The aspartate metabolism pathway is differentiable in human hepatocellular carcinoma: transcriptomics and (13) C-isotope based metabolomics.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than a year following diagnosis. Although bioinformatic analyses have indicated differentially expressed genes and cancer related mutations in HCC, integrated genetic and metabolic pathway analyses remain to be investigated. Herein, gene (i.e. messenger RNA, mRNA) enrichment analysis was performed to delineate significant alterations of metabolic pathways in HCC. The objective of this study was to investigate the pathway of aspartate metabolism in HCC of humans. Coupled with transcriptomic (i.e. mRNA) and NMR based metabolomics of human tissue extracts, we utilized liquid chromatography mass spectrometry based metabolomics analysis of stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine metabolism of HCC cell culture. Our results indicated that aspartate metabolism is a significant and differentiable metabolic pathway of HCC compared with non-tumor liver (p value < 0.0001). In addition, branched-chain amino acid metabolism (p value < 0.0001) and tricarboxylic acid metabolism (p value < 0.0001) are significant and differentiable. Statistical analysis of measurable NMR metabolites indicated that at least two of the group means were significantly different for the metabolites alanine (p value = 0.0013), succinate (p value = 0.0001), lactate (p value = 0.0114), glycerophosphoethanolamine (p value = 0.015), and inorganic phosphate (p value = 0.0001). However, (13) C isotopic enrichment analysis of these metabolites revealed less than 50% isotopic enrichment with either stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine. This may indicate the differential account of total metabolite pool versus de novo metabolites from a (13) C labeled substrate. The ultimate translation of these findings will be to determine putative enzyme activity via (13) C labeling, to investigate targeted therapeutics against these enzymes, and to optimize the in vivo performance of (13) C MRI techniques.

In vivo MRSI of hyperpolarized [1-(13)C]pyruvate metabolism in rat hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than 1 year following diagnosis. Most patients with HCC are diagnosed at an advanced stage, and no efficient marker exists for the prediction of prognosis and/or response(s) to therapy. We have reported previously a high level of [1-(13)C]alanine in an orthotopic HCC using single-voxel hyperpolarized [1-(13)C]pyruvate MRS. In the present study, we implemented a three-dimensional MRSI sequence to investigate this potential hallmark of cellular metabolism in rat livers bearing HCC (n = 7 buffalo rats). In addition, quantitative real-time polymerase chain reaction was used to determine the mRNA levels of lactate dehydrogenase A, nicotinamide adenine (phosphate) dinucleotide dehydrogenase quinone 1 and alanine transaminase. The enzyme levels were significantly higher in tumor than in normal liver tissues within each rat, and were associated with the in vivo MRSI signal of [1-(13)C]alanine and [1-(13)C]lactate after a bolus intravenous injection of [1-(13)C]pyruvate. Histopathological analysis of these tumors confirmed the successful growth of HCC as a nodule in buffalo rat livers, revealing malignancy and hypervascular architecture. More importantly, the results demonstrated that the metabolic fate of [1-(13)C]pyruvate conversion to [1-(13)C]alanine significantly superseded that of [1-(13)C]pyruvate conversion to [1-(13)C]lactate, potentially serving as a marker of HCC tumors.

In vitro design and characterization of the nonviral gene delivery vector iopamidol, protamine, ethiodized oil reagent.

PURPOSE: To demonstrate cellular selectivity toward hepatoma cells and compare the efficiency of gene delivery of a novel nonviral vector of iopamidol, protamine, and ethiodized oil reagents (VIPER). MATERIALS AND METHODS: Rat hepatocellular carcinoma (HCC) cells were transfected in triplicate under varying conditions by using firefly luciferase as a reporter gene. Conditions included variations of a protamine:DNA (P:D) complex (20:1, 50:1, 100:1, 200:1 mass ratios), iopamidol (0%, 10%, 33%), and ethiodized oil (0%, 1%, 2%, 4%, 8%, and 16%). The conditions affording efficient gene transfer and ease of translation to in vivo studies were selected for cell line comparison (HCC cells vs hepatocytes). Adenoviral transduction was compared with nonviral vector transfection. RESULTS: At low concentrations, ethiodized oil increased transfection efficiency regardless of P:D mass ratio. However, high concentrations resulted in significant attenuation. Unexpectedly, the addition of iopamidol to P:D complexes markedly improved transfection efficiency. When using an optimal P:D, iopamidol, and ethiodized oil solution, DNA transfection of normal liver and tumor cells showed significant selectivity for tumor cells. In the context of hepatoma cells, transfection efficiency with the nonviral vector was better than 10(4) pfu adenovirus. CONCLUSIONS: The development and characterization of the VIPER system provides a possible alternative to viral gene therapy of HCC.

Epstein-Barr virus latent membrane protein 1 activates nuclear factor-kappa B in human endothelial cells and inhibits apoptosis.

BACKGROUND: Although vascular changes and transplant vasculopathy have been described with cytomegalovirus, the impact of Epstein-Barr virus (EBV) on the vascular endothelium of the transplanted allograft is largely unknown. We recently reported that EBV (+) patients taken off immunosuppressive medications for periods of time had a low incidence of chronic rejection. In another report, we noted that there was expression of the "protective" antiapoptotic factor Bcl-2 in the vascular endothelium of transplant allografts from EBV (+) patients. In this report, we determined the effect of latent EBV infection on endothelial cell activation and apoptosis. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) were either infected with EBV or transduced with EBV latent membrane protein 1 and examined for apoptosis, nuclear factor (NF)-kappaB activation, and expression of chemokines, cytokines, and adhesion molecules. RESULTS: EBV infection and latent membrane protein 1 expression in HUVEC resulted in NF-kappaB activation and increased expression of the cytokines interleukin (IL)-1alpha, IL-1beta, and IL-6; the chemokines IL-8, monocyte chemotactic protein-1, and RANTES; and the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. There was increased expression of the antiapoptotic genes A1, c-IAP2, and TRAF1; inhibition of caspase 3; and protection from apoptosis. CONCLUSIONS: Latent EBV in HUVEC results in constitutive NF-kappaB activation, protection from apoptosis, and increased basal expression of inflammatory factors. The in vivo effect of latent EBV in the vascular endothelium of the transplanted allograft and its resultant impact on transplant vasculopathy are the subject of further investigations in our laboratory.