Redox Regulation Of Metabolic And Signaling Pathways By Thioredoxin And Glutaredoxin In Nitric Oxide Treated Hepatoblastoma Cells.
NO has an antiproliferative action on HepG2 cells and Thioredoxin (Trx) and Glutaredoxin (Grx) have denitrosilase and deglutathionylase activities.



To ascertain whether Trx and/or Grx systems intermediate the anti-proliferative effect of NO on hepatoblastoma cells by modulating the redox-state of key proteins.



HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) were transfected with specific siRNA to silence Trx1 and Grx1. The expression and thiolic redox state of proteins were determined by Western blot and redox mobility shift assay.



Overexpression of NOS3 increased the levels and activities of proteins of the redoxin systems, Trx1, Grx1, TrxR1 and TxnIP, and the levels of signaling proteins (Akt1, pAkt1-Ser473, MapK, pMapK, Stat3, Fas). The thiolic redox state of Trx1, Grx1 and Akt1 shifted to more oxidized. Increases were also observed in Pro-apoptotic Caspase-3 fragment levels; caspase 3, 8 and 9 activities; antiapoptotic (Bcl-2); mitochondrial energetic (Aco2) and heme (Urod) metabolism; Glycolysis (Pkm2); and pentose phosphate pathway (Tkt). However, two cytosolic proteins related to iron (Aco1) and one carbon (Mat2) metabolism decreased markedly. Moreover, the redox state of Urod and Aco1 shifted to more oxidized cysteines. Trx1 or Grx1 silencing augmented Tyr nitration and diminished cell proliferation in WT cells, but attenuated the antiproliferative effect on NO, the increase of Fas, Akt1 and pAkt1-Ser473 and the oxidative modification of Akt1 in NOS3 cells.



Trx1 and Grx1 exert contradictory influences on HepG2 cells. They are required for proliferation but they also contribute to antiproliferative effect of NO, associated to Akt1 redox changes.
Department of Biochemistry and Molecular Biology, IMIBIC, University of Córdoba, Córdoba, Spain.
C Alicia Padilla
CA
Peña
Department of Biochemistry and Molecular Biology, IMIBIC, University of Córdoba, Córdoba, Spain.
Raúl
R
González
Department of Biochemistry and Molecular Biology, IMIBIC, University of Córdoba, Córdoba, Spain.
María J
MJ
López-Grueso
Department of Biochemistry and Molecular Biology, IMIBIC, University of Córdoba, Córdoba, Spain.
J
J
Antonio Bárcena
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D054477:Glutaredoxins / Q000378:metabolism*; D018197:Hepatoblastoma / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000473:pathology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D013879:Thioredoxins / Q000378:metabolism*
D002352:Carrier Proteins; C516005:GLRX protein, human; D054477:Glutaredoxins; C491203:TXN protein, human; C486715:TXNIP protein, human; D013879:Thioredoxins; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt