F. Jorquera

Melatonin induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cell line

Abstract:  Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in the treatment of hepatocarcinoma are limited. In this study, we examined the effect of melatonin administration on HepG2 human hepatocarcinoma cells, analyzing cell cycle arrest, apoptosis and mitogen‐activated protein kinase (MAPK) signalling pathways. Melatonin was dissolved in the cell culture media in 0.2% dimethyl sulfoxide and administered at different concentrations for 2, 4, 6, 8 and 10 days. Melatonin at concentrations 1000–10,000 μm caused a dose‐ and time‐dependent reduction in cell number. Furthermore, melatonin treatment induced apoptosis with increased caspase‐3 activity and poly(ADP‐ribose) polymerase proteolysis. Proapoptotic effects of melatonin were related to cytosolic cytochrome c release, upregulation of Bax and induction of caspase‐9 activity. Melatonin treatment also resulted in increased caspase‐8 activity, although no significant change was observed in Fas‐L expression. In addition, JNK 1,‐2 and ‐3 and p38, members of the MAPK family, were upregulated by melatonin treatment. Growth inhibition by melatonin altered the percentage or cells in G0–G1 and G2/M phases indicating cell cycle arrest in the G2/M phase. The reduced cell proliferation and alterations of cell cycle were coincident with a significant increase in the expression of p53 and p21 proteins. These novel findings show that melatonin, by inducing cell death and cell cycle arrest, might be useful as adjuvant in hepatocarcinoma therapy.

Malignant gastrointestinal obstruction: endoscopic stenting versus surgical palliation.

BackgroundMalignant gastrointestinal obstruction is a secondary complication of cancers in an advanced state. Treatment has consisted of gastrojejunostomy. However, the endoscopic placement of metallic stents has provided positive results. This study aimed to compare the efficiency of both therapeutic options.MethodsA total of 41 patients with gastrointestinal obstruction caused by inoperable neoplasm were treated endoscopically with enteral stent (24 patients) or gastrojejunostomy (17 patients).ResultsIn the endoscopic group (EG) 24 patients (100%) achieved efficient gastric emptying, as compared with 82.3% in the surgical group (SG). The difference was not significant. The average time for initiating oral food tolerance was 2.4 days for the EG and 5 days for the SG (p < 0.001). The average inpatient time was 7.1 days for the EG and 11.5 days for the SG (p < 0.001). Mortality at 30 days was lower in the EG (16.6%) than in the SG (29.4%) (p < 0.05). The survival time was 20 weeks for the EG and 21.6 weeks for the SG. The difference was not significant. The rate of complications was 4% in the (EG) and 17.6% in the (SG), with the difference was not significant.ConclusionEndoscopic treatment of malignant gastrointestinal obstruction provides an adequate palliation of the symptoms. It is less invasive, avoids the morbidity associated with open gastrojejunostomy, and achieves a faster start to oral food and a shorter hospital stay, leading to a higher quality of life.

Melatonin attenuates apoptotic liver damage in fulminant hepatic failure induced by the rabbit hemorrhagic disease virus.

Abstract:  Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate the antiapoptotic effect of melatonin in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 104 hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 and 20 mg/kg at 0, 12, and 24 hr postinfection. RHDV infection induced liver apoptosis, with increased caspase‐3 immunoexpression and activity and poly(ADP‐ribose)polymerase‐1 (PARP‐1) proteolysis. These effects were attenuated by melatonin in a concentration‐dependent manner. Antia‐poptotic effects of melatonin were related to a reduced expression of Bax and cytosolic cytochrome c release, increased expression of Bcl‐2 and Bcl‐xL, and inhibition of caspase‐9 activity. Increased thiobarbituric reactive acid substances concentration and oxidized‐to‐reduced glutathione ratio were significantly prevented by melatonin administration. Melatonin treatment also resulted in a reduction in caspase‐8 activity, tumor necrosis factor receptor‐1 (TNF‐R1) expression, and phosphorylated Janus kinase (JNK) expression, and increased expression of cellular FLICE‐inhibitory protein (c‐FLIP). Our findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by RHDV and supports a potential hepatoprotective role of melatonin in FHF.

Palliative treatment of malignant obstruction of gastric outlet using an endoscopically placed enteral wallstent

Surgical gastrojejunostomy is the standard treatment for malignant gastric outlet obstruction, although it is associated with significant morbidity and mortality. The aim of this study was to evaluate the efficacy and feasibility of a newly designed expandable metal stent (Wallstent Enteral) to treat malignant gastric outlet obstruction. Six patients (five women, one man; mean age 76 years) underwent stenting. Stents 20–22 mm in diameter and 60–90 mm in length were deployed through a duodenoscope channel under endoscopic and fluoroscopic control, without previous stricture dilation. In all six cases the stent was adequately positioned and food intake was possible in the next 24 h. The mean time for hospital discharge was 2.5 days (1–5 days), without complications related to the procedure. Five patients died in the follow-up from progression of their cancer and one remains alive; none had recurrent obstruction. The median survival time was 9 weeks (95% CI: 3–15 weeks). In conclusion, endoscopic self-expandable stent (Wallstent Enteral) placement is safe and effective palliation for malignant gastric outlet obstruction and appears to be a therapeutic alternative to surgical gastrojejunostomy.

Liver blood flow changes during laparoscopic surgery in pigs. A study of hepatic indocyanine green removal.

AbstractBackground: Physiological effects caused by abdominal insufflation in the course of laparoscopic surgery are partially unknown. The purpose of the present study was to determine if indocyanine green (ICG) pharmacokinetic parameters, as an index of hepatic blood flow, change during laparoscopic surgery in the presence of a CO2 pneumoperitoneum. This effect could cause important alterations in the kinetics of anesthetic drugs. Methods: Eighteen female pigs were anaesthetized under constant ventilation and randomly assigned to three groups undergoing insufflation with CO2 (I), laparoscopic oophorectomy with CO2 pneumoperitoneum (LS), or oophorectomy by open surgery (OS). CO2 pneumoperitoneum was performed at 14 mmHg. ICG (1 mg/kg) was injected into a marginal vein on two separate occasions: 30 min before and 30 min after the start of insufflation or surgery. Blood was sampled from the carotid artery at time intervals after the injection of ICG and after pharmacokinetic parameters were obtained by a computer program. Results: The area under the curve (AUC0–∞) indicated important disfunctions in ICG availability in all three groups of animals, with significant increases of 104%, 82%, and 48% for groups I, LS, and OS, respectively. The ICG apparent half-life did not significantly change in group OS, but it rose in groups I (+17%) and LS (+28%). ICG clearance was significantly reduced by 32% in group OS and to a larger extent in groups I and LS (−45% and −46%, respectively). Conclusion: These findings confirm the contribution of CO2 pneumoperitoneum to decreased liver blood flow during laparoscopic surgery.

Effectiveness, safety and clinical outcomes of direct-acting antiviral therapy in HCV genotype 1 infection: results from a Spanish real world cohort.

BACKGROUND & AIMS Clinical trials evaluating second-generation direct-acting antiviral agents (DAAs) have shown excellent rates of sustained virologic response (SVR) and good safety profiles in patients with chronic hepatitis C virus (HCV) genotype 1 infection. We aimed to investigate the effectiveness and safety of two oral DAA combination regimens, ombitasvir/paritaprevir/ritonavir plus dasabuvir (OMV/PTV/r+DSV) and ledipasvir/sofosbuvir (LDV/SOF), in a real-world clinical practice. METHODS Data from HCV genotype 1 patients treated with either OMV/PTV/r+DSV±ribavirin (RBV) (n=1567) or LDV/SOF±RBV (n=1758) in 35 centers across Spain between April 1, 2015 and February 28, 2016 were recorded in a large national database. Demographic, clinical and virological data were analyzed. Details of serious adverse events (SAEs) were recorded. RESULTS The two cohorts were not matched with respect to baseline characteristics and could not be compared directly. The SVR12 rate was 96.8% with OMV/PTVr/DSV±RBV and 95.8% with LDV/SOF±RBV. No significant differences were observed in SVR according to HCV subgenotype (p=0.321 [OMV/PTV/r+DSV±RBV] and p=0.174 [LDV/SOF]) or degree of fibrosis (c0.548 [OMV/PTV/r/DSV±RBV] and p=0.085 [LDV/SOF]). Only baseline albumin level was significantly associated with failure to achieve SVR (p<0.05) on multivariate analysis. Rates of SAEs and SAE-associated treatment discontinuation were 5.4% and 1.7%, in the OMV/PTV/r+DSV subcohort and 5.5% and 1.5% in the LDV/SOF subcohort, respectively. Hepatocellular carcinoma (HCC) recurred in 30% of patients with a complete response to therapy for previous HCC. Incident HCC was reported in 0.93%. CONCLUSIONS In this large cohort of patients managed in the real-world setting in Spain, OMV/PTV/r+DSV and LDV/SOF achieved high rates of SVR12, comparable to those observed in randomized controlled trials, with similarly good safety profiles. LAY SUMMARY In clinical trials, second-generation direct-acting antiviral agents (DAAs) have been shown to cure over 90% of patients chronically infected with the genotype 1 hepatitis C virus and have been better tolerated than previous treatment regimens. However, patients enrolled in clinical trials do not reflect the real patient population encountered in routine practice. The current study, which includes almost 4,000 patients, demonstrates comparable rates of cure with two increasingly used DAA combinations as those observed in the clinical trial environment, confirming that clinical trial findings with DAAs translate into the real-world setting, where patient populations are more diverse and complex.

Liver X receptor α-mediated regulation of lipogenesis by core and NS5A proteins contributes to HCV-induced liver steatosis and HCV replication.

Molecular mechanisms contributing to hepatitis C virus (HCV)-associated steatosis are not well established, although HCV gene expression has been shown to alter host cell cholesterol/lipid metabolism. As liver X receptors (LXRs) play a role as key modulators of metabolism signaling in the development of steatosis, we aimed to investigate in an HCV in vitro model the effect of HCV NS5A protein, core protein, and viral replication on the intracellular lipid accumulation and the LXRα-regulated expression of lipogenic genes. The effects of LXRα siRNA or agonist GW3965 treatment on lipogenesis and HCV replication capacity in our HCV replicon system were also examined. NS5A- and core-expressing cells and replicon-containing cells exhibited an increase of lipid accumulation by inducing the gene expression and the transcriptional activity of LXRα, and leading to an increased expression of its lipogenic target genes sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor-γ, and fatty acid synthase. Transcriptional induction by NS5A protein, core protein, and viral replication occurred via LXR response element activation in the lipogenic gene promoter. No physical association between HCV proteins and LXRα was observed, whereas NS5A and core proteins indirectly upregulated LXRα through the phosphatidylinositol 3-kinase pathway. Finally, it was found that LXRα knockdown or agonist-mediated LXRα induction directly regulated HCV-induced lipogenesis and HCV replication efficiency in replicon-containing cells. Combined, our data suggest that LXRα-mediated regulation of lipogenesis by core and NS5A proteins may contribute to HCV-induced liver steatosis and to the efficient replication of HCV.

Effects of parenteral nutrition supplemented with glutamine or glutamine dipeptides on liver antioxidant and detoxication systems in rats.

Our aim was to determine the effects of glutamine or alanyl glutamine parenteral supplementation on the liver oxidant/antioxidant balance and on cytochrome-P450-mediated detoxication in rats. Animals were infused for 5 d with standard total parenteral nutrition (TPN), glutamine-enriched TPN, or alanyl glutamine-enriched TPN. The hepatic concentration of glutathione was reduced, and the levels of thiobarbituric-acid-reactive substances (TBARS) were increased in animals receiving standard TPN. Both glutamine and alanyl glutamine supplementation normalized glutathione, but thiobarbituric-acid-reactive substance concentration was only decreased by ananyl glutamine. This effect was parallel to a partial recovery of the activity of antioxidant enzymes. Cytochrome-P450 liver content, cytochrome-P450-dependent monooxygenases, and antipyrine clearance were not modified by glutamine or alanyl glutamine. Our data suggest a better protection against free radicals by alanyl glutamine supplementation and an absence of effects of both glutamine and alanyl glutamine on liver oxidative metabolism.

Usefulness of combined measurement of serum bile acids and ferritin as additional prognostic markers to predict failure to reach sustained response to antiviral treatment in chronic hepatitis C

Aim: To investigate the relationship between serum levels of ferritin and bile acids (BA) and the response to antiviral treatment in chronic hepatitis C (HCV).

Modulation of PI3K-LXRα-dependent lipogenesis mediated by oxidative/nitrosative stress contributes to inhibition of HCV replication by quercetin.

There is experimental evidence that some antioxidant flavonoids show therapeutic potential in the treatment of hepatitis C through inhibition of hepatitis C virus (HCV) replication. We examined the effect of treatment with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin on HCV replication efficiency in an in vitro model. While all flavonoids studied were able to reduce viral replication at very low concentrations (ranging from 0.1 to 5 μM), quercetin appeared to be the most effective inhibitor of HCV replication, showing a marked anti-HCV activity in replicon-containing cells when combined with interferon (IFN)α. The contribution of oxidative/nitrosative stress and lipogenesis modulation to inhibition of HCV replication by quercetin was also examined. As expected, quercetin decreased HCV-induced reactive oxygen and nitrogen species (ROS/RNS) generation and lipoperoxidation in replicating cells. Quercetin also inhibited liver X receptor (LXR)α-induced lipid accumulation in LXRα-overexpressing and replicon-containing Huh7 cells. The mechanism underlying the LXRα-dependent lipogenesis modulatory effect of quercetin in HCV-replicating cells seems to involve phosphatidylinositol 3-kinase (PI3K)/AKT pathway inactivation. Thus, inhibition of the PI3K pathway by LY294002 attenuated LXRα upregulation and HCV replication mediated by lipid accumulation, showing an additive effect when combined with quercetin. Inactivation of the PI3K pathway by quercetin may contribute to the repression of LXRα-dependent lipogenesis and to the inhibition of viral replication induced by the flavonol. Combined, our data suggest that oxidative/nitrosative stress blockage and subsequent modulation of PI3K-LXRα-mediated lipogenesis might contribute to the inhibitory effect of quercetin on HCV replication.