Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches.

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

Watching Subtitled Films Can Help Learning Foreign Languages.

Watching English-spoken films with subtitles is becoming increasingly popular throughout the world. One reason for this trend is the assumption that perceptual learning of the sounds of a foreign language, English, will improve perception skills in non-English speakers. Yet, solid proof for this is scarce. In order to test the potential learning effects derived from watching subtitled media, a group of intermediate Spanish students of English as a foreign language watched a 1h-long episode of a TV drama in its original English version, with English, Spanish or no subtitles overlaid. Before and after the viewing, participants took a listening and vocabulary test to evaluate their speech perception and vocabulary acquisition in English, plus a final plot comprehension test. The results of the listening skills tests revealed that after watching the English subtitled version, participants improved these skills significantly more than after watching the Spanish subtitled or no-subtitles versions. The vocabulary test showed no reliable differences between subtitled conditions. Finally, as one could expect, plot comprehension was best under native, Spanish subtitles. These learning effects with just 1 hour exposure might have major implications with longer exposure times.

Splicing regulator SLU7 preserves survival of hepatocellular carcinoma cells and other solid tumors via oncogenic miR-17-92 cluster expression.

Resisting death is a central hallmark of cancer cells. Tumors rely on a number of genetic mechanisms to avoid apoptosis, and alterations in mRNA alternative splicing are increasingly recognized to have a role in tumorigenesis. In this study, we identify the splicing regulator SLU7 as an essential factor for the preservation of hepatocellular carcinoma (HCC) cells viability. Compared with hepatocytes, SLU7 expression is reduced in HCC cells; however, further SLU7 depletion triggered autophagy-related cellular apoptosis in association with the overproduction of reactive oxygen species. Remarkably, these responses were not observed in primary human hepatocytes or in the well-differentiated HepaRG cell line. Mechanistically, we demonstrate that SLU7 binds the C13orf25 primary transcript in which the polycistronic oncomir miR-17-92 cluster is encompassed, and is necessary for its processing and expression. SLU7 knockdown altered the splicing of the C13orf25 primary transcript, and markedly reduced the expression of its miR-17, miR-20 and miR-92a constituents. This led to the upregulation of CDKN1A (P21) and BCL2L11 (BIM) expression, two bona fide targets of the miR-17-92 cluster and recognized mediators of its pro-survival and tumorigenic activity. Interestingly, altered splicing of miR-17-92 and downregulation of miR-17 and miR-20 were not observed upon SLU7 knockdown in non-transformed hepatocytes, but was found in other (HeLa, H358) but not in all (Caco2) non-hepatic tumor cells. The functional relevance of miR-17-92 dysregulation upon SLU7 knockdown was established when oxidative stress, autophagy and apoptosis were reversed by co-transfection of HCC cells with a miR-17 mimic. Together, these findings indicate that SLU7 is co-opted by HCC cells and other tumor cell types to maintain survival, and identify this splicing regulator as a new determinant for the expression of the oncogenic miR-17-92 cluster. This novel mechanism may be exploited for the development of antitumoral strategies in cancers displaying such SLU7-miR-17-92 crosstalk.

Antitumoral gene-based strategy involving nitric oxide synthase type III overexpression in hepatocellular carcinoma.

Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO) synthase type III (NOS-3) overexpression induces cell death in hepatoblastoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. The first-generation adenoviruses were designed to overexpress NOS-3 or green fluorescent protein, and luciferase complementary DNA under the regulation of murine alpha-fetoprotein (AFP) and Rous Sarcoma Virus (RSV) promoters, respectively. Both adenovirus and Hepa 1-6 cells were used for in vitro and in vivo experiments. Adenoviruses were administered through the tail vein 2 weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8, -9 and -3 activities in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by Nω-nitro-l-arginine methyl ester hydrochloride, p53 and CD95 small interfering RNA. AFP-NOS-3/RSV-luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

An Experimental Study on the Use of Calcium Alginate to Heal Colonic Anastomoses.

BACKGROUND: Anastomotic leak is considered the major complication following abdominal surgery. In recent years, the use of a variety of sealing materials for the prevention of leaks has been analyzed. Different biomaterials have been employed as scaffolds to favour tissue repair and regeneration. Among these materials we must mention alginate, a natural polymer with different applications as temporary supporting matrix. The aim of the present study is to evaluate the behavior of both alginate-impregnated sutures and lyophilized alginate sponges in the healing process of colonic anastomes using an experimental animal model. MATERIAL AND METHODS: A preliminary study was undertaken to select the adequate scaffold. Animals (n = 45) were distributed into three groups: control (colonic anastomosis using non-continuous 5-0 Polyglactin 910 suture), suture (colonic anastomosis using suture impregnated with alginate gel at 4%) and sponge (colonic anastomosis using suture reinforced with lyophilized alginate sponge). The macroscopic and histological variables were assessed at 4, 8 and 12 days after surgical intervention. RESULTS: No statistically significant differences have been observed between the groups during the analysis of macroscopic variables. Animals with sponge implantation showed a greater degree of epithelial reepithalization, less acute and chronic inflammation and greater collagen deposit. CONCLUSIONS: The use of lyophilized alginate sponges to reinforce colonic anastomoses in an animal model reduces inflammation and promotes the earlier formation of greater collagen deposits without increasing the number of adhesions or the incidence of stenosis.

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

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.

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.

The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.

Protein tyrosine phosphatase 1B modulates GSK3ß/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity.

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAP-induced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B(-/-) hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)ß/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B(-/-) cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B(-/-) mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAP-induced liver failure.

Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression.

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 ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). 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.

Effects of nitric oxide synthase-3 overexpression on post-translational modifications and cell survival in HepG2 cells.

Hepatocarcinoma is the fifth most common neoplasm and the third cause of cancer-related death. The development of genetic- and/or molecular-based therapies is urgently required. The administration of high doses of nitric oxide (NO) promotes cell death in hepatocytes. NO contributes to cell signaling by inducing oxidative/nitrosative-dependent post-translational modifications. The aim of the present study was to investigate protein modifications and its relation with alteration of cell proliferation and death in hepatoma cells. Increased intracellular NO production was achieved by stable nitric oxide synthase-3 (NOS-3) overexpression in HepG2 cells. We assessed the pattern of nitration, nitrosylation and carbonylation of proteins by proteomic analysis. The results showed that NOS-3 cell overexpression increased oxidative stress, which affected proteins mainly involved in cell protein folding. Carbonylation also altered metabolism, as well as immune and antioxidant responses. The interaction of nitrosative and oxidative stress generated tyrosine nitration, which affected the tumor marker Serpin B3, ATP synthesis and cytoskeleton. All these effects were associated with a decrease in chaperone activity, a reduction in cell proliferation and an increased cell death. Our study showed that alteration of nitration, nitrosylation and carbonylation pattern of proteins by NO-dependent oxidative/nitrosative stress was related to a reduction of cell survival in a hepatoma cell line.

Expression of p53 during apoptosis induced by D-galactosamine and the protective role of PGE1 in cultured rat hepatocytes.

p53 is a critical player in the prevention of tumor development. It can contribute directly to DNA repair and inhibition of angiogenesis and subsequently to the induction of apoptosis. The regulation of p53 expression is mediated by the transcription factor NF-kappaB. This includes regulation of p53 protein stability, control of its subcellular localization and conformational changes that allow activation of the DNA binding activity of p53. Rat hepatocytes were isolated from male Wistar rats following collagenase perfusion of liver. We examined the change in the expression level of p53 by western blotting in hepatocytes and its effect on apoptosis as a response of treatment with D-galactosamine, prostaglandin E1 and/or the Proteosome Inhibitor (PSI). A kinetic study of the extracellular lactate dehydrogenase activity, NF-kappaB activation, induced nitric oxide synthase expression and nitric oxide production was carried out in hepatocytes. The addition of prostaglandin E1 to control and D-galactosamine-treated hepatocytes increased p53 expression in the cytoplasm during 24 h. While the addition of PSI in the absence of prostaglandin E1 decreased p53 expression at 5 mM D-galactosamine. This inhibition is reversed in the presence of prostaglandin E1 at 5 and 40 mM D-galactosamine. The protective action of prostaglandin E1 against the apoptotic effect of D-galactosamine is mediated by NF-kappaB activation, induced nitric oxide synthase and p53 expression.

Estrategias de optimización del trasplante hepático / No disponible

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The role of hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) in the treatment of peritoneal carcinomatosis in recurrent ovarian cancer

BACKGROUND AND OBJECTIVES: Peritoneal carcinomatosis in women frequently has an ovarian origin. Hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) along with radical surgery/peritonectomy could present a new therapeutic approach with curative intention. The purpose of this research is to evaluate the role of the administration of HIPEC. METHODS: A series of patients (N=26) diagnosed with peritoneal carcinomatosis for recurrent epithelial ovarian cancer (stage III) from January 1997 to December 2004 submitted to radical surgery/peritonectomy with optimal cytoreduction (R0-R1) were included in this study, 14 treated with HIPEC and 12 without HIPEC. RESULTS: The variables age, histologic type, peritonectomy procedures, peritoneal cancer index (PCI) and lymph node affectation were similar in both groups. The 5-year global survival was 58% and 17% (p=0.046), and 67% and 29% in patients with maximal cytoreduction (R0) (p=0.264), in the HIPEC- and non-HIPEC-treated patients, respectively. In patients with optimal cytoreduction and partial peritonectomy, 5-year global survival was also superior in the HIPEC group (75% vs. 11%, p=0.011). Average time free of disease was superior in the HIPEC group (48+/-42 vs. 24+/-21 months), with less reinterventions due to a new reappearance during the first three evolutionary years (2/14 vs. 4/12). Postoperative morbidity did not show substantial differences in both groups and there was no surgical mortality. CONCLUSIONS: HIPEC is a complement to radical surgery/ peritonectomy, which has been shown to be a surgical procedure with high tolerability, low morbimortality, enhanced survival and prolonged disease-free interval in patients with peritoneal carcinomatosis for recurrent ovarian cancer (AU)

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Effect of melatonin on myocardial oxidative stress induced by experimental obstructive jaundice.

OBJECTIVE: melatonin has been demonstrated to have active antioxidant properties in different tissues during experimental cholestasis. The aim of this research was to study myocardial oxidative stress on obstructive jaundice, and to analyze the effect of melatonin on myocardial oxidative lesions. MATERIAL AND METHODS: we achieved cholestasis by ligature and sectioning of the main bile duct. Melatonin was administered intraperitoneally (500 microg/kg/day). We measured malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxydase (GPx) antioxidant enzyme levels in the heart tissue. RESULTS: obstructive cholestasis increased MDA and decreased GSH as well as all antioxidant enzymes. Melatonin administration significantly decreased MDA values, and increased GSH and antioxidant enzymes on the icteric animal myocardium. CONCLUSIONS: melatonin treatment prevents oxidative stress in the cardiac tissue as induced by experimental cholestasis.

Alteration of the renal regulatory hormonal pattern during experimental obstructive jaundice.

OBJECTIVE: The alteration of hormones regulating sodium and water status is related to renal failure in obstructive jaundice (OJ). EXPERIMENTAL DESIGN: OJ was induced by common bile duct ligation. Samples were obtained from the control (SO) and OJ groups at 24 and 72 hours, and at 7 days. Different parameters related to biliary obstruction, liver and renal injury, and vasoactive mediators such as renin, aldosterone, endothelin-1 (ET-1) and prostaglandin E2 (PGE2) were studied. RESULTS: Bile duct ligation caused an increase in total bilirubin (p < 0.001) and alkaline phosphatase (AP) (p < 0.001). The SO and OJ groups had the same values for diuresis, renin, and creatinine clearance at 24 h. However, animals with OJ had a lower sodium concentration in urine than SO animals (p < 0.01), as well as an increase in aldosterone levels (p < 0.03). ANP levels were moderately increased during OJ but did not reach statistical significance when compared to the SO group. In contrast, OJ animals showed a rise in serum ET-1 concentration (p < 0.001) and increased PGE2 in urine (p < 0.001). CONCLUSIONS: Biliary obstruction induced an increase in ET-1 release and PGE2 urine excretion. These hormones might play a role during the renal complications associated with renal disturbances that occur during OJ.

Effect of melatonin on myocardial oxidative stress induced by experimental obstructive jaundice

Objective: melatonin has been demonstrated to have activeantioxidant properties in different tissues during experimentalcholestasis. The aim of this research was to study myocardial oxidativestress on obstructive jaundice, and to analyze the effect ofmelatonin on myocardial oxidative lesions.Material and methods: we achieved cholestasis by ligatureand sectioning of the main bile duct. Melatonin was administeredintraperitoneally (500 ìg/kg/day). We measured malondialdehyde(MDA), reduced glutathione (GSH), catalase (CAT), superoxidedismutase (SOD) and glutathione peroxydase (GPx) antioxidantenzyme levels in the heart tissue.Results: obstructive cholestasis increased MDA and decreasedGSH as well as all antioxidant enzymes. Melatonin administrationsignificantly decreased MDA values, and increased GSH and antioxidantenzymes on the icteric animal myocardium.Conclusions: melatonin treatment prevents oxidative stressin the cardiac tissue as induced by experimental cholestasis(AU)

Alteration of the renal regulatory hormonal pattern during experimental obstructive jaundice

Objective: the alteration of hormones regulating sodium andwater status is related to renal failure in obstructive jaundice (OJ).Experimental design: OJ was induced by common bile ductligation. Samples were obtained from the control (SO) and OJgroups at 24 and 72 hours, and at 7 days. Different parametersrelated to biliary obstruction, liver and renal injury, and vasoactivemediators such as renin, aldosterone, endothelin-1 (ET-1) andprostaglandin E2 (PGE2) were studied.Results: bile duct ligation caused an increase in total bilirubin(p < 0.001) and alkaline phosphatase (AP) (p < 0.001). The SOand OJ groups had the same values for diuresis, renin, and creatinineclearance at 24 h. However, animals with OJ had a lowersodium concentration in urine than SO animals (p < 0.01), as wellas an increase in aldosterone levels (p < 0.03). ANP levels weremoderately increased during OJ but did not reach statistical significancewhen compared to the SO group. In contrast, OJ animalsshowed a rise in serum ET-1 concentration (p < 0.001) and increasedPGE2 in urine (p < 0.001).Conclusions: biliary obstruction induced an increase in ET-1release and PGE2 urine excretion. These hormones might play arole during the renal complications associated with renal disturbancesthat occur during OJ(AU)