Muñoz, J. (Javier); Santamaria, E. (Enrique)Primary liver cancer is the fifth most frequent neoplasm and the third most common cause of cancer-related death, with more than 500,000 new cases diagnosed yearly. The outcome for hepatocellular carcinoma (HCC) patients still remains dismal, partly because of our limited knowledge of its molecular pathogenesis and the difficulty in detecting the disease at its early stages. Therefore, studies aimed at the definition of the mechanisms associated with HCC progression and the identification of new biomarkers leading to early diagnosis and more effective therapeutic interventions are urgently needed. Proteomics is a rapidly expanding discipline that is expected to change the way in which diseases will be diagnosed, treated, and monitored in the near future. In the last few years, HCC has been extensively investigated using different proteomic approaches on HCC cell lines, animal models, and human tumor tissues. In this review, state-of-the-art technology on proteomics is overviewed, and recent advances in liver cancer proteomics and their clinical projections are discussed.
- A signature of six genes highlights defects on cell growth and specific metabolic pathways in murine and human hepatocellular carcinoma(Springer Verlag, 2011) Riezu-Boj, J.I. (José Ignacio); Corrales, F.J. (Fernando José); Schröder, P.C. (Paul C.); Sangro, B. (Bruno); Prieto, J. (Jesús); Segura, V. (Víctor); Mato, J.M. (José María); Santamaria, E. (Enrique)Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Clinical Proteomics.
- Proteomic analysis of human hepatoma cells expressing methionine adenosyltransferase I/III Characterization of DDX3X as a target of S-adenosylmethionine(Elsevier, 2012) Corrales, F.J. (Fernando José); Lu, S.C. (Shelly C.); Schröder, P.C. (Paul C.); Fernandez-Irigoyen, J. (Joaquín); Serna, A. (Antonio); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Mato, J.M. (José María); Bigaud, E. (Emilie)Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Clinical Proteomics.
- Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases(American Chemical Society, 2006) Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Berasain, C. (Carmen); Lu, S.C. (Shelly C.); Avila, M.A. (Matías Antonio); Fernandez-Irigoyen, J. (Joaquín); Prieto, J. (Jesús); Sesma, L. (Laura); Muñoz, J. (Javier); Mato, J.M. (José María); Santamaria, E. (Enrique)S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders.
- Identification of replication-competent HSV-1 Cgal+ strain signaling targets in human hepatoma cells by functional organelle proteomics(American Society for Biochemistry and Molecular Biology, 2009) Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Potel, C. (Corinne); Fernandez-Irigoyen, J. (Joaquín); Carro-Roldan, E. (Elvira); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Santamaria, E. (Enrique)In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.
- Regulation of stathmin phosphorylation in mouse liver progenitor-29 cells during proteasome inhibition(Wiley-VCH Verlag Berlin, 2009) Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Sanchez-Quiles, V. (Virginia); Fernandez-Irigoyen, J. (Joaquín); Prieto, J. (Jesús); Muñoz, J. (Javier); Santamaria, E. (Enrique)Proteasome inhibitors are potential therapeutic agents in the treatment of hepatocarcinoma and other liver diseases. The analysis of alternative protein phosphorylation states might contribute to elucidate the underlying mechanisms of proteasome inhibitor-induced apoptosis. We have investigated the response of mouse liver progenitor-29 (MLP-29) cells to MG132 using a combination of phosphoprotein affinity chromatography, DIGE, and nano LC-MS/MS. Thirteen unique deregulated phosphoproteins involved in chaperone activity, stress response, mRNA processing and cell cycle control were unambiguously identified. Alterations in NDRG1 and stathmin suggest new mechanisms associated to proteasome inhibitor-induced apoptosis in MLP-29 cells. Particularly, a transient modification of the phosphorylation state of Ser(16), Ser(25) and Ser(38), which are involved in the regulation of stathmin activity, was detected in three distinct isoforms upon proteasome inhibition. The parallel deregulation of calcium/calmodulin-activated protein kinase II, extracellular regulated kinase-1/2 and cyclin-dependent kinase-2, might explain the modified phosphorylation pattern of stathmin. Interestingly, stathmin phosphorylation profile was also modified in response to epoxomicin treatment, a more specific proteasome inhibitor. In summary, we report here data supporting that regulation of NDRG1 and stathmin by phosphorylation at specific Ser/Thr residues may participate in the cellular response induced by proteasome inhibitors.
- Identification of replication-competent HSV-1 Cgal+ strain targets in a mouse model of human hepatocarcinoma xenograft(Elsevier, 2009) Greco, A. (Anna); Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Manservigi, R. (Roberto); Fernandez-Irigoyen, J. (Joaquín); Carro-Roldan, E. (Elvira); Prieto, J. (Jesús); Molina, M. (Manuela); Marconi, P. (Peggy); Hernandez-Alcoceba, R. (Rubén); Santamaria, E. (Enrique)Recent studies based on animal models have shown the advantages and potential of oncolytic viral therapy using HSV-1 -based replication-competent vectors in the treatment of liver tumors, but little is known about the cellular targets that are modulated during viral infection. In the present work, we have studied the effects of intratumoral injections of HSV-1 Cgal(+) strain in a murine model of human hepatoma xenografts. Viral replication was assessed for more than 1month, leading to a significant reduction of tumor growth rate mediated, in part, by a cyclin B dependent cell proliferation arrest. Early events resulting in this effect were analyzed using a proteomic approach. Protein extracts from xenografted human hepatomas treated with saline or HSV-1 Cgal(+) strain during 24h were compared by 2-D DIGE and differential spots were identified by nanoLC-ESI-MS/MS. Alterations on glutathione S transferase 1 Omega, and ERp29 suggest novel HSV-1 Cgal(+) targets in solid liver tumors. Additionally, ERp29 showed a complex differential isoform pattern upon HSV-1 Cgal(+) infection, suggesting regulatory mechanisms based on post-translational modification events.
- Prohibitin deficiency blocks proliferation and induces apoptosis in human hepatoma cells: molecular mechanisms and functional implications(Wiley-VCH Verlag Berlin, 2010) Corrales, F.J. (Fernando José); Sanchez-Quiles, V. (Virginia); Prieto, J. (Jesús); Sesma, L. (Laura); Segura, V. (Víctor); Santamaria, E. (Enrique)Prohibitin is a multifunctional protein participating in a plethora of essential cellular functions, such as cell signaling, apoptosis, survival and proliferation. In the liver, deficient prohibitin activity participates in the progression of non-alcoholic steatohepatitis and obesity, according to mechanisms that still must be elucidated. In this study, we have used a combination of transcriptomics and proteomics technologies to investigate the response of human hepatoma PLC/PRF/5 cells to prohibitin silencing to define in detail the biological function of hepatic Phb1 and to elucidate potential prohibitin-dependent mechanisms participating in the maintenance of the transformed phenotype. Abrogation of prohibitin reduced proliferation and induced apoptosis in human hepatoma cells in a mechanism dependent on NF kappaB signaling. Moreover, down-regulation of ERp29 together with down-regulation of Erlin 2 suggests ER stress. In agreement, increased C/EBP homologous protein levels, poly-ADP ribose polymerase cleavage and activation of caspase 12 and downstream caspase 7 evidenced ER stress-induced apoptosis. Down-regulation of proteasome activator complex subunit 2 and stathmin as well as accumulation of ubiquitinated proteins suggest interplay between ER stress and proteasome malfunction. Taken together, our results provide evidences for prohibitin having a central role in the maintenance of the transformed and invasive phenotype of human hepatoma cells and may further support previous studies suggesting prohibitin as a potential clinical target.
- HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells(American Society for Biochemistry and Molecular Biology, 2011) Greco, A. (Anna); Mora, M.I. (María I.); Corrales, F.J. (Fernando José); Epstein, A.L. (Alberto L.); Dayon, L. (Loïc); Sanchez-Quiles, V. (Virginia); Foschini, M.G. (María Giovanna); Prieto, J. (Jesús); Sánchez, J.C. (Jean-Charles); Segura, V. (Víctor); Santamaria, E. (Enrique)Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.
- Different doses of adenoviral vector expressing IL-12 enhance or depress the immune response to a coadministered antigen: the role of nitric oxide(American Association of Immunologists, 1999) Corrales, F.J. (Fernando José); Borras-Cuesta, F. (Francisco); Lopez-Diaz-de-Cerio, A. (Ascensión); Qian, C. (Cheng); Casares, N. (Noelia); Prieto, J. (Jesús); Xie, X. (Xiaoming); Lasarte, J.J. (Juan José)Joint immunization with two recombinant adenoviruses, one expressing hepatitis C virus (HCV) core and E1 proteins and another expressing IL-12 (RAdIL-12), strongly potentiates cellular immune response against HCV Ags in BALB/c mice when RAdIL-12 was used at doses of 1 x 105-1 x 107 plaque-forming units. However, cellular immunity against HCV Ags was abolished when higher doses (1 x 108 plaque-forming units) of RAdIL-12 were used. This immunosuppressive effect was associated with marked elevation of IFN-gamma and nitric oxide in the serum and increased cell apoptosis in the spleen. Administration of N-nitro-L -arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, to mice that received high doses of RAdIL-12 was lethal, whereas no apparent systemic toxicity by L -NAME was observed in those immunized with lower doses of the adenovirus. Interestingly, in mice immunized with recombinant adenovirus expressing core and E1 proteins of HCV in combination with RAdIL-12 at low doses (1 x 107 plaque-forming units), L -NAME inhibited T cell proliferation and CTL activity in response to HCV Ags and also production of Abs against adenoviral proteins. In conclusion, gene transfer of IL-12 can increase or abolish cell immunity against an Ag depending of the dose of the vector expressing the cytokine. IL-12 stimulates the synthesis of NO which is needed for the immunostimulating effects of IL-12, but apoptosis of T cells and immunosuppression ensues when IFN-gamma and NO are generated at very high concentrations.