ABSTRACT
Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO) synthase type III (NOS-3) overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP) and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP) and Rous Sarcoma Virus (RSV) promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.
Subject(s)
Adenoviridae , Carcinoma, Hepatocellular/therapy , Genetic Therapy , Liver Neoplasms, Experimental/therapy , Nitric Oxide Synthase Type III , Animals , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms, Experimental/enzymology , Liver Neoplasms, Experimental/genetics , Mice , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nitric Oxide Synthase Type III/biosynthesis , Nitric Oxide Synthase Type III/geneticsABSTRACT
Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins regulates many processes in the eukaryotic cell. This reaction is similar to ubiquitination and usually requires an E3 ligase for substrate modification. However, only a few SUMO ligases have been described so far, which frequently facilitate sumoylation by bringing together the SUMO-conjugating enzyme Ubc9 and the target protein. Ubc9 is an interaction partner of the transcription factor Krox20, a key regulator of hindbrain development. Here, we show that Krox20 functions as a SUMO ligase for its coregulators--the Nab proteins--and that Nab sumoylation negatively modulates Krox20 transcriptional activity in vivo.
