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El carcinoma hepatocellular (CHC) es reconocido como el principal cáncer primario de hígado y uno de los cánceres con mayor incidencia y mortalidad a nivel mundial. Éste se desarrolla en un contexto de enfermedad hepática crónica donde la aparición, así como la progresión de la enfermedad, está influenciada por numerosos factores de riesgo. Desafortunadamente, el alto grado de heterogeneidad genética intratumoral dificulta el desarrollo de terapias verdaderamente eficaces. Sorafenib fue el primer fármaco aprobado por la FDA (Food and Drug Administration) para su uso como tratamiento en pacientes avanzados de CHC. A pesar de que otras terapias hayan sido recientemente aprobadas para su uso como primera línea de tratamiento en dichos pacientes, Sorafenib sigue siendo la opción recomendada para un porcentaje significativo de ellos. Este fármaco es un inhibidor multiquinasa con efectos anti-proliferativos, anti-angiogénicos y pro-apoptóticos. Desgraciadamente, Sorafenib cuenta con unos beneficios clínicos muy limitados ya que por un lado produce numerosos efectos secundarios y por otro, el desarrollo de resistencia al tratamiento es muy frecuente en esta enfermedad. Otras terapias basadas en diferentes inhibidores multiquinasa han sido estudiadas en ensayo clínico, pero desgraciadamente, han mostrado una eficacia inferior o igual al Sorafenib, sugiriendo que la efectividad clínica del fármaco podrías estar basada en efectos adicionales a su acción inhibidora de la actividad quinasa. El proceso de síntesis de proteínas, también conocido como traducción, consiste en la formación de nuevas cadenas polipeptídicas a partir de la información contenida en una molécula de RNA mensajero (mRNA). Dicho proceso es mediado por el ribosoma que, junto con numerosas proteínas llamadas factores de traducción, sintetizan las proteínas de novo en función de la demanda celular. El proceso de traducción constituye el principal punto de regulación de la expresión génica, donde los niveles de

Introducción. El cáncer de páncreas es un tumor maligno intratable en el que la mayoría de los pacientes no muestran síntomas evidentes hasta que la enfermedad alcanza un estadio avanzado, lo que dificulta su detección precoz y hace que el tratamiento sea tardío. Esta situación dificulta la tendencia hacia la mejoría en la curva de supervivencia de estos pacientes. En caso del cáncer de páncreas metastásico la supervivencia es aún menor. En la actualidad no existen marcadores pronósticos y predictivos que indiquen la presencia de invasión vascular, neural o linfática, lo que dificulta el manejo clínico de los pacientes y, por tanto, su posibilidad de curación. Entre los biomarcadores que podrían evaluarse antes de la operación, las CTCs, como parte de la familia de la BL, podrían ser uno de los marcadores clínicos propuestos. Método. Se han evaluado CTCs y clusters en 35 pacientes con adenocarcinoma pancreático temprano a partir de 7 ml de muestras de sangre venosa portal (VP) y central (CVC) extraídas en el momento de la cirugía. Para el aislamiento de CTCs y clusters se ha utilizado el sistema IsofluxTM, que se basa en un proceso microfluídico de selección positiva inmunomagnética. Las células aisladas se detectaron mediante microscopía de fluorescencia tras tinción inmunocitoquímica (CK+/CD45- /Hoechst33342+) y se cuantificaron utilizando el método del algoritmo de la transformada de Hough. Resultados. Los pacientes con menos de 185 CTCs (p=0,018) y con menos de 15 clusters (p=0,040) en VP mostraron una mayor SG, así como los que no presentaron invasión vascular, linfática y tumor con grado de diferenciación G3. El análisis multivariante mostró que tanto un número de CTCs<185 (HR=4,464; p=0,016) como la ausencia de invasión vascular (HR=3,663; p=0,013) fueron predictores independientes de mejor supervivencia a largo plazo. En cuanto a la supervivencia libre de progresión local, tanto el análisis univariante como el multivariante, determinaron que tan solo la inva

Hepatocellular carcinoma (HCC) is one of the most frequent neoplasias and one of the major causes of cancer-related death worldwide. It usually arises upon chronic liver damage in cirrhotic livers. Several etiologies with different incidence increase the risk of HCC development, including hepatitis B and C virus infection, non-alcoholic liver disease (NAFLD) and alcohol abuse, among others. In the last years, there has been a shift in epidemiology from viral hepatitis to NAFLD and alcoholic liver disease (ALD). Treatment and patient prognosis are stablished according to the Barcelona Clinic Liver Cancer Staging System (BCLC). While early stages might receive curative treatments, intermediate and advanced stages of the disease rely on palliative treatments. The standard of care for intermediate patients at BCLC-B stages is transartherial chemoembolization (TACE), that consists in the embolization of the arteries that irrigate the tumor and the delivery of doxorubicin eluting beads. The landscape of treatments for advanced stages (BCLC-C) and patients at BCLC-B with untreatable progressive disease has profoundly changed. Nowadays, the combined treatment of Atezolizumab + Bevacizumab is the first line treatment. For patients in whom immunotherapy is not feasible, the tyrosine kinase inhibitors (TKIs) Sorafenib or Lenvatinib are still suitable therapeutic options. Regorafenib might be used upon Sorafenib progression in Sorafenib tolerant patients, and Cabozantinib has shown to be similar to Regorafenib. Given the limited efficacy of Sorafenib and other tyrosine kinase inhibitors (TKIs) in the clinical setting, new biomarkers of patient prognosis are urgently needed. In particular, liquid biopsy such as circulating tumor cells (CTCs), miRNAs, and extracellular vesicles (EVs). Therefore, the current thesis focuses on the impact of CTCs, EVs and miRNAs in the prognosis of patients with HCC at intermediate or advanced stages treated with TACE, Sorafenib or Lenvatinib. Circu

Hepatocellular carcinoma (HCC) is one of the most frequent and essentially incurable cancers in its advanced stages. The tyrosine kinase inhibitor Sorafenib (Sfb) remains the globally accepted treatment for advanced HCC. However, the extent of its therapeutic benefit is limited. Sfb exerts antitumor activity through its cytotoxic, anti-proliferative and pro-apoptotic roles in HCC cells. To better understand the molecular mechanisms underlying these effects, we used RNA sequencing to generate comprehensive transcriptome profiles of HepG2 and SNU423, hepatoblastoma-(HB) and HCC-derived cell lines, respectively, following a Sfb treatment at a pharmacological dose. This resulted in similar alterations of gene expression in both cell lines. Genes functionally related to membrane trafficking, stress-responsible and unfolded protein responses, circadian clock and activation of apoptosis were predominantly upregulated, while genes involved in cell growth and cycle, DNA replication and repair, ribosome biogenesis, translation initiation and proteostasis were downregulated. Our results suggest that Sfb causes primary effects on cellular stress that lead to upregulation of selective responses to compensate for its negative effect and restore homeostasis. No significant differences were found specifically affecting each cell line, indicating the robustness of the Sfb mechanism of action despite the heterogeneity of liver cancer. We discuss our results on terms of providing rationalization for possible strategies to improve Sfb clinical outcomes.

Debido a la multitud de funciones que a día de hoy se conocen que desempeñan los carbohidratos dentro del entorno celular y de la matriz extracelular (MEC) de los tejidos y, aprovechando los conocimientos en química orgánica sintética y supramolecular adquiridos por el grupo de investigación donde se enmarca la presente Tesis Doctoral; se han diseñado y sintetizados una serie de compuestos anfifílicos basados en carbohidratos, de bajo peso molecular, capaces de autoensamblarse por sí mismos en agua y alrededor de nanomateriales para su funcionalización, y obtener nanosistemas 1D y 3D (nanofibras de lípidos, micelas e hidrogeles) que expongan carbohidratos en su superficie de forma multivalente (efecto glicocluster). Para, de esta forma contribuir al entendimiento de la glicobiología celular, simulando el ambiente de la MEC, al avance en la química supramolecular de anfífilos y, en última instancia, posibilitar su aplicación en diferentes áreas de la biomedicina. Para el desarrollo de este objetivo global se plantearon diferentes objetivos parciales, los cuales corresponden a los tres capítulos en los que se ha estructurado la presente Tesis Doctoral (Figura 1). - (Capítulo 1): Diseño y síntesis de anfífilos basados en carbohidratos (neoglicolípidos) de bajo peso molecular, autoensamblables y gelificantes en medio acuoso, para la formación de hidrogeles supramoleculares, y el estudio de sus propiedades físico-químicas y posibles aplicaciones biológicas. En el capitulo 1, se han diseñado racionalmente y sintetizado 9 neoglicolípidos diacetilénicos fotopolimerizables, cuya autoasociación supramolecular en agua ha generado geles supramoleculares funcionales. Estos geles son capaces de responder a un estímulo externo, cambiando su coloración del azul al rojo, demostrando su utilidad como biosensores. Debido a la exposición homomultivalente y heteromultivalente de los carbohidratos en la superficie de sus constituyentes fibrilares, los geles son capaces de establecer inte

Purpose: Lack of diagnostic and prognostic biomarkers in hepatocellular carcinoma impedes stratifying patients based on their risk of developing cancer. The aim of this study was to evaluate phenotypic and genetic heterogeneity of circulating epithelial cells (CECs) based on asialoglycoprotein receptor 1 (ASGR1) and miR-122-5p expression as potential diagnostic and prognostic tools in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Methods: Peripheral blood samples were extracted from LC and HCC patients at different disease stages. CECs were isolated using positive immunomagnetic selection. Genetic and phenotypic characterization was validated by double immunocytochemistry for cytokeratin (CK) and ASGR1 or by in situ hybridization with miR-122-5p and CECs were visualized by confocal microscopy. Results: The presence of CECs increased HCC risk by 2.58-fold, however, this was only significant for patients with previous LC (p = 0.028) and not for those without prior LC (p = 0.23). Furthermore, the number of CECs lacking ASGR1 expression correlated significantly with HCC incidence and absence of miR-122-5p expression (p = 0.014; r = 0.23). Finally, overall survival was significantly greater for patients at earlier cancer stages (p = 0.018), but this difference was only maintained in the group with the presence of CECs (p = 0.021) whereas progression-free survival was influenced by the absence of ASGR1 expression. Conclusion: Identification and characterization of CECs by ASGR1 and/or miR- 122-5p expression may be used as a risk-stratification tool in LC patients, as it was shown to be an independent prognostic and risk-stratification marker in LC and early disease stage HCC patients.

Macroautophagy (autophagy herein) is a cellular stress response and a survival pathway involved in self-renewal and quality control processes to maintain cellular homeostasis. The alteration of autophagy has been implicated in numerous diseases such as cancer where it plays a dual role. Autophagy serves as a tumor suppressor in the early phases of cancer formation with the restoration of homeostasis and eliminating cellular altered constituents, yet in later phases, autophagy may support and/or facilitate tumor growth, metastasis and may contribute to treatment resistance. Key components of autophagy interact with either pro- and anti-apoptotic factors regulating the proximity of tumor cells to apoptotic cliff promoting cell survival. Autophagy is regulated by key cell signaling pathways such as Akt (protein kinase B, PKB), mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) involved in cell survival and metabolism. The expression of critical members of upstream cell signaling, as well as those directly involved in the autophagic and apoptotic machineries are regulated by microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Consequently, non-coding RNAs play a relevant role in carcinogenesis and treatment response in cancer. The review is an update of the current knowledge in the regulation by miRNA and lncRNA of the autophagic components and their functional impact to provide an integrated and comprehensive regulatory network of autophagy in cancer.

Background: Sorafenib constitutes a suitable treatment alternative for patients with advanced hepatocellular carcinoma (HCC) in whom atezolizumab + bevacizumab therapy is contraindicated. The aim of the study was the identification of a miRNA signature in liquid biopsy related to sorafenib response. Methods: miRNAs were profiled in hepatoblastoma HepG2 cells and tested in animal models, extracellular vesicles (EVs), and plasma from HCC patients. Results: Sorafenib altered the expression of 11 miRNAs in HepG2 cells. miR-200c-3p and miR-27a-3p exerted an anti-tumoral activity by decreasing cell migration and invasion, whereas miR-122-5p, miR-148b-3p, miR-194-5p, miR-222-5p, and miR-512-3p exerted pro-tumoral properties by increasing cell proliferation, migration, or invasion, or decreasing apoptosis. Sorafenib induced a change in EVs population with an increased number of larger EVs, and promoted an accumulation of miR-27a-3p, miR-122-5p, miR-148b-3p, miR-193b-3p, miR-194-5p, miR-200c-3p, and miR-375 into exosomes. In HCC patients, circulating miR-200c-3p baseline levels were associated with increased survival, whereas high levels of miR-222-5p and miR-512-3p after 1 month of sorafenib treatment were related to poor prognosis. The RNA sequencing revealed that miR-200c-3p was related to the regulation of cell growth and death, whereas miR-222-5p and miR-512-3p were related to metabolic control. Conclusions: The study showed that Sorafenib regulates a specific miRNA signature in which miR-200c-3p, miR-222-5p, and miR-512-3p bear prognostic value and contribute to treatment response.

Nitric oxide (NO) has been identified and described as a dual mediator in cancer according to dose-, time- and compartment-dependent NO generation. The present review addresses the different epigenetic mechanisms, such as histone modifications and non-coding RNAs (ncRNAs), miRNA and lncRNA, which regulate directly or indirectly nitric oxide synthase (NOS) expression and NO production, impacting all hallmarks of the oncogenic process. Among lncRNA, HEIH and UCA1 develop their oncogenic functions by inhibiting their target miRNAs and consequently reversing the inhibition of NOS and promoting tumor proliferation. The connection between miRNAs and NO is also involved in two important features in cancer, such as the tumor microenvironment that includes key cellular components such as tumor-associated macrophages (TAMs), cancer associated fibroblasts (CAFs) and cancer stem cells (CSCs).

Type 2 diabetes (T2D) is a very prevalent, multisystemic, chronic metabolic disorder closely related to atherosclerosis and cardiovascular diseases. It is characterised by mitochondrial dysfunction and the presence of oxidative stress. Metformin is one of the safest and most effective anti-hyperglycaemic agents currently employed as first-line oral therapy for T2D. It has demonstrated additional beneficial effects, unrelated to its hypoglycaemic action, on weight loss and several diseases, such as cancer, cardiovascular disorders and metabolic diseases, including thyroid diseases. Despite the vast clinical experience gained over several decades of use, the mechanism of action of metformin is still not fully understood. This review provides an overview of the existing literature concerning the beneficial mitochondrial and vascular effects of metformin, which it exerts by diminishing oxidative stress and reducing leukocyte-endothelium interactions. Specifically, we describe the molecular mechanisms involved in metformin's effect on gluconeogenesis, its capacity to interfere with major metabolic pathways (AMPK and mTORC1), its action on mitochondria and its antioxidant effects. We also discuss potential targets for therapeutic intervention based on these molecular actions.

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis,cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adaptcell metabolism and integrate several intracellular and redox signaling to promote cell survival in an in-flammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administrationof tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies atadvanced stages.There are important interrelationships between cell stress, redox status, mitochondrial function, metabolismand cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrestwidely related to the antitumoral properties of TKIs result from tightly controlled events involving differentcellular compartments and signaling pathways. The aim of the present review is to update the most relevantstudies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER)stress and Ca2+disturbances, leading to alteration of mitochondrial function, redox status and phosphatidyli-nositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activatedprotein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will becovered. Emphasis will be given to studies that identify key components of the integrated molecular patternincluding receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events thatappear to be involved in the resistance of cancer cells to TKI treatments.

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples

Background/Aims: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. Methods: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. Results: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15 P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172 P-Cdk4/ Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors in

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.

Type 2 diabetes (T2D) is one of the main current threats to human health. Both T2D and its numerous clinical complications are related to mitochondrial dysfunction and oxidative stress. Over the past decade, great progress has been made in extending our knowledge about the signaling events regulated by mitochondria. However, the links among mitochondrial impairment, oxidative stress, autophagy, endoplasmic reticulum (ER) stress, and activation of the inflammasome still need to be clarified. In light of this deficit, we aim to provide a review of the existing literature concerning the complicated crosstalk between mitochondrial impairment, autophagy, ER stress, and the inflammasome in the molecular pathogenesis of T2D.

Sorafenib and Regorafenib are the recommended first- and second-line therapies in patients with advanced hepatocellular carcinoma (HCC). Lenvatinib and Cabozantinib have shown non-inferior antitumoral activities compared with the corresponding recommended therapies. The clinical trials have established recommended doses for each treatment that lead different blood concentrations in patients for Sorafenib (10 µM), Regorafenib (1 µM), Lenvatinib (0.1 µM), and Cabozantinib (1 µM). However, very low response rates are observed in patients attributed to intrinsic resistances or upregulation of survival signaling. The aim of the study was the comparative dose-response analysis of the drugs (0-100 µM) in well-differentiated (HepG2, Hep3B, and Huh7), moderately (SNU423), and poorly (SNU449) differentiated liver cancer cells in 2D/3D cultures. Cells harbors wild-type p53 (HepG2), non-sense p53 mutation (Hep3B), inframe p53 gene deletion (SNU423), and p53 point mutation (Huh7 and SNU449). The administration of regular used in vitro dose (10 µM) in 3D and 2D cultures, as well as the dose-response analysis in 2D cultures showed Sorafenib and Regorafenib were increasingly effective in reducing cell proliferation, and inducing apoptosis in well-differentiated and expressing wild-type p53 in HCC cells. Lenvatinib and Cabozantinib were particularly effective in moderately to poorly differentiated cells with mutated or lacking p53 that have lower basal oxygen consumption rate (OCR), ATP, and maximal respiration capacity than observed in differentiated HCC cells. Sorafenib and Regorafenib downregulated, and Lenvatinib and Cabozantinib upregulated epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor receptor (c-Met) in HepG2 cells. Conclusions: Sorafenib and Regorafenib were especially active in well-differentiated cells, with wild-type p53 and increased mitochondrial respiration. By contrast, Lenvatinib and Cabozantinib appeared more effective in moder

Sorafenib is the first-line recommended therapy for patients with advanced hepatocarcinoma (HCC) in de-differentiation stage (presenting epithelial–mesenchymal transition, EMT). We studied the role of the thioredoxin system (Trx1/TrxR1) in the sensitivity or resistance of HCC cells to the treatment with Sorafenib. As a model, we used a set of three established HCC cell lines with different degrees of de-differentiation as occurs in metastasis. By quantitative proteomics, we found that the expression levels of Trx1 and TrxR1 followed the same trend as canonical EMT markers in these cell lines. Treatment with Sorafenib induced thiol redox reductive changes in critical elements of oncogenic pathways in all three cell lines but induced drastic proteome reprograming only in HCC cell lines of intermediate stage. Trx1 downregulation counteracted the thiol reductive effect of Sorafenib on Signal Transducer and Activator of Transcription 3 (STAT3) but not on Mitogen-Activated Protein Kinase (MAPK) or Protein Kinase B (Akt) and transformed advanced HCC cells into Sorafenib-sensitive cells. Ten targets of the combined Sorafenib–siRNATrx1 treatment were identified that showed a gradually changing expression trend in parallel to changes in the expression of canonical EMT markers, likely as a result of the activation of Hippo signaling. These findings support the idea that a combination of Sorafenib with thioredoxin inhibitors should be taken into account in the design of therapies against advanced HCC.

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin‐agarose hydrogel patch, with (c‐NFAH) or without cells (a‐NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post‐operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a‐NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a‐NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a‐NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures.

Hepatitis C virus (HCV) infection has been related to increased risk of development of hepa tocellular carcinoma (HCC) while metformin (M) and statins treatment seemed to protect against HCC development. In this work, we aim to identify the mechanisms by which metfor min and simvastatin (S) could protect from liver cancer. Huh7.5 cells were infected with HCV particles and treated with M+S. Human primary hepatocytes were treated with M+S. Treatment with both drugs inhibited Huh7.5 cell growth and HCV infection. In non-infected cells S increased translational controlled tumor protein (TCTP) and phosphatase and tensin homolog (PTEN) proteins while M inhibited mammalian target of rapamycin (mTOR) and TCTP. Simvastatin and metformin co-administered down-regulated mTOR and TCTP, while PTEN was increased. In cells infected by HCV, mTOR, TCTP, p62 and light chain 3B II (LC3BII) were increased and PTEN was decreased. S+M treatment increased PTEN, p62 and LC3BII in Huh7.5 cells. In human primary hepatocytes, metformin treatment inhibited mTOR and PTEN, but up-regulated p62, LC3BII and Caspase 3. In conclusion, simvastatin and metformin inhibited cell growth and HCV infection in vitro. In human hepatocytes, met formin increased cell-death markers. These findings suggest that M+S treatment could be useful in therapeutic prevention of HCV-related hepatocellular carcinoma.

Los tumores periampulares y especialmente el adenocarcinoma de páncreas presentan en la actualidad una supervivencia a los 5 años del 5%. La cirugía sigue siendo el tratamiento de elección, pero no ha conseguido mejorar los datos de supervivencia en los últimos 20 años. Los tratamientos sistémicos con fármacos quimioterápicos presentan unos resultados limitados y la investigación en ese campo es escasa. El objetivo del estudio es desarrollar un modelo experimental de tumor periampular mediante xenoinjerto subcutáneo en ratón y comprobar los posibles cambios histológicos de dicho tumor en los sucesivos pases en ratones, para que sea la base de nuevos proyectos de investigación sobre tumores periampulares. Existen modelos experimentales previos en ratones realizados con xenoinjertos de metástasis hepáticas y otros en el que se han utilizado tóxicos que no han tenido los resultados esperados con índices de desarrollo tumoral cercanos al 40%.

Debido al mal pronóstico de los tumores del área periampular y especialmente de adenocarcinoma de páncreas (ADCP), resulta fundamental la búsqueda de biomarcadores para su detección precoz y de moléculas que permitan el desarrollo urgente de nuevas estrategias terapéuticas. Para ello, los modelos animales han demostrado ser muy útiles en la evolución de los avances en el cáncer de páncreas. Han contribuido al estudio de nuevos quimioterápicos que posteriormente han sido trasladados a ensayos clínicos con humanos que han demostrado un aumento en la supervivencia. El desarrollo de los quimioterápicos y otras moléculas antitumorales se encuentra íntimamente relacionado con los modelos experimentales. Durante los últimos 50 años, los xenoinjertos subcutáneos derivados de líneas celulares cultivadas in vitro han sido ampliamente utilizados. Multitud de quimioterápicos han sido testados utilizando estos modelos. Sin embargo, fármacos que funcionan bien en el ratón no son necesariamente efectivos en el ser humano. Desde los años 70, se han utilizado muestras de cánceres humanos tomados mediante biopsia o mediante cirugía que posteriormente han sido implantados directamente en ratones. Estos xenoinjertos, también conocidos como “tumorgraft” mantienen la morfología tumoral inicial, así como las mutaciones genéticas que le acompañan. Sin embargo, estos modelos fueron inicialmente desechados debido a su alto índice de rechazos. Recientemente, se ha retomado esta línea de investigación gracias al desarrollo de nuevas cepas de ratones inmunodeprimidos modificadas genéticamente que han minimizado las tasas rechazo. De esta manera, ha sido posible que este modelo sea competitivo a la hora de analizar la eficacia de las distintas drogas para el estudio de ADCP. Siguiendo esta línea de trabajo hemos desarrollados tres modelos experimentales mediante el uso de xenoinjertos: subcutáneo, intraabdominal y pancreático. El objetivo principal de este estudio es valorar la viabilidad de los

The NLRP3-inflammasome complex has emerged as an important component of inflammatory processes in metabolic dysfunction induced by high-caloric diets. In this study, we investigate the molecular mechanisms by which NLRP3 inhibition may attenuate diet-induced cardiac injury. Here we show the cardiac damage induced by high sugar diet (HSD), high fat diet (HFD) or high sugar/fat diet (HSFD) over 15 weeks. Genetic ablation of NLRP3 protected against this damage by autophagy induction and apoptotic control. Furthermore, NLRP3 inhibition by the selective small molecule MCC950 resulted in similar autophagy induction and apoptotic control in hearts after diets. These data were reproduced in THP-1 cells treated with MCC950 and cultured in media supplemented with serum from mice dosed with MCC950 and fed with diets. NLRP3 inhibition exerted beneficial metabolic, and autophagic adaptations in hearts from obesogenic diets. The inhibition of NLRP3 activation may hold promise in the treatment of metabolic and cardiovascular diseases.

The NLRP3-inflammasome complex has emerged as an important component of inflammatory processes in metabolic dysfunction induced by high-caloric diets. In this study, we investigate the molecular mechanisms by which NLRP3 inhibition may attenuate diet-induced cardiac injury. Here we show the cardiac damage induced by high sugar diet (HSD), high fat diet (HFD) or high sugar/fat diet (HSFD) over 15 weeks. Genetic ablation of NLRP3 protected against this damage by autophagy induction and apoptotic control. Furthermore, NLRP3 inhibition by the selective small molecule MCC950 resulted in similar autophagy induction and apoptotic control in hearts after diets. These data were reproduced in THP-1 cells treated with MCC950 and cultured in media supplemented with serum from mice dosed with MCC950 and fed with diets. NLRP3 inhibition exerted beneficial metabolic, and autophagic adaptations in hearts from obesogenic diets. The inhibition of NLRP3 activation may hold promise in the treatment of metabolic and cardiovascular diseases.

Objetivos: Evaluar el efecto del drenaje biliar interno y los anticuerpos antiTNF-a en las alteraciones de la inmunidad celular, citoquinas y endotoxemia en la ictericia obstructiva experimental; Evaluar las alteraciones de la inmunidad celular en la IO experimental determinando en sangre periférica la respuesta proliferativa a mitógenos linfocitarios, la capacidad fagocítica y oxidativa de neutrófilos y macrófagos y las proporciones entre las diversas subpoblaciones leucocitarias y su grado de activación; Evaluar el estado de las citoquinas y endotoxemia en animales con ligadura de la vía biliar; Comparar el efecto del drenaje biliar interno y de los anticuerpos antiTNF-a en las alteraciones anteriormente descritas. Metodología experimental: Ratas Wistar macho adultas (200-290 g), en jaulas individuales y con libre acceso a pienso standard y agua, y cuyo manejo se hará conforme a los protocolos de la Guía de cuidados y uso de animales de laboratorio del National Institute of Health ( publicación nº86-23, revisado en 1985 ). Las ratas se repartirán al azar en series de 6 animales, uno por cada grupo de trabajo, repitiendo las series hasta incluir 5 animales en cada grupo. Previamente a la cirugía, la anestesia se realizará mediante inyección intraperitoneal de Ketamina (8 mg / 100 g) y Midazolán (0’4 mg / 100 g). Los procedimientos se llevarán a cabo en condiciones de esterilidad y sobre una mesa termorregulada con fuente de luz propia. Ligadura del conducto biliar (LCB): Los animales se colocarán en decúbito supino más Pillet. La vía de abordaje será por laparotomía media desde xifoides al pubis. Según la técnica standard de IO en ratas modificada 14, realizando una tracción suave del duodeno se expone el conducto biliar común, que es disecado y ligado mediante doble ligadura con seda de 3/0. El cierre de la pared abdominal se hará en dos planos con sutura continua de seda de 3/0. Plan de trabajo: Antes de realizar cualquier procedimiento se recogerá el peso de los

Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with potential portal hypertension and/or bilirubinemia, but without vascular-associated diseases. The patients are receiving immunosuppressive therapy to reduce graft rejection, but differential side effects have been related to calcineurin and mTOR inhibitor administration regarding tumor recurrence and nephrotoxicity. The in vitro studies showed that Tacrolimus exerted a more potent pro-apoptotic effect than Evero- limus (Huh 7>Hep 3B>HepG2), being sirolimus only active in Hep3B cell line. Tacrolimus and Everolimus exerted potent antiproliferative properties in Huh 7 and Hep3B in which cells Sirolimus was inactive. Interestingly, Tacrolimus- and Everolimus-dependent G 0 /G 1 cell accumulation occurred as a consequence of drastic reduction in S, as well as in S and G 2+M phases, respectively. The in vivo studies support data on the more effective antitu- moral properties of Everolimus, eventual risk of pro-angiogenic tumoral properties and nephrotoxicity of Tacrolimus, and pro-proliferative properties of Sirolimus in tumors developed in nude mice.

Obstructive Jaundice (OJ) is associated with a significant risk of developing acute renal failure (ARF). The involvement of oxidative stress in the development of cholestasis has been demonstrated in different experimental models. However, its role in the morbidity of human cholestasis is far to be elucidated. The aim of the study was the evaluation of oxidative stress markers in blood from patients with OJ and its relation to complications and benign/malignant evolution of cholestasis. Methods: A prospective cross-sectional study of 105 patients with OJ and 34 control subjects were included. Several markers of liver function and oxidative stress, such as lipoperoxides (LPO), as well as reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were assessed. Results: The patients with OJ showed a marked increase in plasma levels of LPO, SOD and GSH, while GSH-Px levels were decreased. The increase in lipid peroxidation products and the depletion of SOD activity in blood were also related to renal dysfunction. The highest level of LPO was associated with malignant etiology of the disease. The logistic regression analysis showed that the age of the patient and the levels of LPO in blood were predictors of renal dysfunction in OJ patients. Conclusions: This study demonstrates a correlation between oxidative stress and renal dysfunction patients with OJ.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultra-performance liquid chromatography coupled to mass spectrometry (UPLCMS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual’s level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values = 0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention.