title: S-adenosyl-L-methionine modifies antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells.
abstract: OBJECTIVE
  To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression.
  
  
  METHODS
  We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent.
  
  
  RESULTS
  SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the transcriptional and translational level.
  
  
  CONCLUSIONS
  A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: Sonia Amelia
authors.initials: SA
authors.lastname: Lozano-Sepulveda
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Bautista-Osorio
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: Jose Angel
authors.initials: JA
authors.lastname: Merino-Mascorro
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: Marta
authors.initials: M
authors.lastname: Varela-Rey
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: Linda Elsa
authors.initials: LE
authors.lastname: Muñoz-Espinosa
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: Paula
authors.initials: P
authors.lastname: Cordero-Perez
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: María Luz
authors.initials: ML
authors.lastname: Martinez-Chantar
authors.affiliation: Sonia Amelia Lozano-Sepulveda, Eduardo Bautista-Osorio, Jose Angel Merino-Mascorro, Ana Maria Rivas-Estilla, Department of Biochemistry and Molecular Medicine, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Nuevo Leon 64460, Mexico.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Rivas-Estilla
mesh_terms: D000975:Antioxidants / Q000378:metabolism*; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005978:Glutathione / Q000096:biosynthesis*; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D006526:Hepatitis C / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D054884:Host-Pathogen Interactions; D006801:Humans; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D012367:RNA, Viral / Q000096:biosynthesis; D012436:S-Adenosylmethionine / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000975:Antioxidants; D000998:Antiviral Agents; D012367:RNA, Viral; D012436:S-Adenosylmethionine; C506577:MAT1A protein, human; C527161:MAT2A protein, human; D008716:Methionine Adenosyltransferase; D005978:Glutathione
keywords: Antioxidants; Hepatitis C virus; Hepatitis C virus-RNA; NS5A; Oxidative stress; Pyrrolidine dithiocarbamate; Reactive oxygen species; Replication; S-adenosyl-L-methionine; Superoxide dismutase 1; Superoxide dismutase 2; Viral proteins