title: Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism.
abstract: Ultraviolet (UV) light-induced activation of activator protein-1 (AP-1), resulting at least in part from oxidative stress, promotes skin carcinogenesis. It has not yet been determined whether elevating cellular phase II enzymes and glutathione (GSH) levels inhibits the AP-1 activation. We have, therefore, examined the effects of two well-known inducers of phase II enzymes, sulforaphane (SF) and tert-butylhydroquinone (tBHQ), on UVB-induced AP-1 activation, with an AP-1-luciferase reporter plasmid that was stably transfected into human HaCaT keratinocytes (HCL14 cells). Exposure of HCL14 cells to SF or tBHQ led to the induction of quinone reductase-1 (QR-1), a marker of global cellular phase II enzymes, as well as elevation of cellular GSH levels. Incubation of the cells with 1-10 microM SF or 11-45 microM tBHQ for 24 h resulted in up to 1.4-fold and 1.7-fold increase of QR-1 activity, respectively, and up to 1.5-fold and 1.6-fold increases in cellular GSH levels, respectively. AP-1 activation was dramatically enhanced by irradiating HCL14 cells with 250 J/m(2) of UVB. While the above SF treatment dose-dependently reduced the UVB-induced AP-1 activation in HCL14 cells, the tBHQ treatment did not, suggesting that elevating cellular phase II enzymes and GSH levels may not lead to inhibition of UVB-induced AP-1 activation. Indeed, depleting cellular GSH by 80% did not affect UVB-induced AP-1 activation either. Subsequent electrophoretic mobility shift assays (EMSA) showed that SF added directly to the EMSAs inhibited AP-1 DNA binding activity, whereas tBHQ was ineffective. Taken together, our results indicated that elevating phase II enzymes and GSH levels in human keratinocytes does not lead to significant inhibition of UVB-induced AP-1 activation. The inhibitory effect of SF on UVB-induced AP-1 activation appears to be at least partly due to the direct inhibition of AP-1 DNA binding activity. This direct effect of SF on AP-1 DNA binding is a novel mechanism for the action of a drug inhibitor of AP-1 activation.
authors.affiliation: Arizona Cancer Center, The University of Arizona, Tucson, Arizona 85724-5024, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhu
authors.forename: Yuesheng
authors.initials: Y
authors.lastname: Zhang
authors.forename: Simon
authors.initials: S
authors.lastname: Cooper
authors.forename: Ewa
authors.initials: E
authors.lastname: Sikorski
authors.forename: John
authors.initials: J
authors.lastname: Rohwer
authors.forename: G Timothy
authors.initials: GT
authors.lastname: Bowden
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D017879:Isothiocyanates; D015603:Keratinocytes / Q000201:enzymology / Q000528:radiation effects*; D008156:Luciferases / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays
chemical_list: D016588:Anticarcinogenic Agents; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; D018808:Transcription Factor AP-1; C018855:2-tert-butylhydroquinone; D008156:Luciferases; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D017364:Collagenases; D000252:Calcium-Transporting ATPases; C016766:sulforaphane; D005978:Glutathione