Regulación transcripcional de cyba (p22 phox ), inducida por el factor de crecimiento de hepatocitos (HGF) acoplado a su receptor c - Met en hepatocitos de ratón

It is well known that signaling mediated by the hepatocyte growth factor (HGF) and its receptor c - Met is involved in the control of cellular redox status and oxidative stress, particularly through its ability to induce hepatoprotective gene expression by activating survival pathways in the hepatocyte. We recently showed that HGF can regulate the expression of many members of the NADPH oxidase family in liver cells, particularly the catalytic subunits and p22 phox. In the present work we were focused to figure out the mechanism mediated by HGF/c - Met in the expres sion and protein content of p22 phox in primary mouse hepatocytes. The analys is of the expression of cyba by qRT - PCR under HGF treatment revealed the repression of this messenger in a time dependent manner. To identify the most relevant cis elements in the cyba promoter, different regions were cloned into a vector to measure the pr omoter activity, the most relevant region was - 662 / - 1362 (B - C). While this effect was related with p22 phox protein content, the timing of the effect suggested an independent process that could be involved in p22 phox levels. In order to figure out the pos sible p22 phox degradation by 26S proteasome induced by HGF, we treated cells with epoxomicin (200 nM) for 30 min before HGF (50 ng/ml) treatment for 12h. Result showed that p22 phox degradation was abrogated. To gain more evidence regarding this mechanism, we used wortmannin (2 mM) to inhibit PI3K/Akt, due to some reports linking PI3K/Akt to the mdm2 ubiquitin ligase. We found that also p22 phox degradation was revoked. Finally, as we previously found we confirmed the interaction of p22 phox and c - Met by immun oprecipitation, suggesting an additional post - translational mechanism of regulation of NADPH oxidase and HGF/c - Met. In conclusion, our data clearly show that HGF/c - Met exerts a wide - range of molecular mechanisms tending to control the expression and activity of those NADPH oxidases that use p22 phox as a key component.La señalización mediada por el factor de crecimiento de hepatocitos (HGF) y su receptor c - Met está implicada en el control de estado redox celular y el estrés oxidante, en particular a través de su capacidad para inducir la expresión de genes hepatoprotector es mediante la activación de las vías de supervivencia en el hepatocito. Recientemente, nuestro grupo de trabajo ha reportado que el HGF regula la expresión de subunidades que forman parte del complejo multiproteico de la enzima NADPH oxidasa en hepatocitos de ratón, especialmente las subunidades catalíticas Nox2 y Nox4 y la reguladora p22 phox. En el presente trabajo nos centramos en investigar el mecanismo mediado por HGF/c - Met en la regulación de la expresión del gen cyba que codifica para la proteína p22 phox en cultivo primario de hepatocitos de ratón. El análisis de la expresión de cyba por q RT - PCR en hepatocitos trata dos con HGF, reveló la repr esión de este mensajero de manera dependiente del tiempo. Para identificar los elementos cismás relevantes del promotor de cyba se clon aron diferentes regiones en un vector para medir la actividad promotora, la región má s relevante fue – 662/ - 1362 (B - C). Y mediante el análisis in silico de este promotor se logró identificar regiones consenso para NF - kB. Posteriormente se evaluó el contenido proteico de p22 phox mediante inmunode te cciones y se pudo observar el mismo efecto que en el mRNA. Con el fin de averig uar sí el proteosoma 26S está involucrado en la degradación de p22 phox inducid o por el HGF, se pre - trataron las células con epoxomicina (200 nM) durante 30 minutos e inmediatamente después se trataron con HGF (50 ng/ml) durante 12 h. El resultado mostró qu e la degradación de p22 phox fue abrogada. Para obtener más pruebas con respecto a este mecanismo, se utilizó wortmani na (2 mM) para inhibir PI3K/Akt/MDM2, y t ambién encontramos que la degradación de p22 phox fue revocada. Por último, hemos encontrado un a in teracción entre p22 phox y c - Met, lo que sugiere un mecanismo post - traduccional adicional de regulación de la NADPH oxidasa y HGF/c - Met. En conclusión, nuestros datos muestran claramente que HGF/c - Met ejerce una amplia gama de mecanismos moleculares que tienden a controlar la expresión y la actividad de las NADPH oxidasas que utiliza a p22 phox como un componente clave

Tumor immune microenvironment modulation by cholesterol in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is considered one of the most aggressive tumors worldwide. The consumption of lipid-enriched diets, mainly high cholesterol, induces oxidative stress and chronic inflammation, leading to HCC progression. Moreover, fatty acids and cholesterol could display differential responses on immune cells inside the tumor immune microenvironment (TIME). Tumor-associated macrophages (TAMs) represent one of the most critical leukocytes in the tumor microenvironment (TME) displaying pro-tumoral responses and one of the mainly cholesterol donors to cancer cells. Immunotherapy or cholesterol regulators, alone or combined, would represent an effective strategy for HCC treatment. Nonetheless, steatotic etiology from non-alcoholic fatty liver disease (NAFLD)-HCC tumors has been unexpectedly resulting in highly aggressive behavior

Mediterranean-like mix of fatty acids induces cellular protection on lipid-overloaded hepatocytes from western diet fed mice

Introduction and objective. Non-alcoholic fatty liver disease remains as one of the main liver disorders worldwide. It is widely accepted that is the kind of lipid, rather than the amount deposited in the cells that determines cell damage. Cholesterol and saturated free fatty acids are deleterious lipids when accumulated but, in contrast, there are some valuable lipids that could counteract those with harmful properties. Much of this knowledge arises from studies using a single fatty acid, but the effects of a combination of fatty acids, as obtained by diet has been poorly addressed. In the present work, we were focused to figure out the cellular effect of two different mixes of fatty acids, one with high proportion of saturated fatty acids, and another one with high proportion of unsaturated fatty acids (Mediterranean-like) in a cellular model of steatosis. Material and methods. Primary mouse hepatocytes from animals fed with a western diet (high fat and carbohydrates diet), were treated with both mixes of fatty acids for 24 h. Results. Our data clearly show that only the high unsaturated fatty acid mix induced a decrease in triglycerides (47.5%) and cholesterol (59%) content in steatotic hepatocytes mediating cellular protection associated to the decrement of ROS and oxidative damage. The mixture of high saturated fatty acids exhibited no effects, preserving high levels of cholesterol and triglycerides and oxidative damage. In conclusion, our results show that Mediterranean-like mix of fatty acids exerts cellular protection in steatosis by decreasing triglycerides, cholesterol, ROS content and oxidative damage

Cholangiocyte death in ductopenic cholestatic cholangiopathies: Mechanistic basis and emerging therapeutic strategies

Among hepatic diseases, cholestatic ductopenic cholangiopathies are poorly studied, and they are rarely given the importance they deserve, especially considering their high incidence in clinical practice. Although cholestatic ductopenic cholangiopathies have different etiologies and pathogenesis, all have the same target (the cholangiocyte) and similar mechanistic basis of cell death. Cholestatic cholangiopathies are characterized, predominantly, by obstructive or functional damage in the biliary epithelium, resulting in an imbalance between proliferation and cholangiocellular death; this leads to the progressive disappearance of bile ducts, as has been shown to occur in primary sclerosing cholangitis, primary biliary cholangitis, low-phospholipid-associated cholelithiasis syndrome, cystic fibrosis-related liver disease, and drug-induced ductopenia, among other biliary disorders. This review summarizes the features of the more common ductopenic syndromes and the cellular mechanisms involved in cholengiocellular death, with focus on the main forms of cholangiocyte death described so far, namely apoptosis, autophagy, necrosis, and necroptosis. It also emphasizes the importance to study in depth the molecular mechanisms of cholengiocyte death to make possible to counteract them with therapeutic purposes. These therapeutic strategies are limited in number and efficacy at present, and this is why it is important to find complementary, safe strategies to stimulate cholangiocellular proliferation in order favor bile duct replenishment as well. Successful in finding appropriate treatments would prevent the patient from having liver transplantation as the only therapeutic alternative.Fil: Salas Silva, Soraya. Universidad Autónoma Metropolitana; MéxicoFil: Simoni Nieves, Arturo. Universidad Autónoma Metropolitana; MéxicoFil: Lopez Ramirez, Jocelyn. Universidad Autónoma Metropolitana; MéxicoFil: Bucio, Leticia. Universidad Autónoma Metropolitana; México. Instituto Nacional de Cardiología Ignacio Chavez; México. Universidad Nacional Autónoma de México; MéxicoFil: Gómez Quiroz, Luis E.. Universidad Autónoma Metropolitana; México. Instituto Nacional de Cardiología Ignacio Chavez; México. Universidad Nacional Autónoma de México; MéxicoFil: Gutiérrez Ruiz, María Concepción. Universidad Autónoma Metropolitana; México. Instituto Nacional de Cardiología Ignacio Chavez; México. Universidad Nacional Autónoma de México; MéxicoFil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentin

HGF induces protective effects in α-naphthylisothiocyanate-induced intrahepatic cholestasis by counteracting oxidative stress

Cholestasis is a clinical syndrome common to a large number of hepatopathies, in which either bile production or its transit through the biliary tract is impaired due to functional or obstructive causes; the consequent intracellular retention of toxic biliary constituents generates parenchyma damage, largely via oxidative stress-mediated mechanisms. Hepatocyte growth factor (HGF) and its receptor c-Met represent one of the main systems for liver repair damage and defense against hepatotoxic factors, leading to an antioxidant and repair response. In this study, we evaluated the capability of HGF to counteract the damage caused by the model cholestatic agent, α-naphthyl isothiocyanate (ANIT). HGF had clear anti-cholestatic effects, as apparent from the improvement in both bile flow and liver function test. Histology examination revealed a significant reduction of injured areas. HGF also preserved the tight-junctional structure. These anticholestatic effects were associated with the induction of basolateral efflux ABC transporters, which facilitates extrusion of toxic biliary compounds and its further alternative depuration via urine. The biliary epithelium seems to have been also preserved, as suggested by normalization in serum GGT levels, CFTR expression and cholangyocyte primary cilium structure our results clearly show for the first time that HGF protects the liver from a cholestatic injury.Fil: Salas Silva, Soraya. Universidad Autónoma Metropolitana; MéxicoFil: Simoni Nieves, Arturo. Universidad Autónoma Metropolitana; MéxicoFil: Razori, María Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: López Ramirez, Jocelyn. Universidad Autónoma Metropolitana; MéxicoFil: Barrera Chimal, Jonatan. Universidad Nacional Autónoma de México; MéxicoFil: Lazzarini, Roberto. Universidad Autónoma Metropolitana; MéxicoFil: Bello, Oscar. Universidad Autónoma Metropolitana; MéxicoFil: Souza, Verónica. Universidad Autónoma Metropolitana; MéxicoFil: Miranda Labra, Roxana U.. Universidad Autónoma Metropolitana; MéxicoFil: Gutiérrez Ruiz, María Concepción. Universidad Autónoma Metropolitana; MéxicoFil: Gomez Quiroz, Luis Enrique. Universidad Autónoma Metropolitana; MéxicoFil: Roma, Marcelo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Bucio Ortiz, Leticia. Universidad Autónoma Metropolitana; Méxic

The Consumption of Cholesterol-Enriched Diets Conditions the Development of a Subtype of HCC with High Aggressiveness and Poor Prognosis

International audienceNon-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH) result as a consequence of diverse conditions, mainly unbalanced diets. Particularly, high-fat and cholesterol content, as well as carbohydrates, such as those commonly ingested in Western countries, frequently drive adverse metabolic alterations in the liver and promote NAFLD development. Lipid liver overload is also one of the main risk factors for initiation and progression of hepatocellular carcinoma (HCC), but detailed knowledge on the relevance of high nutritional cholesterol remains elusive. We were aimed to characterize HCC development in mice fed with a Western diet (high in lipids and cholesterol) and to identify molecular alterations that define a subtype of liver cancer induced by lipid overload. Mice under western or high cholesterol diets more frequently developed tumors with a more aggressive phenotype than animals fed with a chow diet. Associated changes involved macrophage infiltration, angiogenesis, and stemness features. RNA-seq revealed a specific gene expression signature ( and ) resembling the adverse phenotypic features and poor clinical outcomes seen in patients with HCC. In conclusion; consumption of lipid enriched diets; particularly cholesterol; could accelerate HCC development with an aggressive phenotype and poor prognosis

GDF11 exhibits tumor suppressive properties in hepatocellular carcinoma cells by restricting clonal expansion and invasion

International audienceGrowth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24 h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72 h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5 days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells

GDF11 restricts aberrant lipogenesis and changes in mitochondrial structure and function in human hepatocellular carcinoma cells

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features. Recently, it has been reported that GDF11 exerts tumor‐suppressive properties in hepatocellular carcinoma cells, decreasing clonogenicity, proliferation, spheroid formation, and cellular function, all associated with a decrement in stemness features, resulting in mesenchymal to epithelial transition and loss of aggressiveness. The aim of the present work was to investigate the mechanism associated with the tumor‐suppressive properties displayed by GDF11 in liver cancer cells. Hepatocellular carcinoma‐derived cell lines were exposed to GDF11 (50 ng/ml), RNA‐seq analysis in Huh7 cell line revealed that GDF11 exerted profound transcriptomic impact, which involved regulation of cholesterol metabolic process, steroid metabolic process as well as key signaling pathways, resembling endoplasmic reticulum‐related functions. Cholesterol and triglycerides determination in Huh7 and Hep3B cells treated with GDF11 exhibited a significant decrement in the content of these lipids. The mTOR signaling pathway was downregulated, and this was associated with a reduction in key proteins involved in the mevalonate pathway. In addition, real‐time metabolism assessed by Seahorse technology showed abridged glycolysis as well as glycolytic capacity, closely related to an impaired oxygen consumption rate and decrement in adenosine triphosphate production. Finally, transmission electron microscopy revealed mitochondrial abnormalities, such as cristae disarrangement, consistent with metabolic changes. Results provide evidence that GDF11 impairs cancer cell metabolism targeting lipid homeostasis, glycolysis, and mitochondria function and morphology.This study was partially funded by a grant from the Consejo Nacionalde Ciencia y Tecnología (CONACYT): CB252942, Fronteras de laCiencia1320, Apoyo al Fortalecimiento y Desarrollo de la Infra-estructura 2013205941 and 2017280788, and Universidad Autonoma Metropolitana. We thank the confocal core unit of the Universidad Autonoma Metropolitana Iztapalapa. SH, MGR, ASN arescholarship holders from Conacyt. We acknowledge the support fromgrants: PID2019111669RB, and SAF201785877R from PlanNacional de IþD, Spain, and by the CIBEREHD; the center grantP50AA011999 Southern California Research Center for ALPD andCirrhosis funded by the National Institute on Alcohol Abuse andAlcoholism/National Institutes of Health (NIH); as well as supportfrom AGAUR of the Generalitat de Catalunya SGR20171112,European Cooperation in Science & Technology (COST) ACTIONCA17112 Prospective European DrugInduced Liver Injury Network,and the Fundación BBVA. RED Nacional 2018102799Tdeenfermedades metabólicas y Cáncer y Proyecto 201916/31 DeFundacion Marató TV3Peer reviewe

AXL and error-prone DNA replication confer drug resistance and offer strategies to treat EGFR-mutant lung cancer.

Anti-cancer therapies have been limited by emergence of mutations and other adaptations. In bacteria, antibiotics activate the SOS response, which mobilizes error-prone factors that allow for continuous replication at the cost of mutagenesis. We investigated whether treatment of lung cancer with EGFR inhibitors (EGFRi) similarly engages hypermutators. In cycling drug-tolerant persister (DTP) cells and in EGFRi-treated patients presenting residual disease we observed upregulation of GAS6, while ablation of GAS6's receptor, AXL, eradicated resistance. Reciprocally, AXL overexpression enhanced DTP survival and accelerated the emergence of T790M, an EGFR mutation typical to resistant cells. Mechanistically, AXL induces low-fidelity DNA polymerases and activates their organizer, RAD18, by promoting neddylation. Metabolomics uncovered another hypermutator, AXL-driven activation of MYC and increased purine synthesis that is unbalanced by pyrimidines. Aligning anti-AXL combination treatments with the transition from DTPs to resistant cells cured patient-derived xenografts. Hence, similar to bacteria, tumors tolerate therapy by engaging pharmacologically targetable endogenous mutators