Oleocanthal Exerts Antitumor Effects on Human Liver and Colon Cancer Cells Through ROS Generation

The beneficial health properties of the Mediterranean diet are well recognized. The principle source of fat in Mediterranean diet is extra-virgin olive oil (EVOO). Oleocanthal (OC) is a naturally occurring minor phenolic compound isolated from EVOO, which has shown a potent anti-inflammatory activity, by means of its ability to inhibit the cyclooxygenase (COX) enzymes COX-1 and COX-2. A large body of evidence indicates that phenols exhibit anticancer activities. The aim of the present study was to evaluate the potential anticancer effects of OC in hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) models. A panel of human HCC (HepG2, Huh7, Hep3B and PLC/PRF/5) and CRC (HT29, SW480) cell lines was used. Cells were treated with OC, and cell viability and apoptosis were evaluated. Compared with classical commercially available COX inhibitors (ibuprofen, indomethacin, nimesulide), OC was more effective in inducing cell growth inhibition in HCC and CRC cells. Moreover, OC inhibited colony for mation and i nduced ap optosis, as confirmed by PARP cleavage, activation of caspases 3/7 and chromatin condensation. OC treatment in a dose dependent-manner induced expression of \uce\ub3H2AX, a marker of DNA damage, increased intracellular ROS production and caused mitochondrial depolarization. Moreover, the effects of OC were suppressed by the ROS scavenger N-acetyl-L-cysteine. Finally, OC was not toxic in primary normal human hepatocytes. In conclusion, OC treatment was found to exert a potent anticancer activity against HCC and CRC cells. Taken together, our findings provide preclinical support of the chemotherapeutic potential of EVOO against cancer

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective. Keywords: CRISPR; FRGN; ex vivo; genome editing; hepatocyte transplantation; liver-humanized mouse; primary hepatocytes; urea cycle disorder

Special Section on Pediatric Drug Disposition and Pharmacokinetics Role of Chromatin Structural Changes in Regulating Human CYP3A Ontogeny s

ABSTRACT Variability in drug-metabolizing enzyme developmental trajectories contributes to interindividual differences in susceptibility to chemical toxicity and adverse drug reactions, particularly in the first years of life. Factors linked to these interindividual differences are largely unknown, but molecular mechanisms regulating ontogeny are likely involved. To evaluate chromatin structure dynamics as a likely contributing mechanism, age-dependent changes in modified and variant histone occupancy were evaluated within known CYP3A4 and 3A7 regulatory domains. Chromatin immunoprecipitation using fetal or postnatal human hepatocyte chromatin pools followed by quantitative polymerase chain reaction DNA amplification was used to determine relative chromatin occupancy by modified and variant histones. Chromatin structure representing a poised transcriptional state (bivalent chromatin), indicated by the occupancy by modified histones associated with both active and repressed transcription, was observed for CYP3A4 and most 3A7 regulatory regions in both postnatal and fetal livers. However, the CYP3A4 regulatory regions had significantly greater occupancy by modified histones associated with repressed transcription in the fetal liver. Conversely, some modified histones associated with active transcription exhibited greater occupancy in the postnatal liver. CYP3A7 regulatory regions also had significantly greater occupancy by modified histones associated with repressed transcription in the fetus. The observed occupancy by modified histones is consistent with chromatin structural dynamics contributing to CYP3A4 ontogeny, although the data are less conclusive regarding CYP3A7. Interpretation of the latter data may be confounded by celltype heterogeneity in the fetal liver

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

Over the last decade, pioneering liver-directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno-associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver-directed gene therapy. AAV-derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large-scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk-benefit analysis for each indication, especially in paediatrics

Translation of amnion stem cells to the clinic

Cellular therapy for liver disease has been available in the clinic for more than 20 years, yet remarkably few patients receive this experimental therapy. Reasons for the small number of transplants performed are partially related to access to useful liver tissue and the difficulty with the isolation of viable cells. Stem cell sources of hepatocytes could theoretically relieve these obstacles to therapy if large numbers of functional hepatocytes could be generated and transplanted without risk of tumorigenicity. To date, there are no reports of stem cell sources with all of these characteristics, despite claims otherwise. Here we report the results of preclinical studies with appropriate animals models of metabolic liver disease and acute liver failure, and their correction by the transplantation of human amnion epithelial stem cells. The encouraging results of the preclinical studies have motivated the movement of isolation and banking of these cells to good manufacturing practice conditions so that the cells can be used in the clinic for transplantation of patients with liver disease

Melatonin expresses powerful anti-inflammatory and antioxidant activities resulting in complete improvement of acetic-acid-induced colitis in rats

Introduction: Increased free-radical production, decreased antioxidant capacity, and excessive inflammation are well-known features in the pathogenesis of inflammatory bowel disease. Melatonin is a powerful antioxidant and a scavenger of hydroxyl radicals. Melatonin has also been shown to have anti-inflammatory activities in tissues. Our study objective is to investigate the effects of melatonin on tissue inflammatory activities using an ulcerative colitis (UC) model induced by acetic acid (AA) in rats. Methods: Wistar rats (n = 32) were divided into four groups. AA-induced colitis was performed in two of the groups, while the other two groups were injected with saline intrarectally. One of the AA-induced colitis groups and one of the control groups were administered 100 mg/kg/day melatonin intraperitoneally, and the pair groups were given saline. After 4 days, colonic changes were evaluated biochemically by measuring proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6], myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) levels in tissue homogenates and by histopathological examination. Results: AA caused colonic mucosal injury, whereas melatonin suppressed these changes in the AA-induced colitis group (P < 0.001). AA administration resulted in increased TNF-α, IL-1β, IL-6, MPO, and MDA levels, and decreased GSH and SOD levels, whereas melatonin administration reversed these effects (all P < 0.001). Conclusions: The present study proposes that melatonin has a dual action as an effective anti-inflammatory and an antioxidant, and may be a hopeful therapeutic agent for UC

The history and use of human hepatocytes for the treatment of liver diseases: the first 100 patients

Orthotopic liver transplantation remains the only curative treatment for many end-stage liver diseases, yet the number of patients receiving liver transplants remains limited by the number of organs available for transplant. There is a need for alternative therapies for liver diseases. The transplantation of isolated hepatocytes (liver cells) has been used as an experimental therapy for liver disease in a limited number of cases. Recently, the 100(th) case of hepatocyte transplantation was reported. This review discusses the history of the hepatocyte transplant field, the major discoveries that supported and enabled the first hepatocyte transplants, and reviews the cases and outcomes of the first 100 clinical transplants. Some of the problems that limit the application or efficacy of hepatocyte transplantation are discussed, as are possible solutions to these problems. In conclusion, hepatocyte transplants have proven effective particularly in cases of metabolic liver disease where reversal or amelioration of the characteristic symptoms of the disease is easily quantified. However, no patients have been completely corrected of a metabolic liver disease for a significant amount of time by hepatocyte transplantation alone. It is likely that future developments in new sources of cells for transplantation will be required before this cellular therapy can be fully implemented and available for large numbers of patients. © 2014 by John Wiley & Sons, Inc