Risk versus Benefit of Tyrosine Kinase Inhibitors for Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

Although the treatment landscape has rapidly evolved over the last years, hepatocellular carcinoma (HCC) is one of the most lethal cancers. With recent advances, both immunotherapy and tyrosine kinase inhibitors (TKIs)-based chemotherapy constitute the standard treatment for advanced HCC. A systematic search of randomized clinical trials employing TKIs was performed in 17 databases, obtaining 25 studies evaluating the prognosis, tumor response, and presence of adverse events (AEs) related to TKIs in HCC. Overall effect sizes were estimated for the hazard ratios (HR) and odds ratios (OR) with 95% confidence interval (CI), either extracted or calculated with the Parmar method, employing STATA 16. Heterogeneity was assessed by Chi-square-based Q-test and inconsistency (I2) statistic; source of heterogeneity by meta-regression and subgroup analysis; and publication bias by funnel plot asymmetry and Egger's test. The research protocol was registered in PROSPERO (CRD42023397263). Meta-analysis revealed a correlation between survival and tumor response parameters and TKI treatment vs. placebo, despite detecting high heterogeneity. Combined TKI treatment showed a significantly better objective response rate (ORR) with no heterogeneity, whereas publication bias was only detected with time to progression (TTP). Few gastrointestinal and neurological disorders were associated with TKI treatment vs. placebo or with combined treatment. However, a higher number of serious AEs were related to TKI treatment vs. sorafenib alone. Results show positive clinical benefits from TKI treatment, supporting the approval and maintenance of TKI-based therapy for advanced HCC, while establishing appropriate strategies to maximize efficacy and minimize toxicity.

Correction: Crespo et al. Protective Effect of Protocatechuic Acid on TNBS-Induced Colitis in Mice Is Associated with Modulation of the SphK/S1P Signaling Pathway. Nutrients 2017, 9, 288.

In the original publication [...].

Neuropilins as potential biomarkers in hepatocellular carcinoma: a systematic review of basic and clinical implications.

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers worldwide and is characterized by complex molecular carcinogenesis. Neuropilins (NRPs) NRP1 and NRP2 are the receptors of multiple proteins involved in key signaling pathways associated with tumor progression. We aimed to systematically review all the available findings on their role in HCC. We searched the Scopus, Web of Science (WOS), PubMed, Cochrane and Embase databases for articles evaluating NRPs in preclinical or clinical HCC models. This study was registered in PROSPERO (CRD42022349774) and include 49 studies. Multiple cellular and molecular processes have been associated with one or both NRPs, indicating that they are potential diagnostic and prognostic biomarkers in HCC patients. Mainly NRP1 has been shown to promote tumor cell survival and progression by modulating several signaling pathways. NRPs mainly regulate angiogenesis, invasion and migration and have shown to induce invasion and metastasis. They also regulate the immune response and tumor microenvironment, showing a crucial interplay with the hypoxia response and microRNAs in HCC. Altogether, NRP1 and NRP2 are potential biomarkers and therapeutic targets, providing novel insight into the clinical landscape of HCC patients.

Hepatocellular carcinoma cells loss lenvatinib efficacy in vitro through autophagy and hypoxia response-derived neuropilin-1 degradation.

Despite pharmacological advances such as lenvatinib approval, therapeutic failure of hepatocellular carcinoma (HCC) remains a big challenge due to the complexity of its underlying molecular mechanisms. Neuropilin-1 (NRP1) is a co-receptor involved in several cellular processes associated to chemoresistance development. Since both the double-edged process of autophagy and hypoxia-derived response play crucial roles in the loss of therapeutic effectiveness, herein we investigated the interplay among NRP1, autophagy and hypoxia in development of lenvatinib resistance in HCC cell lines. We first analyzed NRP1 expression levels in human HCC samples from public databases, found significantly increased NRP1 expression in human HCC samples as well as its correlation with advanced tumor and metastasis stages. Among 3 HCC cell lines (HepG2, Huh-7 and Hep3B), Hep3B and Huh-7 cells showed significantly increased NRP1 expression levels and cell migration ability together with higher susceptibility to lenvatinib. We demonstrated that NRP1 gene silencing significantly enhanced the anticancer effects of lenvatinib on Hep3B and Huh-7 cells. Furthermore, lenvatinib suppressed NRP1 expression through promoting autophagy in Hep3B and Huh-7 cells; co-treatment with bafilomycin A1 attenuated the antitumor effects of lenvatinib, and NRP1 silencing prevented this loss of in vitro effectiveness of lenvatinib even in the presence of bafilomycin A1. In addition, exposure to a hypoxic microenvironment significantly decreased NRP1 expression through autophagy in Hep3B and Huh-7 cells. Under hypoxia, HIF-1α directly modulated NRP1 expression; HIF-1α silencing not only enhanced the anticancer effects of combined lenvatinib and hypoxia, but also prevented the loss of effectiveness caused by bafilomycin A1, highlighting the potential role of HIF-1α-derived hypoxia response in the adaptive cellular response to lenvatinib and promoting resistance acquisition by autophagy modulation. Overall, NRP1 may constitute a potential therapeutic target to prevent lenvatinib failure derived from a hypoxia-associated modulation of autophagy in advanced HCC.

Neuropilin-1 as a Potential Biomarker of Prognosis and Invasive-Related Parameters in Liver and Colorectal Cancer: A Systematic Review and Meta-Analysis of Human Studies.

Neuropilin-1 (NRP1) is a transmembrane protein involved in numerous cellular functions which has had increasing interest from cancer researchers. Liver cancer and colorectal cancer (CRC) are two of the most frequent and deadly tumors with a complex pharmacological framework. Here, we assessed the prognostic, diagnostic and clinicopathological value of NRP1 in liver cancer and CRC patients. We searched PubMed, Scopus, Web of Science, Embase and Cochrane Library databases for articles evaluating the NRP1 correlation with survival parameters, tumor development or clinicopathological features. Hazard ratios and odds ratios with 95% confidence intervals were extracted or estimated by Parmar method and pooled to evaluate the overall effect size with STATA 16 software. Heterogeneity was analyzed by chi-square-based Q test and I2 statistic, along with meta-regression and subgroup analysis, and publication bias was assessed by funnel plot asymmetry and Egger's test. The study protocol was registered in PROSPERO (CRD42022307062). NRP1 overexpression was significantly correlated with lower survival in liver cancer patients and with tumor development in hepatocarcinoma patients, and was strongly correlated with an increased risk of vascular invasion in liver cancer and metastasis in CRC and liver tumors. These results support the role of NRP1 as a potential and useful biomarker in both types of cancer.

Beneficial effects of melatonin on liver fibrosis: A systematic review of current biological evidence.

Hepatic fibrosis is a reversible response to either acute or chronic cellular injury from a wide variety of etiologies, characterized by excessive deposition of extracellular matrix resulting in liver dysfunction and cirrhosis. Melatonin (N-acetyl-5-methoxytryptamine), the main product secreted by the pineal gland, is a multitasking indolamine with important physiological functions such as anti-inflammatory and antioxidant actions, modulation of circadian rhythms, and immune system enhancement. Among the numerous biological activities of melatonin, its antifibrotic effects have received increasingly more attention. In this study, we performed a systematic review of publications of the last 10 years evaluating the mechanisms of action of melatonin against liver fibrosis. The study protocol was registered at PROSPERO (CRD42022304744). Literature research was performed employing PubMed, Scopus, and Web of Science (WOS) databases, and after screening, 29 articles were included. Results from the selected studies provided denoted the useful actions of melatonin on the development, progression, and evolution of liver fibrosis. Melatonin antifibrotic effects in the liver involved the reduction of profibrogenic markers and modulation of several cellular processes and molecular pathways, mainly acting as an antioxidant and anti-inflammatory agent. In addition, the indolamine influenced different molecular processes, such as hepatocyte apoptosis, modulation of autophagy and mitophagy, restoration of circadian rhythms, and modulation of microRNAs, among others. Although some limitations have been found regarding variability in the study design, the findings here summarized display the potential role of melatonin in ameliorating the development of liver fibrosis and its possible progression to liver cirrhosis and hepatocarcinoma.

Melatonin modulates mitophagy, innate immunity and circadian clocks in a model of viral-induced fulminant hepatic failure.

The haemorrhagic disease virus (RHDV) is a non-cultivable virus that promotes in rabbits an acute disease which accomplishes many characteristics of an animal model of fulminant hepatic failure (FHF). Beneficial effects of melatonin have been reported in RHDV-infected rabbits. This study investigated whether protection against viral-derived liver injury by melatonin is associated with modulation of mitophagy, innate immunity and clock signalling. Rabbits were experimentally infected with 2 × 104 haemagglutination units of a RHDV isolate and killed at 18, 24 and 30 hours after infection (hpi). Melatonin (20 mg/kg body weight ip) was administered at 0, 12 and 24 hpi. RHDV infection induced mitophagy, with the presence of a high number of mitophagosomes in hepatocytes and increased expression of mitophagy genes. Greater expression of main innate immune intermediaries and inflammasome components was also found in livers with RHDV-induced FHF. Both mitophagy and innate immunity activation was significantly hindered by melatonin. FHF induction also elicited an early dysregulation in clock signalling, and melatonin was able to prevent such circadian disruption. Our study discloses novel molecular routes contributing to RHDV-induced damage progression and supports the potential of melatonin as a promising therapeutic option in human FHF.

Melatonin Attenuates Dysregulation of the Circadian Clock Pathway in Mice With CCl4-Induced Fibrosis and Human Hepatic Stellate Cells.

Dysregulation of the circadian clock machinery is a critical mechanism in the pathogenesis of fibrosis. This study aimed to investigate whether the antifibrotic effect of melatonin is associated with attenuation of circadian clock pathway disturbances in mice treated with carbon tetrachloride (CCl4) and in human hepatic stellate cells line LX2. Mice received CCl4 5 µL/g body weight i.p. twice a week for 4 or 6 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p., beginning 2 weeks after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of α-smooth muscle actin (α-SMA) positive cells and a significant decrease of peroxisome proliferator-activated receptor (PPARα) expression. CCl4 led to a lower expression of brain and muscle Arnt-like protein 1 (BMAL1), circadian locomotor output cycles kaput (CLOCK), period 1-3 (PER1, 2, and 3), cryptochrome 1 and 2 (CRY1 and 2) and the retinoic acid receptor-related orphan receptor (RORα). The expression of the nuclear receptor REV-ERBα showed a significant increase. Melatonin significantly prevented all these changes. We also found that melatonin (100 or 500 µM) potentiated the inhibitory effect of REV-ERB ligand SR9009 on α-SMA and collagen1 expression and increased the expression of PPARα in LX2 cells. Analysis of circadian clock machinery revealed that melatonin or SR9009 exposure upregulated BMAL1, CLOCK, PER2, CRY1, and RORα expression, with a higher effect of combined treatment. Findings from this study give new insight into molecular pathways accounting for the protective effect of melatonin in liver fibrosis.

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine-induced hepatocellular carcinoma.

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long-term diseases including hepatocellular carcinoma (HCC). Melatonin has been reported to alleviate promotion and progression of HCC, but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg-1 d-1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK1ε, Rev-erbα, and Rev-erbß decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT1 receptor and reduced levels of the hypoxia-inducible factors Hif-1α and Hif-2α. An increased expression of p21, p53, and PARP1/2, a higher Bax/Bcl-2 ratio, and a lower expression of Cyclin D1, CDK6, HSP70, HSP90, and GRP78 proteins were also observed in melatonin-treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV-ERB agonist SR9009 in human Hep3B cells, and BMAL1 knocking down attenuated the pro-apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.

Effects Of Oral Glutamine on Inflammatory and Autophagy Responses in Cancer Patients Treated With Abdominal Radiotherapy: A Pilot Randomized Trial.

Background and Aims: Abdominal radiotherapy (RT) causes harm to the mid gastrointestinal mucosa by release of pro-inflammatory cytokines and promotes autophagic changes in tumor cells. This study was aimed to measure the effect of glutamine administration on markers of inflammation and autophagy in cancer patients treated with RT. Methods: In this double-blind, randomized, controlled pilot trial 43 patients under abdominal RT diagnosed of pelvic or abdominal malignancies receiving glutamine (30 g/d) or placebo (casein, 30 g/d). Patient recruitment took place in the Complejo Asistencial Universitario of León (CAULE), Spain. Patient evaluation took place at three different time points during the study: before RT (pre-treatment), in the middle of the RT period (mid-treatment), and after finishing RT (post-treatment). Data were compared by analysis of variance and the Newmann Keuls test. Significance was accepted at p < 0.05. Results Abdominal RT increased whole blood mRNA levels of inflammatory and autophagic markers, but glutamine administration showed significantly lower expression of toll-like receptor 4 (TLR4), CD36, interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-9 (MMP-9). Moreover, glutamine reduced the expression of the transcription factors nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1). Glutamine also inhibited the autophagic response, with changes in expression of beclin-1, UV radiation resistance associated gene (UVRAG), autophagy-related protein-5 (Atg5), protein 1 light chain 3 (LC3), sequestosome 1 (p62/SQSTM1) and lysosome-associated membrane protein (LAMP)-1. Conclusions Findings provide evidence that glutamine decreases the inflammatory response and abolishes the changes of the autophagy machinery in patients receiving abdominal RT. The protective effect of glutamine must continue being investigated to disclose further molecular pathways.

Protective Effect of Protocatechuic Acid on TNBS-Induced Colitis in Mice Is Associated with Modulation of the SphK/S1P Signaling Pathway.

(1) Background: The present study aimed to investigate whether beneficial effects of protocatechuic acid (PCA) are associated with inhibition of the SphK/S1P axis and related signaling pathways in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) model of inflammatory bowel disease; (2) Methods: Colitis was induced in male Balb/c mice by intracolonic administration of 2 mg of TNBS. PCA (30 or 60 mg/kg body wt) was given intraperitoneally daily for five days; (3) Results: Administration of PCA prevented the macroscopic and microscopic damage to the colonic mucosa, the decrease in body weight gain and the increase in myeloperoxidase activity induced by TNBS. PCA-treated mice exhibited a lower oxidized/reduced glutathione ratio, increased expression of antioxidant enzymes and Nrf2 and reduced expression of proinflammatory cytokines. Following TNBS treatment mRNA levels, protein concentration and immunohistochemical labelling for SphK1 increased significantly. S1P production and expression of S1P receptor 1 and S1P phosphatase 2 were significantly elevated. However, there was a decreased expression of S1P lyase. Furthermore, TNBS-treated mice exhibited increased phosphorylation of AKT and ERK, and a higher expression of pSTAT3 and the NF-κB p65 subunit. PCA administration significantly prevented those changes; (4) Conclusions: Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the anti-inflammatory effect of PCA.

Melatonin prevents deregulation of the sphingosine kinase/sphingosine 1-phosphate signaling pathway in a mouse model of diethylnitrosamine-induced hepatocellular carcinoma.

The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway is involved in multiple biological processes, including carcinogenesis. Melatonin shows beneficial effects in cell and animal models of hepatocellular carcinoma, but it is unknown if they are associated with the modulation of the SphK/S1P system, along with different downstream signaling pathways modified in cancer. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight i.p) once a week for 8 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p. beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Melatonin alleviated the distortion of normal hepatic architecture, lowered the incidence of preneoplastic/neoplastic lesions, and inhibited the expression of proliferative/cell cycle regulatory proteins (Ki67, PCNA, cyclin D1, cyclin E, CDK4, and CDK6). S1P levels and expression of SphK1, SphK2, and S1P receptors (S1PR1/S1PR3) were significantly elevated in DEN-treated mice. However, there was a decreased expression of S1P lyase. These effects were significantly abrogated in a time- and dose-dependent manner by melatonin, which also increased S1PR2 expression. Following DEN treatment, mice exhibited increased phosphorylation of PI3K, AKT, mTOR, STAT3, ERK, and p38, and a higher expression of NF-κB p50 and p65 subunits. Melatonin administration significantly inhibited those changes. Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the protective effects of melatonin in hepatocarcinogenesis.

Inhibition of the SphK1/S1P signaling pathway by melatonin in mice with liver fibrosis and human hepatic stellate cells.

The sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) system is involved in different pathological processes, including fibrogenesis. Melatonin abrogates activation of hepatic stellate cells (HSCs) and attenuates different profibrogenic pathways in animal models of fibrosis, but it is unknown if protection associates with its inhibitory effect on the SphK1/S1P axis. Mice in treatment groups received carbon tetrachloride (CCl4 ) 5 µL g-1 body wt i.p. twice a week for 4 or 6 weeks. Melatonin was given at 5 or 10 mg kg-1  day-1 i.p, beginning 2 weeks after the start of CCl4 administration. At both 4 and 6 weeks following CCl4 treatment, liver mRNA levels, protein concentration and immunohistochemical labelling for SphK1 increased significantly. S1P production, and expression of S1P receptor (S1PR)1, S1PR3 and acid sphingomyelinase (ASMase) were significantly elevated. However, there was a decreased expression of S1PR2 and S1P lyase (S1PL). Melatonin attenuated liver fibrosis, as shown by a significant inhibition of the expression of α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-ß and collagen (Col) Ι. Furthermore, melatonin inhibited S1P production, lowered expression of SphK1, S1PR1, SP1R3, and ASMase, and increased expression of S1PL. Melatonin induced a reversal of activated human HSCs cell line LX2, as evidenced by a reduction in α-SMA, TGF-ß, and Col I expression. Melatonin-treated cells also exhibited an inhibition of the SphK1/S1P axis. Antifibrogenic effect of SphK1 inhibition was confirmed by treatment of LX2 cells with PF543. Abrogation of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in liver fibrogenesis. © 2016 BioFactors, 43(2):272-282, 2017.

Melatonin inhibits the sphingosine kinase 1/sphingosine-1-phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin.

The sphingosine kinase (SphK)1/sphingosine-1-phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV-infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll-like receptor (TLR)4, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, nuclear factor-kappa B (NF-κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.

Melatonin inhibits autophagy and endoplasmic reticulum stress in mice with carbon tetrachloride-induced fibrosis.

This study aimed to investigate whether inhibition of autophagy and endoplasmic reticulum (ER stress) associates with the antifibrogenic effect of melatonin in mice treated with carbon tetrachloride (CCl4 ). Mice received CCl4 5 µL/g body wt i.p. twice a week for 4 wk or 6 wk. Melatonin was given at 5 or 10 mg/kg/day i.p, beginning 2 wk after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of α-smooth muscle actin (α-SMA)-positive cells. CCl4 induced an autophagic response measured as the presence of autophagic vesicles, protein 1 light chain 3 (LC3) staining, conversion of LC3-I to autophagosome-associated LC3-II, changes in expression of beclin-1, UV radiation resistance-associated gene (UVRAG), ubiquitin-like autophagy-related (Atg5), Atg12, Atg16L1, sequestosome 1 (p62/SQSTM1), and lysosome-associated membrane protein (LAMP)-2, and increased phosphorylation of the mammalian target of rapamycin (mTOR). There was an increase in the expression of the ER stress chaperones CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin-heavy-chain-binding protein (BiP/GRP78), and 94-kDa glucose-regulated protein (GRP94), and in the mRNA levels of pancreatic ER kinase (PERK), activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1 (IRE1), and spliced X-box-binding protein-1 (XBP1). Phospho-IRE1, ATF6, and phospho-PERK protein concentration also increased significantly. Immunohistochemical staining of α-SMA indicated an abrogation of hepatic stellate cells activation by melatonin. Furthermore, treatment with the indole resulted in significant inhibition of the autophagic flux and the unfolded protein response. Findings from this study give new insight into molecular pathways accounting for the protective effect of melatonin in fibrogenesis.

Melatonin limits the expression of profibrogenic genes and ameliorates the progression of hepatic fibrosis in mice.

We investigated whether melatonin ameliorates fibrosis and limits the expression of fibrogenic genes in mice treated with carbon tetrachloride (CCl4). Mice in treatment groups received CCl4 5 µL/g body weight intraperitoneally twice a week for 4 or 6 weeks. Melatonin was given at 5 or 10 mg/kg/d intraperitoneally, beginning 2 weeks after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of Van Gieson and α-smooth muscle actin (α-SMA) positive cells in the liver. At both 4 and 6 weeks, CCl4 induced an increase in the messenger RNA levels of collagens I and III, transforming growth factor (TGF)-ß, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), amphiregulin, matrix metalloproteinase (MMP)-9, and tissue inhibitor of metalloproteinase (TIMP)-1. Protein concentrations of CTGF, amphiregulin, MMP-9, TIMP-1, and phospho-Smad3 were also significantly augmented in fibrotic mice. Melatonin successfully attenuated liver injury, as shown by histopathology and decreased levels of serum transaminases. Immunohistochemical staining of α-SMA indicated an abrogation of hepatic stellate cell activation by the indol. Furthermore, melatonin treatment resulted in significant inhibition of the expression of collagens I and III, TGF-ß, PDGF, CTGF, amphiregulin, and phospho-Smad3. The MMP-9 activity decreased and the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) increased in mice receiving melatonin. Data obtained suggest that attenuation of multiple profibrogenic gene pathways contributes to the beneficial effects of melatonin in mice with CCl4-induced liver fibrosis.

Autophagic response in the Rabbit Hemorrhagic Disease, an animal model of virally-induced fulminant hepatic failure.

The Rabbit Hemorrhagic Disease Virus (RHDV) induces a severe disease that fulfils many requirements of an animal model of fulminant hepatic failure. However, a better knowledge of molecular mechanisms contributing to liver damage is required, and it is unknown whether the RHDV induces liver autophagy and how it relates to apoptosis. In this study, we attempted to explore which signalling pathways were involved in the autophagic response induced by the RHDV and to characterize their role in the context of RHDV pathogenesis. Rabbits were infected with 2 × 104 hemmaglutination units of a RHDV isolate. The autophagic response was measured as presence of autophagic vesicles, LC3 staining, conversion of LC3-I to autophagosome-associated LC3-II and changes in expression of beclin-1, UVRAG, Atg5, Atg12, Atg16L1 and p62/SQSTM1. RHDV-triggered autophagy reached a maximum at 24 hours post-infection (hpi) and declined at 30 and 36 hpi. Phosphorylation of mTOR also augmented in early periods of infection and there was an increase in the expression of the endoplasmic reticulum chaperones BiP/GRP78, CHOP and GRP94. Apoptosis, measured as caspase-3 activity and expression of PARP-1, increased significantly at 30 and 36 hpi in parallel to the maximal expression of the RHDV capsid protein VP60. These data indicate that RHDV infection initiates a rapid autophagic response, perhaps in an attempt to protect liver, which associates to ER stress development and is independent from downregulation of the major autophagy suppressor mTOR. As the infection continues and the autophagic response declines, cells begin to exhibit apoptosis.

Melatonin modulates the autophagic response in acute liver failure induced by the rabbit hemorrhagic disease virus.

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin-1, ubiquitin-like autophagy-related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3-I to autophagosome-associated LC3-II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome-associated membrane protein (LAMP)-1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase-3 activity, and immunoglobulin-heavy-chain-binding protein (BiP) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.

Melatonin treatment reduces endoplasmic reticulum stress and modulates the unfolded protein response in rabbits with lethal fulminant hepatitis of viral origin.

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress and unfolded protein response (UPR) inhibition is an underlying mechanism of melatonin anti-apoptotic effects in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 mg/kg and 20 mg/kg at 0 hr, 12 hr and 24 hr postinfection. RHDV infection induced increased expression of CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin heavy chain binding protein (BiP/GRP78), glucose-regulated protein 94 (GRP94), phospho-c-Jun N-terminal kinase (JNK) and caspase-12. These effects were attenuated by melatonin. Double immunofluorescence staining showed colocalization of CHOP and cleaved caspase-3 in liver sections of RHDV-infected rabbits, while immunostaining decreased markedly with melatonin treatment. RHDV infection resulted in significant increases in the mRNA levels of activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1 (IRE1), spliced X-box binding protein-1 (XBP1s) and tumor necrosis factor receptor-associated factor 2 (TRAF2). Melatonin attenuated the extent of the changes. Data obtained provide evidence that in rabbits with experimental infection by RHDV, reduction in apoptotic liver damage by melatonin is associated with attenuation of ER stress through a modulation of the three arms of UPR signaling and further support a potential hepatoprotective role of melatonin in FHF.

Glutamine treatment attenuates endoplasmic reticulum stress and apoptosis in TNBS-induced colitis.

Endoplasmic reticulum (ER) stress and apoptotic cell death play an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). We aimed to explore the potential of glutamine to reduce ER stress and apoptosis in a rat model of experimental IBD. Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/dL) was given by rectal route daily for 2 d or 7 d. Both oxidative stress (TBARS concentration and oxidised/reduced glutathione ratio) and ER stress markers (CHOP, BiP, calpain-1 and caspase-12 expression) increased significantly within 48 h of TNBS instillation, and glutamine attenuated the extent of the changes. Glutamine also inhibited the significant increases of ATF6, ATF4 and spliced XBP-1 mRNA levels induced by TNBS instillation. TNBS-colitis resulted in a significant increase in p53 and cytochrome c expression, and a reduced Bcl-xL expression and Bax/Bcl-2 ratio. These effects were significantly inhibited by glutamine. Treatment with the amino acid also resulted in significant decreases of caspase-9, caspase-8 and caspase-3 activities. Double immunofluorescence staining showed co-localization of CHOP and cleaved caspase-3 in colon sections. Phospho-JNK and PARP-1 expression was also significantly higher in TNBS-treated rats, and treatment with glutamine significantly decreased JNK phosphorylation and PARP-1 proteolysis. To directly address the effect of glutamine on ER stress and apoptosis in epithelial cells, the ER stress inducers brefeldin A and tunicamycin were added to Caco-2 cells that were treated with glutamine (5 mM and 10 mM). The significant enhancement in PERK, ATF6 phosphorylated IRE1, BiP and cleaved caspase-3 expression induced by brefeldin A and tunicamycin was partly prevented by glutamine. Data obtained indicated that modulation of ER stress signalling and anti-apoptotic effects contribute to protection by glutamine against damage in TNBS-induced colitis.