Glutathione species and metabolomic prints in subjects with liver disease as biological markers for the detection of hepatocellular carcinoma.

BACKGROUND: The incidence of liver disease is increasing in USA. Animal models had shown glutathione species in plasma reflects liver glutathione state and it could be a surrogate for the detection of hepatocellular carcinoma (HCC). METHODS: The present study aimed to translate methods to the human and to explore the role of glutathione/metabolic prints in the progression of liver dysfunction and in the detection of HCC. Treated plasma from healthy subjects (n = 20), patients with liver disease (ESLD, n = 99) and patients after transplantation (LTx, n = 7) were analyzed by GC- or LC/MS. Glutathione labeling profile was measured by isotopomer analyzes of 2H2O enriched plasma. Principal Component Analyzes (PCA) were used to determined metabolic prints. RESULTS: There was a significant difference in glutathione/metabolic profiles from patients with ESLD vs healthy subjects and patients after LTx. Similar significant differences were noted on patients with ESLD when stratified by the MELD score. PCA analyses showed myristic acid, citric acid, succinic acid, l-methionine, d-threitol, fumaric acid, pipecolic acid, isoleucine, hydroxy-butyrate and glycolic, steraric and hexanoic acids were discriminative metabolites for ESLD-HCC+ vs ESLD-HCC- subject status. CONCLUSIONS: Glutathione species and metabolic prints defined liver disease severity and may serve as surrogate for the detection of HCC in patients with established cirrhosis.

PIAS3 expression in squamous cell lung cancer is low and predicts overall survival.

Unlike lung adenocarcinoma, little progress has been made in the treatment of squamous cell lung carcinoma (SCC). The Cancer Genome Atlas (TCGA) has recently reported that receptor tyrosine kinase signaling pathways are altered in 26% of SCC tumors, validating the importance of downstream Signal Transducers and Activators of Transcription 3 (STAT3) activity as a prime therapeutic target in this cancer. In the present report we examine the status of an endogenous inhibitor of STAT3, called Protein Inhibitor of Activated STAT3 (PIAS3), in SCC and its potential role in this disease. We examine PIAS3 expression in SCC tumors and cell lines by immunohistochemistry of a tissue microarray and western blotting. PIAS3 mRNA expression and survival data are analyzed in the TCGA data set. SCC cell lines are treated with curcumin to regulate PIAS3 expression and cell growth. PIAS3 protein expression is decreased in a majority of lung SCC tumors and cell lines. Analysis of PIAS3 mRNA transcript levels demonstrated that low PIAS3 levels predicted poor survival; Cox regression analysis revealed a hazard ratio of 0.57 (95% CI: 0.37-0.87), indicating a decrease in the risk of death by 43% for every unit elevation in PIAS3 gene expression. Curcumin treatment increased endogenous PIAS3 expression and decreased cell growth and viability in Calu-1 cells, a model of SCC. Our results implicate PIAS3 loss in the pathology of lung SCC and raise the therapeutic possibility of upregulating PIAS3 expression as a single target that can suppress signaling from the multiple receptor tyrosine kinase receptors found to be amplified in SCC.

Polyethylene glycol modified-albumin enhances the cold preservation properties of University of Wisconsin solution in rat liver and a hepatocyte cell line.

Liver grafts preserved in cold storage undergo changes mainly manifested by morphological modifications of the sinusoidal endothelium that result in poor graft function upon reperfusion. The present studies aimed to determine if the addition of polyethylene glycol-albumin to University of Wisconsin (Peg-AlbUW) solution ameliorates the cold preservation injuries of liver grafts. Rat livers were preserved cold with various preservation solutions and evaluated for weight changes and endothelial morphology. Solutions that preserved graft weight and endothelial morphology were tested in the isolated perfused rat liver model to assess graft function. A rat hepatocyte cell line was evaluated for both viability and glutathione concentrations emulating cold preservation and reperfusion conditions. Liver grafts preserved with Peg-AlbUW maintained their initial weight and showed a conserved endothelial morphology compared with liver grafts preserved in UW for 30 h (P<0.05). Liver grafts preserved with Peg-AlbUW had improved portal blood flow and bile secretion compared with liver grafts preserved in UW for 30 h (P<0.05). In vitro we noted comparable hepatocyte viability when cells were preserved in Peg-AlbUW versus UW under similar preservation conditions (P>0.05); glutathione concentrations (reduced and total) were significantly increased in hepatocytes preserved in 3% Peg-AlbUW compared with other preservation solutions (P<0.05). The addition of Peg-Alb to UW preservation solution ameliorated the cold preservation injuries of rat liver grafts as shown by stable liver graft weight, a better preservation of the endothelial morphology, improved portal vein blood flow, and increased bile secretion. Peg-Alb-UW solution improved the integrity of the glutathione redox buffer system of a hepatocyte cell line after cold storage and reperfusion.

Dynamics of glutathione and ophthalmate traced with 2H-enriched body water in rats and humans.

We developed a LC-MS-MS assay of the (2)H labeling of free glutathione (GSH) and bound glutathione [GSSR; which includes all DTT-reducible forms, primarily glutathione disulfide (GSSG) and mixed disulfides with proteins] and ophthalmate (an index of GSH depletion) labeled from (2)H-enriched body water. In rats whose body water was 2.5% (2)H enriched for up to 31 days, GSH labeling follows a complex pattern because of different rates of labeling of its constitutive amino acids. In rats infused with [(13)C(2),(15)N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 micromol.min(-1).kg(-1), and the half-life of plasma GSH/GSSR was 6-8 min. In healthy humans whose body fluids were 0.5% (2)H enriched, the (2)H labeling of GSH/GSSR and ophthalmate can be precisely measured after 4 h, with GSH being more rapidly labeled than GSSR. Since plasma GSH/GSSR derives mostly from liver, this technique opens the way to 2) probe noninvasively the labeling pattern and redox status of the liver GSH system in humans and 2) assess the usefulness of ophthalmate as an index of GSH depletion.