Investigación básica sobre hepatocarcinoma en la Universidad de León

Estudio de nuevas estrategias quimioterapéuticas en el tratamiento del hepatocarcinoma human

On-line surface plasmon resonance biosensing of vascular endothelial growth factor signaling in intact-human hepatoma cell lines

[EN] Surface plasmon resonance (SPR) monitoring of biorecognition events at intracellular levels is a valuable tool for studying the angiogenic response of carcinoma living cells during tumor growth and proliferation. We report here a comparative study of two different strategies to detect human hepatoma cell interactions between transmembrane vascular endothelial growth factor receptor (VEGFR2) and vascular endothelial growth factor (VEGF). To monitor VEGFR2 activation after VEGF stimulation, intact hepatocellular carcinoma HepG2 or Huh7 cells (2 x 105 cells mL1) were directly immobilized on the sensor chip. Distinguishable SPR sensorgrams were obtained for each cell line depending on the time required for VEGFR2 activation. SPR signals for VEGF/VEGFR2 binding were inhibited by VEGFR inhibitor, CBOP11. The SPR response after VEGF stimulation/inhibition was in good agreement with the results observed by immunoblotting analysis. In a second approach we used intact cell lines as analytes. SPR analysis was done by injecting HepG2 and HuH7 cell suspensions (24 x 104 cells mL1) onto a sensor surface previously immobilized with VEGF via thiol selfassembled monolayer (SAM). Specificity and reproducibility were evaluated reusing the same chip surface over more than 60 complete regeneration cycles. Comparison between both methods yielded differences in terms of reliability, making the last strategy more effective for the analysis of real samples. The investigation of VEGF signaling in intact human hepatoma living cells by SPR monitoring comprises a novel and promising design for the study of tumor angiogenesis via downregulation of VEGF and VEGFR2 pathways. Further investigation on VEGFR activation and vascular function could contribute to establish a robust and meaningful tool for early cancer diagnostics.S

On-line surface plasmon resonance biosensing of vascular endothelial growth factor signaling in intact-human hepatoma cell lines

Surface plasmon resonance (SPR) monitoring of biorecognition events at intracellular levels is a valuable tool for studying the angiogenic response of carcinoma living cells during tumor growth and proliferation. We report here a comparative study of two different strategies to detect human hepatoma cell interactions between transmembrane vascular endothelial growth factor receptor (VEGFR2) and vascular endothelial growth factor (VEGF). To monitor VEGFR2 activation after VEGF stimulation, intact hepatocellular carcinoma HepG2 or Huh7 cells (2 × 10 cells per mL) were directly immobilized on the sensor chip. Distinguishable SPR sensorgrams were obtained for each cell line depending on the time required for VEGFR2 activation. SPR signals for VEGF-VEGFR2 binding were inhibited by the VEGFR inhibitor, CBO-P11. The SPR response after VEGF stimulation/inhibition was in good agreement with the results observed by immunoblotting analysis. In a second approach we used intact cell lines as analytes. SPR analysis was done by injecting HepG2 and HuH7 cell suspensions (2-4 × 10 cells per mL) onto a sensor surface previously immobilized with VEGF via a thiol self-assembled monolayer (SAM). Specificity and reproducibility were evaluated reusing the same chip surface over more than 60 complete regeneration cycles. Comparison between both methods yielded differences in terms of reliability, making the latter strategy more effective for the analysis of real samples. The investigation of VEGF signaling in intact human hepatoma living cells by SPR monitoring comprises a novel and promising design for the study of tumor angiogenesis via downregulation of VEGF and VEGFR2 pathways. Further investigation on VEGFR activation and vascular function could contribute to establish a robust and meaningful tool for early cancer diagnostics

Effect of exercise on gut microbiota and metabolic status modulation in an in vivo model of early obesity and NAFLD

1 p.Childhood obesity is one of the most serious public health concerns from this century, associated with metabolic syndrome, nonalcoholic fatty liver disease (NAFLD) and gut microbiota alterations. Physical exercise improves obesity and NAFLD progression, modulating the gut microbial balance. We aim to investigate the effect of physical exercise on gut microbiota and the metabolic status of an in vivo model of early obesity, metabolic syndrome, and NAFLD. Resumen de un trabajo resultado del proyecto de investigación financiado por la Consejería de Educación de la Junta de Castilla y León (referencia LE063U16)S

Beneficial effects of exercise on gut microbiota functionality and barrier integrity, and gut-liver axis crosstalk in an "in vivo" model of early obesity and non-alcoholic fatty liver disease

[EN]Childhood obesity has reached epidemic levels, representing one of the most serious public health concerns associated with metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). There is limited clinical experience concerning pediatric NAFLD patients, and thus the therapeutic options are scarce. The aim of this study was to evaluate the benefits of exercise on gut microbiota composition and functionality balance, and consequent effects on early obesity and NAFLD onset in an in vivo model. Juvenile (21-day-old) male Wistar rats fed a control diet or a high-fat diet (HFD) were subjected to a combined aerobic and resistance training protocol. Fecal microbiota was sequenced by an Illumina MiSeq system, and parameters related to metabolic syndrome, fecal metabolome, intestinal barrier integrity, bile acid metabolism and transport, and alteration of the gut-liver axis were measured. Exercise decreased HFD-induced body weight gain, metabolic syndrome and hepatic steatosis, as a result of its lipid metabolism modulatory capacity. Gut microbiota composition and functionality were substantially modified as a consequence of diet, age and exercise intervention. In addition, the training protocol increased Parabacteroides, Bacteroides and Flavobacterium genera, correlating with a beneficial metabolomic profile, whereas Blautia, Dysgonomonas and Porphyromonas showed an opposite pattern. Exercise effectively counteracted HFD-induced microbial imbalance, leading to intestinal barrier preservation, which, in turn, prevented deregulation of the gut-liver axis and improved bile acid homeostasis, determining the clinical outcomes of NAFLD. In conclusion, we provide scientific evidence highlighting the benefits of gut microbiota composition and functionality modulation by physical exercise protocols in the management of early obesity and NAFLD development.SIThis work was supported by grants from Ministerio de Economıa y Competitividad ́ (BFU2017-87960-R), Junta de Castilla y León and the European Regional Development Fund (FEDER) (LE063U16 and GRS1888/A/18). D.P. and S.C.-P. were supported by a fellowship from Junta de Castilla y León, co-financed by the European Social Fund. E.N. was supported by Fundación de Investigación Sanitaria of León. M.V.G.-M. was supported by contracts from the CIBERehd, which is funded by Instituto de Salud Carlos III

Efecto del ejercicio físico en la modulación de la microbiata intestinal y el desarrollo de síndrome metabólico y NAFLD en un modelo in vivo de obesidad temprana

2 p.Este trabajo pretende investigar el efecto del ejercicio físico sobre la microbiota intestinal y el estado metabólico en un modelo in vivo de obesidad temprana, síndrome metabólico y NAFLD. Resumen de un trabajo resultado del proyecto de investigación financiado por la Consejería de Educación de la Junta de Castilla y León (referencia LE063U16)S

mTOR signalling, embryogenesis and the control of lung development

The existence of a nutrient sensitive “autocatakinetic” regulator of embryonic tissue growth has been hypothesised since the early 20th century, beginning with pioneering work on the determinants of foetal size by the Australian physiologist, Thorburn Brailsford-Robertson. We now know that the mammalian target of rapamycin complexes (mTORC1 and 2) perform this essential function in all eukaryotic tissues by balancing nutrient and energy supply during the first stages of embryonic cleavage, the formation of embryonic stem cell layers and niches, the highly specified programmes of tissue growth during organogenesis and, at birth, paving the way for the first few breaths of life. This review provides a synopsis of the role of the mTOR complexes in each of these events, culminating in an analysis of lung branching morphogenesis as a way of demonstrating the central role mTOR in defining organ structural complexity. We conclude that the mTOR complexes satisfy the key requirements of a nutrient sensitive growth controller and can therefore be considered as Brailsford-Robertson's autocatakinetic centre that drives tissue growth programmes during foetal development

Efecto de la melatonina sobre la proliferación, apoptosis y angiogénesis en un modelo in vitro de hepatocarcinoma = Effect of melatonin on proliferation, apoptosis and angiogenesis in an in vitro model of hepatocarcinoma

130 p.El objetivo principal de la tesis doctoral, consistió en analizar los efectos oncostáticos, antiproliferativos y anti-angiogénicos de la administración in vitro de la melatonina en un modelo celular de hepatocarcinoma humano, profundizando así en los mecanismos moleculares implicados en dichos efectos y aportando más información para el posible uso terapéutico de esta hormona en el tratamiento del hepatocarcinoma humano. Del trabajo realizado durante estos cuatro años, se pueden extraer las siguientes conclusiones: 1. Los efectos de la melatonina sobre la viabilidad celular y la proliferación en las células HepG2 están parcialmente mediados por el receptor de membrana MT1, que actúa como modulador de los niveles de AMP cíclico y activador de la ruta de protein quinasas ERK. 2. La sobre-expresión de NQO2 inducida por la melatonina cuando se bloquea MT1 competitivamente con luzindol, podría explicarse como una respuesta celular que permite la unión de la melatonina presente en el medio de cultivo, y pone de relieve una nueva interacción entre los receptores MT1 y NQO2 en células tumorales hepáticas. 3. Nuestros resultados confirman las propiedades oncostáticas de la melatonina y muestran la alta selectividad de esta molécula entre los hepatocitos sanos y tumorales. 4. La melatonina puede inducir la apoptosis en células HepG2 induciendo la expresión de la proteína pro-apoptótica Bim, a través de la translocación nuclear y la activación del factor de transcripción FoxO3a. 5. La melatonina ejerce una actividad anti-angiogénica en células HepG2, inhibiendo la activación transcripcional de VEGF, a través de Hif1alpha y STAT3. En base a los efectos de la melatonina sobre la proliferación, la apoptosis y la angiogénesis en un modelo in vitro de HCC, junto con sus propiedades beneficiosas ya documentadas en varios tumores, y su falta de toxicidad, demuestran el potencial interés de esta molécula, al menos como adyuvante, en la terapia del HCC. Sin embargo, se requiere más investigación para confirmar la utilidad de esta molécula en la prevención y el tratamiento de pacientes con cánce