Redox modulation of NF-kappaB nuclear translocation and DNA binding in metastatic melanoma. The role of endogenous and gamma-glutamyl transferase-dependent oxidative stress.

Abstract

The transcription factor NF-kappaB is implicated in the expression of genes involved in cell proliferation, apoptosis and metastasis. In melanoma, high constitutive levels of NF-kappaB activation are usually observed. NF-kappaB is regulated by oxidation/reduction (redox) processes, and the occurrence of constitutive oxidative stress in melanoma cells has been documented. Recent studies of our laboratories showed that the membrane-bound gamma-glutamyl transferase (GGT) enzyme activity--expressed by a number of malignancies, including melanoma--can act as a basal source of superoxide, hydrogen peroxide and other prooxidants. In the present study we utilized the 2/60 clone of Me665/2 human metastatic melanoma, which displays high levels of GGT activity, in order to verify if the presence of this enzyme--through the promotion of redox processes--may influence the activation status of NF-kappaB. The latter was evaluated by determining the nuclear translocation of the p65 subunit (by immunoblot), the DNA binding of NF-kappaB (by electrophoretic mobility shift assay) and its transcriptional activity (by gene transactivation studies). Me665/2/60 cells displayed a basal production of hydrogen peroxide. Stimulation of GGT activity by its substrates glutathione and glycyl-glycine caused additional production of hydrogen peroxide, up to levels approx. double the basal levels. Nuclear translocation of the NF-kappaB p65 subunit, DNA-binding and gene transactivation were thus investigated in Me665/2/60 cells whose GGT activity was modulated by means of substrates or inhibitors. Stimulation of GGT activity resulted in increased nuclear translocation of p65, while on the other hand NF-kappaB DNA binding and gene transactivation were paradoxically decreased. NF-kappaB DNA binding could be restored by treating cell lysates with the thiol-reducing agent dithiothreitol (DTT). Treatment of cells with exogenous hydrogen peroxide did not affect NF-kappaB activation status. Altogether, the data obtained indicate that GGT activity may impair the redox status of thiols that is critical for NF-kappaB DNA binding and gene transactivation, through the production of prooxidant species allegedly distinct from hydrogen peroxide. GGT activity therefore appears to be an additional factor in modulation of NF-kappaB transcriptional activity in melanoma, capable of hindering NF-kappaB DNA binding even in conditions where continuous oxidative stress would favor NF-kappaB nuclear translocation.