Redox Modulation of NF-κB Nuclear Translocation and DNA Binding in Metastatic Melanoma. The Role of Endogenous and γ-Glutamyl Transferase-Dependent Oxidative Stress
Restricted accessResearch articleFirst published online July, 2003
Redox Modulation of NF-κB Nuclear Translocation and DNA Binding in Metastatic Melanoma. The Role of Endogenous and γ-Glutamyl Transferase-Dependent Oxidative Stress
The transcription factor NF-κB is implicated in the expression of genes involved in cell proliferation, apoptosis and metastasis. In melanoma, high constitutive levels of NF-κB activation are usually observed. NF-κB 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.
Methods
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-κB. The latter was evaluated by determining the nuclear translocation of the p65 subunit (by immunoblot), the DNA binding of NF-kB (by elec-trophoretic mobility shift assay) and its transcriptional activity (by gene transactivation studies).
Results
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-κB 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-κB DNA binding and gene transactivation were paradoxically decreased. NF-κB DNA binding could be restored by treating cell lysates with the thiol-re-ducing agent dithiothreitol (DTT). Treatment of cells with exogenous hydrogen peroxide did not affect NF-κB activation status.
Conclusions
Altogether, the data obtained indicate that GGT activity may impair the redox status of thiols that is critical for NF-κB 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-κB transcriptional activity in melanoma, capable of hindering NF-κB DNA binding even in conditions where continuous oxidative stress would favor NF-κB nuclear translocation.
TakeshitaH., YoshizakiT., MillerW.E., SatoH., FurukawaM., PaganoS., Raab-TraubN.: Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus latent membrane protein 1 C-terminal activation regions 1 and 2.J Virol,73: 5548–5555, 1999.
2.
ReuningU., WilhelmO., NishiguchiT., GuerriniL., BlasiF., GraeffH., SchmittM.: Inhibition of NF-κB-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1.Nucl Acids Res,23: 3887–3893, 1995.
3.
DongG., ChenZ., KatoT., Van WaesC.: The host environment promotes the constitutive activation of nuclear factor-kappa B and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma.Cancer Res,59: 3495–3504, 1999.
4.
BoursV., DejardinE., Goujon-LetaweF., MervilleM-P, Cas-tronovoV.: The NF-κB transcription factor and cancer: high expression of NF-κB- and I-κB-related proteins in tumor cell lines.Biochem Pharmacol,47: 145–149, 1994.
YangC.R., Wilson-Van PattenC., PlanchonS.M., Wuerzberger-DavisS.M., DavisT.W., CuthillS., MiyamotoS., BoothmanD.A.: Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation.FASEB J,14: 379–390, 2000.
7.
HigginsK.A., PerezJ.R., ColemanT.A., DorshkindK., McCo-masW.A., SarmientoU.M., RosenC.A., NarayananR.: Antisense inhibition of the p65 subunit of NF-κB blocks tumorigenicity and causes tumor regression.Proc Natl Acad Sci USA,90: 9901–9905, 1993.
8.
PatelP.S., VarneyM.L., DaveB.J., SinghR.K.: Regulation of constitutive and induced NF-kappaB activation in malignant melanoma cells by capsaicin modulates interleukin-8 production and cell proliferation.J Interferon Cytokine Res,22: 427–435, 2002.
9.
MeyskensF.L.Jr., BuckmeierJ.A., TohidianN.B.: Constitutive activation of NF-kappaB in human melanoma cells and the effect of oxidative stress.Clin Cancer Res,5: 1197–1202, 1999.
10.
YangJ., RichmondA.: Constitutive IkappaB kinase activity correlates with nuclear factor-kappaB activation in human melanoma cells.Cancer Res,61: 4901–4909, 2001.
11.
BakkerT.R., ReedD., RennoT., JongeneelC.V.: Efficient adenoviral transfer of Nf-κB inhibitor sensitizes melanoma to tumor necrosis factor-mediated apoptosis.Int J Cancer,80: 320–323, 1999.
12.
FrancoA.V., ZhangX.D., Van BerkelE., SandersJ.E., ZhangX.Y., ThomasW.D., NguyenT., HerseyP.: The role of NF-kappa B in TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of melanoma cells.J Immunol,166: 5337–5345, 2001.
13.
Shattuck-BrandtR.L., RichmondA.: Enhanced degradation of I-κB alpha contributes to endogenous activation of NF-κB in Hs294T melanoma cells.Cancer Res,57: 3032–3039, 1997.
14.
SchreckR., RieberP., BaeuerleP.A.: Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1.EMBO J,10: 2247–2258, 1991.
15.
FlohéL., Brigelius-FlohéR., SaliouC., TraberM.G., PackerL.: Redox regulation of NF-kappa B activation.Free Rad Biol Med,22: 1115–1126, 1997.
ToledanoM.B., LeonardW.J.: Modulation of transcription factor NF-kappaB binding activity by oxidation-reduction in vitro.Proc Natl Acad Sci USA,88: 4328–4332, 1991.
18.
Kretz-RemyC., MehlenP., MiraultM-E, ArrigoA-P: Inhibition of IκB-a phosphorylation and degradation and subsequent NF-κB activation by glutathione peroxidase overexpression.J Cell Biol133: 1083–1093, 1994.
19.
BrarS.S., KennedyT.P., WhortonA.R., SturrockA.B., Hueck-steadtT.P., GhioA.J., HoidalJ.R.: Reactive oxygen species from NAD(P)H: quinone oxidoreductase constitutively activate NF-kappaB in malignant melanoma cells.Am J Physiol Cell Physiol,280: C659–676, 2001.
20.
MaellaroE., DominiciS., Del BelloB., ValentiniM.A., PieriL., PeregoP., SupinoR., ZuninoF., LorenziniE., PaolicchiA., ComportiM., PompellaA.: Membrane gamma-glutamyl transpeptidase activity of melanoma cells: effects on cellular H2O2 production, cell surface protein thiol oxidation and NF-κB activation status.J Cell Sci,113: 2671–2678, 2000.
21.
MeyskensF.L.Jr., McNultyS.E., BuckmeierJ.A., TohidianN.B., SpillaneT.J., KahlonR.S., GonzalezR.I.: Aberrant redox-regulation in human metastatic melanoma cells compared to normal melanocytes.Free Rad Biol Med,31: 799–808, 2001.
22.
TateT.M., MeisterA.: γ-glutamyl transpeptidase from kidney.Meth Enzymol,113: 400–419, 1985.
23.
LuS.C., SunW.M., YiJ., OokhtensM., SzeG., KaplowitzN.: Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cells.J Clin Invest,97: 1488–1496, 1996.
24.
StarkA-A, ZeigerE., PaganoD.A.: Glutathione metabolism by γ-glutamyl transpeptidase leads to lipid peroxidation: characterization of the system and relevance to hepatocarcinogene-sis.Carcinogenesis,14: 183–189, 1993.
25.
PompellaA., PaolicchiA., DominiciS., ComportiM., TongianiR.: Selective colocalization of lipid peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of y-glutamyl transpeptidase activity.His-tochem Cell Biol,106: 275–282, 1996.
26.
PaolicchiA., TongianiR., TonarelliP., ComportiM., PompellaA.: Gamma-glutamyl transpeptidase-dependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells.Free Rad Biol Med,22: 853–860, 1997.
27.
DominiciS., ValentiniM., MaellaroE., Del BelloB., PaolicchiA., LorenziniE., TongianiR., ComportiM., PompellaA.: Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of gamma-glutamyl transpeptidase-dependent H2O2 production and S-thiolation.Free Rad Biol Med,27: 623–635, 1999.
28.
Del BelloB., PaolicchiA., ComportiM., PompellaA., MaellaroE.: Hydrogen peroxide produced during gamma-glutamyl-transpeptidase activity is involved in prevention of apoptosis and maintenance of proliferation in U937 cells.FASEB J,13: 69–79, 1999.
MurataJ., Ricciardi-CastagnoliP., Dessous L'Eglise MangeP., MartinF., Juillerat-JeanneretL.: Microglial cells induce cytotoxic effects toward colon carcinoma cells: measurement of tumor cytotoxicity with a gamma-glutamyl transpeptidase assay.Int J Cancer,70: 169–174, 1997.
31.
BlairS.L., HeerdtP., SacharS., AbolhodaA., HochwaldS., ChengH., BurtM.: Glutathione metabolism in patients with nonsmall cell lung cancers.Cancer Res,57: 152–155, 1997.
32.
TsutsumiM., SakamuroD., TakadaA., ZangS.C., FurukawaT., TaniguchiN.: Detection of a unique gamma-glutamyl transpeptidase messenger RNA species closely related to the development of hepatocellular carcinoma.Hepatology,23: 1093–1097, 1996.
33.
TägerM., IttensonA., FrankeA., FreyA., GassenH.G., AnsörgeS.: γ-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias.Ann Hematol,70: 237–242, 1995.
34.
SupinoR., MapelliE., SanfilippoO., SilvestroL.: Biological and enzymatic features of human melanoma cell clones with different invasive potential.Melanoma Res,2: 377–384, 1992.
35.
HusebyN.E., StrömmeJ.H.: Practical points regarding routine determination of gamma-glutamyl transferase in serum with a kinetic method at 37 °C.Scand J Clin Lab Invest,34: 357–361, 1974.
36.
AndrewsN.C., FallerD.V.: A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells.Nucleic Acids Res,19: 2499–2504, 1991.
37.
MohantyJ.G., JaffeJ.S., ShulmanE.S., RaibleD.G.: A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.J Immunol Methods,202: 133–141, 1997.
38.
GriskO., KüsterU., AnsörgeS.: The activity of gamma-glutamyl transpeptidase (y-GT) in populations of mononuclear cells from human peripheral blood.Biol Chem Hoppe-Seyler,374: 287–290, 1993.
39.
PeregoP., PaolicchiA., TongianiR., PompellaA., TonarelliP., CareniniN., RomanelliS., ZuninoF.: The cell-specific antiproliferative effect of reduced glutathione is mediated by gamma-glutamyl transpeptidase-dependent extracellular prooxidant reactions.Int J Cancer,71: 246–250, 1997.
40.
DaubeufS., LeroyP., PaolicchiA., PompellaA., Well manM., GalteauM.M., VisvikisA.: Enhanced resistance of HeLa cells to cisplatin by overexpression of gamma-glutamyltransferase.Biochem Pharmacol,64: 207–216, 2002.
41.
PaolicchiA., DominiciS., PieriL., MaellaroE., PompellaA.: Glutathione catabolism as a signalling mechanism.Biochem Pharmacol,64: 1029–1037, 2002.
42.
AccaouiM-J, EnoiuM., MergnyM., MassonC., DominiciS., WellmanM., VisvikisA.: Gamma-glutamyltranspeptidase dependent glutathione catabolism results in activation of NF-κB.Biochem Biophys Res Comm,276: 1062–1067, 2000.
MeyerM., PahlH.L., BaeuerleP.A.: Regulation of the transcription factors NF-kappa B and AP-1 by redox changes.Chem Biol Interact,91: 91–100, 1994.
45.
AndersonM.T., StaalF.J.T., GitlerC., HerzenbergL.A., HerzenbergL.A.: Separation of oxidant-initiated and redox-regulated steps in the NF-κB signal transduction pathway.Proc Natl Acad Sci USA,91: 11527–11531, 1994.
46.
ImbertV., RupecR.A., LivolsiA., PahlH.L., TraencknerB.M., Mueller-DieckmannC., FarahifarD., RossiB., AubergerP., BaeuerleP.A., PeyronJ.F.: Tyrosine phosphorylation of IKB activates NF-κB without proteolytic degradation of IκB-a.Cell,86: 787–798, 1996.
47.
SchoonbroodtS., PietteJ.: Oxidative stress interference with the nuclear factor κB activation pathways.Biochem Pharmacol,60: 1075–1083, 2000.
48.
MatthewsJ.R., WakasugiN., VirelizierJ.L., YodoiJ., HayR.T.: Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62.Nucleic Acids Res,20: 3821–3830, 1992.
49.
MeyskensF.L.Jr., ChauH.V., TohidianN., BuckmeierJ.: Lumi-nol-enhanced chemiluminescent response of human melanocytes and melanoma cells to hydrogen peroxide stress.Pigment Cell Res,3: 184–189, 1997.
50.
PicardoM., GrammaticoP., RoccellaF., RoccellaM., GrandinettiM., Del PortoG., PassiS.: Imbalance in the antioxidant pool in melanoma cells and normal melanocytes from patients with melanoma.J Invest Dermatol,107: 322–326, 1996.
51.
OberleyT.D., OberleyL.W.: Antioxidant enzyme levels in cancer.Histol Histopathol,12: 525–535, 1997.
52.
PaolicchiA., LorenziniE., PeregoP., SupinoR., ZuninoF., ComportiM., PompellaA.: Extracellular thiol metabolism in clones of human metastatic melanoma with different gamma-glutamyl transpeptidase expression: implications for cell response to platinum-based drugs.Int J Cancer,97: 740–745, 2002.
53.
Del BelloB., ValentiniM.A., ZuninoF., ComportiM., MaellaroE.: Cleavage of Bcl-2 in oxidant- and cisplatin-induced apoptosis of human melanoma cells.Oncogene,20: 4591–4595, 2001.
54.
VoehringerD.W., McConkeyD.J., McDonnellT.J., BrisbayS., MeynR.E.: Bcl-2 expression causes redistribution of glutathione to the nucleus.Proc Natl Acad Sci USA,95: 2956–2960, 1998.
55.
LahdenpohjaN., SavinainenK., HurmeM.: Pre-exposure to oxidative stress decreases the nuclear factor-κB-dependent transcription in T lymphocytes.J Immunol,160: 1354–1358, 1998.
