Angiogenin secretion from hepatoma cells activates hepatic stellate cells to amplify a self-sustained cycle promoting liver cancer

Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC managementThis study was funded by grants from the Instituto de Salud Carlos III (FIS PI12/00110, PI09/00056 to A.M., FIS PI10/02114, PI13/00374 to M.M., PI12/01265 to J.C. and PI11/0325 to J.F.C.), Ministerio de Economía y Competitividad (SAF 2012/34831 to J.F.C. and SAF2011-23031 to C.G.R.) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea. “Una manera de hacer Europa”); center grant P50-AA-11999 from Research Center for Liver and Pancreatic Diseases, US NIAAA to J.F.C.); Fundació la Marató de TV3 to J.F.C., Mutua Madrilea (AP103502012) to C.G.R., and by CIBERehd from the Instituto de Salud Carlos IIIPeer Reviewe

Optimisations and challenges involved in the creation of various bioluminescent and fluorescent influenza a virus strains for in vitro and in vivo applications

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses. Copyright

Detailed characterization of multiple myeloma circulating tumor cells shows unique phenotypic, cytogenetic, functional, and circadian distribution profile

[EN]Circulating myeloma tumor cells (CTCs) as defined by the presence of peripheral blood (PB) clonal plasma cells (PCs) are a powerful prognostic marker in multiple myeloma (MM). However, the biological features of CTCs and their pathophysiological role in MM remains unexplored. Here, we investigate the phenotypic, cytogenetic, and functional characteristics as well as the circadian distribution of CTCs vs paired bone marrow (BM) clonal PCs from MM patients. Our results show that CTCs typically represent a unique subpopulation of all BM clonal PCs, characterized by downregulation (P < .05) of integrins (CD11a/CD11c/CD29/CD49d/CD49e), adhesion (CD33/CD56/CD117/CD138), and activation molecules (CD28/CD38/CD81). Fluorescence in situ hybridization analysis of fluorescence-activated cell sorter-sorted CTCs also unraveled different cytogenetic profiles vs paired BM clonal PCs. Moreover, CTCs were mostly quiescent and associated with higher clonogenic potential when cocultured with BM stromal cells. Most interestingly, CTCs showed a circadian distribution which fluctuates in a similar pattern to that of CD34(+) cells, and opposite to stromal cell-derived factor 1 plasma levels and corresponding surface expression of CXC chemokine receptor 4 on clonal PCs, suggesting that in MM, CTCs may egress to PB to colonize/metastasize other sites in the BM during the patients' resting period

Análisis estructural del polimorfismo cuaternario en las helicasas replicativas y del complejo DnaB-DnaC de Escherichia coli

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 04-07-200

El arte de curar según Averroes

An explanation of the medica! treatises written by the great Muslim philosopher of Cardaba, during the arabic period of Spain, exactly in the XI century, and an analysis of his medica! contribution

El rol de enfermería y su influencia en la motivación: ¿un mito o una realidad?

Màster en Administració i Gestió en Cures d'Infermeria de l'E.U. Santa Madrona, 2004, Director: Esteve Pon

Gas6/Axl pathway is activated in chronic liver disease and its targeting reduces fibrosis via hepatic stellate cell inactivation

© 2015 European Association for the Study of the Liver. Background & Aims Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase and its ligand Gas6, are involved in cell differentiation, immune response and carcinogenesis. Methods HSCs were obtained from WT and Axl-/- mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated WT and Axl-/- mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients. Results In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl-/- mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality. Conclusions The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management.This study was funded by grants from the Instituto de Salud Carlos III (FIS PI12/00110, PI09/00056 to A.M., FIS PI13/00374 to M.M., PI12/01265 to J.C., PI14/00320 to P.S-B. and PI11/0325 to J.F.C.), Ministerio de Economía y Competitividad (BFU2010-22185 to P.G.F., SAF2012/34831 to J.F.C. and SAF2011-23031 to C.G.R.) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea. “Una manera de hacer Europa”); center grant P50-AA-11999 from Research Center for Liver and Pancreatic Diseases, (US NIAAA to J.F.C.); National Institutes of Health (R01 AI089824 to C.V.R); Fundació la Marató de TV3 to J.F.C.; Fundación Mutua Madrileña (AP103502012 to C.G.R.) and by CIBERehd from the Instituto de Salud Carlos IIIPeer Reviewe

HCC-Secreted angiogenin promotes HSC activation providing profibrogenic environment for tumor growth

Resumen del póster presentado a The International Liver Congress – 48th Annual meeting of the European Association for the Study of the Liver celebrado en Paises Bajos en abril de 2013.[Background]: Hepatocellular carcinoma (HCC) frequently grows on a background of inflamed and/or fibrotic liver. Recent data point to the cross-talk between tumor cells and the surrounding microenviroment as critical in HCC progression. Hepatic stellate cell (HSC) activation plays a key role in fibrogenesis and extracellular matrix deposition. Better knowledge of the interlink among HCC, adjacent cells and their derived components, that integrate the peri-tumoral stroma, will provide rationale for future therapies. Methods: Primary mouse hepatic stellate cells (HSCs), obtained after collagenase digestion, and activated human HSC cell line (LX2) were exposed to conditioned medium from HCC cell lines (HepsG2 and Hep3B), recombinant proteins (e.g. angiogenin) and inhibitors (e.g. neomycin). Western blots and qPCRs were performed to follow HSC phenotypic changes. Protein microarray was analyzed to search for specific HSC activators in medium from HCC cells. Angiogenin nuclear translocation was visualized in LX2 cells by immunofluorescence. Tumor growth was determined subcutaneously and orthotopically after co-injection of LX2 and HepG2 cells on the flanks and liver of nude mice, respectively. Angiogenin and serum levels were detected by western blot and ELISA. [Results]: HSC activation was observed after exposure to conditioned media from HepG2 and Hep3B cells. Release of angiogenic factors was analyzed in these media and several HCC-derived proteins identified. Among them, angiogenin, a protein secreted by several tumor types with ribonuclease activity and translational control, was able to induce HSC activation in primary mouse and human cell line LX2 after its extracellular caption and nuclear interaction. Blockade of nuclear translocation with neomycin diminished HSC activation by recombinant angiogenin or by HCC-conditioned medium. In vivo, increased angiogenin serum levels were detected in nude mice orthotopically co-injected with LX2 and HepG2 cells. In accordance, higher alpha-SMA mRNA levels were observed in subcutaneous tumors with upper angiogenin levels; while reduced angiogenin signaling, as after neomycin administration, was associated with decreased tumor growth and vascularization. [Conclusion]: Angiogenin release by HCC promotes a profibrogenic environment by activating surrounding HSCs, pointing to strategies directed against angiogenin production, internalization or nuclear interaction as of potential interest in HCC management.Peer reviewe

Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma

Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated.This study was funded by grants from the Instituto de Salud Carlos III (FIS PI12/00110, PI09/00056 to A.M., PI13/00374 to M.M, PI13/01339 to A.V., and SAF2015-69944-R and PI11/0325 to J.F.C.), Ministerio de Economía y Competitividad (SAF2012/34831 to J.F.C., SAF2014-57674-R to C.G.R. and SAF2013-47246-R to A.C.) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea. “Una manera de hacer Europa”); center grant P50-AA-11999 from Research Center for Liver and Pancreatic Diseases, US NIAAA to J.F.C.); Fundació la Marató de TV3 to J.F.C. and A.C., Mutua Madrileña (AP103502012) to C.G.R., and by CIBEREHD from the Instituto de Salud Carlos III. We also want to thank the support of the AGAUR (2014SGR785) from the Generalitat de Catalunya.Peer Reviewe