Nitric Oxide Exposure of CC531 Rat Colon Carcinoma Cells Induces γ-glutamyltransferase which May Counteract Glutathione Depletion and Cell Death

Abstract

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.

Associations Between Total Serum Ggt Activity and Metabolic Risk: MESA

“…GGT and its relationship to redox balance in vivo has been thoroughly summarized by Whitfield [15], and several specific research findings provide a convincing rationale that increased GGT activity represents increased oxidative stress, including: GSH depletion appears to be a prerequisite condition to induce GGT [16];NADPH oxidase-produced reactive oxygen species and reactive nitrogen species both induce GGT expression [17,18];Mitochondria of GGT -knockout mice have depleted GSH, increased reactive oxygen species formation, depleted energy stores and impaired oxidative phosphorylation (thus impaired ATP production), which can be attenuated by N -acetylcysteine, a GSH precursor [19]; GGT -knockout mice die prematurely with complications associated with increased oxidative stress that can be attenuated with N -acetylcysteine [20]. …”

“…NADPH oxidase-produced reactive oxygen species and reactive nitrogen species both induce GGT expression [17,18];…”

Effect of Arsenite on Nitrosative Stress in Human Breast Cancer Cells and Its Modulation by Flavonoids

“…This may be the case of ZR-75-1 cells treated with S and Q, depending on GSH availability. Statistical association found between GSH-related and NO-related pathways might be because NO induces GGT to restore GSH and to prevent nitrosative death, as a feedback response (24). Thus, RNS/RSS balance might be decisive for antioxidant and cytoprotective effects of NO and GSH, respectively, with their reactive derivatives possibly interacting each other forming S-nitroso-metabolites and reciprocally cancelling their oxidative potential.…”

Up-regulation of γ-glutamyl transpeptidase activity following glutathione depletion has a compensatory rather than an inhibitory effect on mitochondrial complex I activity: implications for Parkinson's disease

“…Previous studies with nitric oxide (NO) donors have demonstrated that the activity of the GGT enzyme in colon carcinoma cells is induced after exposure to NO and that the increase in GGT helped maintain intracellular GSH levels in cells grown in cystine-depleted medium by increasing substrate available for uptake into the cells [21]. Increased GGT activity thus acted to protect these cells against nitrosative stress.…”