Estudio de la regulación y del papel oncogénico de lisis oxidasa (LOX) en los tumores de Ewing

Tesis doctoral inédita realizada en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 19 Julio de 2010.Bibliografía, p. 111-12

Lysyl oxidase is downregulated by the EWS/FLI1 oncoprotein and its propeptide domain displays tumor supressor activities in Ewing sarcoma cells

This study was funded by grants of the Ministerio de Ciencia e Innovación (SAF2009-10158), the Fundación Científica de la Científica Española Contra el Cáncer and the Fundación María Francisca de Roviralta. N. Agra was supported by the Fundación General de la U.A.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica

Regulation of 15-hydroxyprostaglandin dehydrogenase expression in hepatocellular carcinoma

11 páginas, 6 figuras.Cyclooxygenase-2 (COX-2), a rate limiting step in arachidonic acid cascade, plays a key role in the biosynthesis of prostaglandin E2 (PGE2) upon inflammatory stimuli, growth factors, hormones and other cellular stresses. Overproduction of PGE2 stimulates proliferation of various cancer cells, confers resistance to apoptosis and favors metastasis and angiogenesis. The steady-state level of PGE2 is maintained by interplay between the biosynthetic pathway including COX and PGE2 synthases and the catabolic pathways involving nicotinamide adenine dinucleotide (NAD(+))-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 15-PGDH is a crucial enzyme responsible for the biological inactivation of PGE2. Adult hepatocytes fail to induce COX-2 expression regardless of the pro-inflammatory factors used. COX-2 is induced in hepatocytes after partial hepatectomy (PH), in animal models of cirrhosis, in human hepatoma cell lines, in human HCC and after HBV and HCV infection. However, no data are available regarding 15-PGDH expression in HCC. Our results show that 15-PGDH is downregulated in human hepatoma cells with a high COX-2 expression, in chemical and genetic murine models of HCC and in human HCC biopsies. Moreover, 15-PGDH expression is suppressed by EGF (epidermal growth factor) and HGF (hepatocyte growth factor) mainly involving PI3K (phosphatidylinositol-3-kinase), ERK (extracellular signal-regulated kinase) and p38MAPK (mitogen-activated protein kinase) activation. Conversely, ectopic expression of 15-PGDH induces apoptosis in hepatoma cells and decreases the growth of hepatoma cells in nude mice whereas the silencing of 15-PGDH increases the tumor formation. These data suggest a potential therapeutic application of 15-PGDH in HCCL C-S is supported by a Post-Doctoral fellowship from CONACYT, Mexico. NA is supported by Red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares RECAVA. Instituto de Salud Carlos III (RD12/0042/0019). This work was supported by Ministry of Science and Innovation (SAF2010-16037, SAF2012-39732 and BFU2011-24760) and by Comunidad de Madrid (P2010/BMD-2378).Peer reviewe

Devazepide, a nonpeptide antagonist of CCK receptors, induces apoptosis and inhibits Ewing tumor growth

The Ewing family of tumors is a group of highly malignant tumors that mainly arise in bone and most often affect children and young adults in the first two decades of life. Despite the use of multimodal therapy, the long-term disease-free survival rate of patients with Ewing tumors is still disappointingly low, making the discovery of innovative therapeutic strategies all the more necessary. We have recently shown that cholecystokinin (CCK), a neuroendocrine peptide, involved in many biological functions, including cell growth and proliferation, is a relevant target of the EWS/FLI1 oncoprotein characteristic of Ewing tumors. CCK silencing inhibits cell proliferation and tumor growth in vivo, suggesting that CCK acts as an autocrine growth factor for Ewing cells. Here, we analyzed the impact of two CCK receptor antagonists, devazepide (a CCK1-R antagonist) and L365-260 (a CCK2-R antagonist), on the growth of Ewing tumor cells. Devazepide (10μmol/l) inhibited cell growth of four different Ewing tumor cells in vitro (range 85-88%), whereas the effect of the CCK2-R antagonist on cell growth was negligible. In a mouse tumor xenograft model, devazepide reduced tumor growth by 40%. Flow cytometry experiments showed that devazepide, but not L365-260, induced apoptosis of Ewing tumor cells. In summary, devazepide induces cell death of Ewing tumor cells, suggesting that it could represent a new therapeutic approach in the management of Ewing's tumor patients. © 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.Peer Reviewe

Evaluation of epigenetic modulation of cyclooxygenase-2 as a prognostic marker for hepatocellular carcinoma

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivative Works 3.0 Unported License.Cyclooxygenases (COX-1 and 2) catalyze the first step in prostanoid biosynthesis. They are implicated in homeostatic processes with an important role in inflammation and carcinogenesis. In the liver, COX-2 expression is restricted to proliferation or dedifferentiation situations. The COX-2 promoter contains numerous CpG islands that, when hypermethylated, result in transcriptionally silencing thus regulating the growth of carcinoma cells. In this work, we investigated whether a correlation exists between COX-2 expression and methylation signatures at the 5′region of the gene in hepatoma cell lines and human hepatocellular carcinoma (HCC). We also examined the acetylation status of the COX-2 promoter and the effects of histone deacetylase (HDAC) inhibitors on COX-2 expression. Our results suggest a significant association between reduced COX-2 expression and promoter hypermethylation of COX-2 and histone deacetylation in some hepatoma cell lines and in HCC. Treatment with demethylating agents or HDAC inhibitors restored the expression of COX-2. Moreover, in an HCC cohort, a statistically significant inverse association was observed between COX-2 mRNA levels and promoter methylation. In agreement with these data, a reduction of overall survival of the patients was observed after decreased COX-2 expression by promoter hypermethylation and histone H3 hypoacetylation.This work was supported by the Ministry of Science and Innovation (SAF2010-16037, SAF2009-12602 and BFU2011-24760) and by Comunidad de Madrid (P2010/BMD-2378). CIBERehd is funded by the Instituto de Salud Carlos III. AF-A and NA were supported by the Carlos III Health Institute (Red de Centros FIS-RECAVA RD06/0014/0025).Peer Reviewe

Cyclooxygenase-2 Is a target of microRNA-16 in human hepatoma cells

This is an open-access article distributed under the terms of the Creative Commons Attribution License.Cyclooxygenase-2 (COX-2) expression has been detected in human hepatoma cell lines and in human hepatocellular carcinoma (HCC); however, the contribution of COX-2 to the development of HCC remains controversial. COX-2 expression is higher in the non-tumoral tissue and inversely correlates with the differentiation grade of the tumor. COX-2 expression depends on the interplay between different cellular pathways involving both transcriptional and post-transcriptional regulation. The aim of this work was to assess whether COX-2 could be regulated by microRNAs in human hepatoma cell lines and in human HCC specimens since these molecules contribute to the regulation of genes implicated in cell growth and differentiation. Our results show that miR-16 silences COX-2 expression in hepatoma cells by two mechanisms: a) by binding directly to the microRNA response element (MRE) in the COX-2 39-UTR promoting translational suppression of COX-2 mRNA; b) by decreasing the levels of the RNA-binding protein Human Antigen R (HuR). Furthermore, ectopic expression of miR-16 inhibits cell proliferation, promotes cell apoptosis and suppresses the ability of hepatoma cells to develop tumors in nude mice, partially through targeting COX-2. Moreover a reduced miR-16 expression tends to correlate to high levels of COX-2 protein in liver from patients affected by HCC. Our data show an important role for miR-16 as a post-transcriptional regulator of COX-2 in HCC and suggest the potential therapeutic application of miR-16 in those HCC with a high COX-2 expression.This work was supported by Ministry of Science and Innovation [SAF2010-16037, SAF2009-12602 and BFU2011-24760] and by Comunidad de Madrid [P2010/BMD-2378].Peer reviewe

The EWS/FLI1 oncogenic protein inhibits expression of the Wnt inhibitor DICKKOPF-1 gene and antagonizes β-catenin/TCF-mediated transcription

Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and β-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and β-catenininduced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on β-catenin/TCF-mediated transcription since it can block transactivation of a consensus β-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage β-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores β-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that β-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to β-catenin, which could explain its negative effect on β-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as β-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family. © The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] de Educación y Ciencia (BFU2007-63038/BMC to J.M.G-S., SAF2007-62101 to J.A.); Fundación Científica de la Asociación Española Contra el Cáncer and Fundación Roviralta (to J.A.).Peer Reviewe

Lysyl oxidase is downregulated by the EWS/FLI1 oncoprotein and its propeptide domain displays tumor supressor activities in Ewing sarcoma cells.

Ewing sarcoma is the second most common bone malignancy in children and young adults. It is driven by oncogenic fusion proteins (i.e. EWS/FLI1) acting as aberrant transcription factors that upregulate and downregulate target genes, leading to cellular transformation. Thus, identificating these target genes and understanding their contribution to Ewing sarcoma tumorigenesis are key for the development of new therapeutic strategies. In this study we show that lysyl oxidase (LOX), an enzyme involved in maintaining structural integrity of the extracellular matrix, is downregulated by the EWS/FLI1 oncoprotein and in consequence it is not expressed in Ewing sarcoma cells and primary tumors. Using a doxycycline inducible system to restore LOX expression in an Ewing sarcoma derived cell line, we showed that LOX displays tumor suppressor activities. Interestingly, we showed that the tumor suppressor activity resides in the propeptide domain of LOX (LOX-PP), an N-terminal domain produced by proteolytic cleavage during the physiological processing of LOX. Expression of LOX-PP reduced cell proliferation, cell migration, anchorage-independent growth in soft agar and formation of tumors in immunodeficient mice. By contrast, the C-terminal domain of LOX, which contains the enzymatic activity, had the opposite effects, corroborating that the tumor suppressor activity of LOX is mediated exclusively by its propeptide domain. Finally, we showed that LOX-PP inhibits ERK/MAPK signalling pathway, and that many pathways involved in cell cycle progression were significantly deregulated by LOX-PP, providing a mechanistic explanation to the cell proliferation inhibition observed upon LOX-PP expression. In summary, our observations indicate that deregulation of the LOX gene participates in Ewing sarcoma development and identify LOX-PP as a new therapeutic target for one of the most aggressive paediatric malignancies. These findings suggest that therapeutic strategies based on the administration of LOX propeptide or functional analogues could be useful for the treatment of this devastating paediatric cancer