Nitric oxide exposure of CC531 rat colon carcinoma cells induces gamma-glutamyltransferase which may counteract glutathione depletion and cell death.
Gamma-glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 5S-radiolabeled GSH. The enzyme was also induced by interferon-gamma and tumor necrosis factor-alpha, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.
Huseby
Nils-Erik
NE
Department of Medical Biochemistry, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway. nilseh@fagmed.uit.no
Asare
Nana
N
Wetting
Silje
S
Mikkelsen
Idun Merete
IM
Mortensen
Bente
B
Sveinbjørnsson
Baldur
B
Wellman
Maria
M
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D003545:Cysteine / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009589:Nitrogen Oxides; D018384:Oxidative Stress; D051381:Rats; D011994:Recombinant Proteins; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis*
D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D007371:Interferon-gamma; D005723:gamma-Glutamyltransferase; D005978:Glutathione; D003545:Cysteine