Sulforaphane is an isothiocyanate derived from cruciferous vegetables that has been linked to decreased risk of certain cancers. Although the role of sulforaphane in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor activator protein-1 (AP-1) may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that sulforaphane is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinoma in a mouse model using cotreatment with the compound and the carcinogen. We also show that sulforaphane pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by sulforaphane. Electrophoretic mobility shift assay analysis of nuclear proteins also shows that sulforaphane and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun, we show that mutation of critical cysteines in the DNA-binding domain of these proteins (Cys154 in cFos and Cys272 in cJun) results in loss of sensitivity to both sulforaphane and diamide in electrophoretic mobility shift assay analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of squamous cell carcinoma by sulforaphane. [Cancer Res 2009;69(17):7103–10]

UV radiation is a known contributor to skin aging and carcinogenesis. Of the two types of UV light that penetrate our atmosphere, UVB (280-320 nm) and UVA (320-400 nm), UVB is much less abundant but exponentially more potent at inducing DNA damage and cell signaling events. The transcription factor activator protein-1 (AP-1) is known to be a key mediator of UV-induced nonmelanoma skin cancer, particularly squamous cell carcinoma. AP-1 is composed of homodimers of Jun family members (cJun, JunD, and JunB) or heterodimers of Jun and Fos family members (cFos, FosB, Fra1, and Fra2). In cultured keratinocytes, UVB treatment leads to dramatic up-regulation of cFos protein levels and an increase in cFos/JunD binding to the 12-O-tetradecanoylphorbol-13-acetate response element (TRE), a binding site in the promoters of AP-1 target genes (1). Binding of AP-1 to the TRE can be prompted by a variety of stimuli and its activation regulates responses such as proliferation, apoptosis, and differentiation. Inhibition of AP-1 activity through genetic or pharmacologic means has been shown to greatly reduce UVB-induced skin carcinogenesis in mice (24). AP-1 is therefore an intriguing target for chemoprevention of nonmelanoma skin cancer using topical agents.

Recent evidence indicates that UVB irradiation causes increased levels of reactive oxygen species in the cell, which contributes to damage and signaling events. These reactive oxygen species have been linked to UVB-induced AP-1 activation (5, 6). Abate and colleagues showed that DNA binding of the AP-1 dimer depends on a conserved lysine-cysteine-arginine motif present in the DNA-binding domain of each subunit of the transcription factor (7). The cysteine amino acid of this motif was shown to be the site of redox-mediated control of AP-1 TRE binding and therefore a major transcriptional control. Replacement of this cysteine with serine resulted in increased TRE binding and loss of sensitivity to thiol-reactive agents (7). Thus, the cysteine of the lysine-cysteine-arginine motif must be maintained in a reduced state in order for AP-1 to bind DNA. Because many stimulants such as UVB actually increase oxidant levels in the cell, the reduction of the AP-1 DNA-binding cysteine is maintained by the redox-sensitive Ref-1 protein (8).

We are interested in characterizing the chemopreventive mechanisms of natural products that are potentially useful for preventing the development of squamous cell carcinoma. Sulforaphane is an isothiocyanate compound found in cruciferous vegetables, especially broccoli and broccoli sprouts. Sulforaphane or sulforaphane-containing extracts are effective at inhibiting lung adenocarcinomas (9), colon polyps (10), and skin cancer (1113) in mouse models, but the molecular chemopreventive mechanism(s) employed by sulforaphane is poorly understood. One major mechanism through which sulforaphane mediates chemopreventive effects is through its ability to react with thiol groups such as those found on cysteine in target proteins. Interaction of sulforaphane with cysteines in the Keap1 protein is thought to be responsible for activation of the Nrf2 transcription factor and therefore up-regulation of antioxidant and detoxification genes in the cell (14, 15). Cells treated with sulforaphane also up-regulate rate-limiting enzymes in the biosynthesis of the thiol-based antioxidant glutathione and develop enhanced abilities to scavenge reactive oxygen species or react to xenobiotic stress (16, 17).

Recent work on the effects of sulforaphane on AP-1 activity suggests that UVB-stimulated AP-1 might be inhibited by sulforaphane due to changes in the redox potential of the cell (18). However, although sulforaphane significantly reduces AP-1 luciferase activity induced by UVB treatment, this occurs independently of glutathione levels in the cell (18). Therefore, we hypothesize that inhibition of AP-1 by sulforaphane is dependent on a chemical modification of the transcription factor by the compound and not simply due to a change in cellular redox status. Although sulforaphane is known to modify cysteines such as those known to be crucial for AP-1/DNA binding (7, 14), the exact mechanism of its inhibition of AP-1 remains uncertain.

Reagents.R,S-sulforaphane [1-isothiocyanato-(4R,S)-(methylsulfinyl)butane] was purchased from LKT Laboratories and diluted in acetonitrile or acetone from Sigma-Aldrich. Diamide (diazenedicarboxylic acid bis[N,N-dimethylamide]) was purchased from Sigma.

UVB skin carcinogenesis. SKH-1 hairless female mice were purchased from Charles River Laboratories and housed in accordance with The University of Arizona Animal Care and Use Committee standards. Mice were split into four groups of 20 each: UVB alone, acetone + UVB, 1 μmol sulforaphane + UVB, and 2.5 μmol sulforaphane + UVB. Mice were exposed to UVB using six FS40T12 UVB lamps (National Biological Corporation) three times weekly for 25 weeks. Fluence was determined using a UVX radiometer (Ultraviolet Products). The UVB dose was initiated at 0.54 kJ/m2 and increased 25% each week until the maximal dose of 1.65 kJ/m2 was reached at week 5 and maintained for the remainder of the experiment. Mice were pretreated with drug or vehicle for 1 week before initiation of UVB exposure and then 1 h before each irradiation. Tumors were measured weekly, and the experiment was terminated at week 25. Tumor burden was calculated by multiplying diameter by height in millimeters (19). Average tumor burden was calculated by dividing the sum of individual tumor burdens each week by the number of mice in the treatment group.

Mouse epidermal luciferase assay. SKH-1 mice expressing TRE-driven luciferase (AP-1 luciferase mice; refs. 2, 20) were separated into two groups of 10 sex and age-matched mice and pretreated with 0.3 μmol sulforaphane/ear or vehicle. This dosage approximates the exposure of 1 μmol per back used in the above carcinogenesis experiment, given that the surface area of a mouse ear (front and back) is roughly one third the size of the mouse back treatment area. Both ears of each mouse were pretreated four times (Monday, Wednesday, Friday, and Monday) before an acute UVB treatment of 2.75 kJ/m2 48 h later (Wednesday). Mice were sacrificed 48 h post-UVB and three 1.5 mm punches were collected from each left ear, snap frozen, and stored at −80°C. Control ear punches were obtained from the right ear of each mouse 1 day before UVB irradiation. Ear punch samples were processed for luciferase assay as described previously (21).

Plasmid cloning and mutation. Full-length cFos (His-tagged) and cJun-expressing plasmids in a pET vector were generously provided by Dr. J.A. Goodrich (University of Colorado). Truncated His-tagged cFos (tFos) and cJun (tJun) were created by PCR amplifying the sequence of interest and cloning back into pET19b at the BamHI and NdeI sites. A Stratagene QuikChange site-directed mutagenesis kit was used to specifically mutate cysteine 49 to serine on tFos and cysteine 58 to serine on tJun. Primer sequences and PCR conditions are available upon request.

Recombinant protein expression and isolation. tFos and tJun proteins were expressed in Escherichia coli BL21:DE3 (Promega). Efficient expression of the cFos and tFos proteins requires cotransformation with a helper plasmid, pSBET (22). Proteins were isolated using Ni-NTA resin (Qiagen) under native conditions as described (QIAexpressionist handbook, June 2003) with optimization for recovery from inclusion bodies. Eluate fractions containing protein were pooled, concentrated, evaluated using SDS-PAGE, and quantified using the Bio-Rad Protein Assay (Bio-Rad Laboratories).

Dialysis to form dimerized proteins. This protocol for forming tFos/tJun heterodimers (tAP-1) was based on work by Ferguson and Goodrich (23). To form tAP-1, concentrated tFos protein was mixed in 2 molar excess with tJun protein. This 2:1 ratio, combined with the fact that Fos proteins cannot form homodimers and the fact that Fos/Jun interactions are more stable than Jun/Jun interactions, results in ideal conditions for heterodimer formation. Mixed proteins were denatured and renatured by sequential dialysis (>4 h each) using buffers Bi, Bii, and Biii as described (23) followed by two exchanges of final buffer [25 mmol/L sodium phosphate (pH 7.5), 1 mmol/L DTT, 5% glycerol], one of which was overnight. Mutant dimers were formed using an identical protocol.

Chromatin immunoprecipitation. Human keratinocyte HaCaT cells at 80% confluence were treated with 10 μmol/L sulforaphane or vehicle (acetonitrile) in serum-free medium for 16 to 24 h. Cells were then exposed to 250 J/m2 UVB (FS20T12 bulbs; National Biological Corporation) and post-treated until harvest 6 h later as described (24). All solutions/buffers used were based on the Upstate Cell Signaling Technology EZ ChIP Kit. Samples containing 106 cells were dispersed in 400 μL SDS lysis buffer plus protease inhibitors, incubated on ice for 10 min, and stored at −80°C before sonication. To quantify the chromatin, uncrosslinked DNA was treated with RNase A and proteinase K before purification using a Qiaquick PCR Purification Kit (Qiagen).

Twenty-five micrograms of sonicated chromatin in equal volumes of SDS lysis buffer were used for each immunoprecipitation including controls. These were diluted 10-fold using ChIP Dilution Buffer plus protease inhibitors and precleared using protein A agarose/salmon sperm DNA blocked beads (Millipore) with rotation at 4°C for 1 h. Supernatants were divided into separate tubes for each immunoprecipitation. Aliquots of each tube were removed as “Input” control. Each immunoprecipitation tube received 8 μg of the appropriate antibody (RNA polymerase II: Upstate 05-623, cFos and IgG: Santa Cruz Biotechnology, sc-53 and sc-2027, respectively) or had no more additions (No Antibody control) and was rotated at 4°C overnight. Next, 100 μL of fresh beads were added to each tube and rotated at 4°C for 2 h. Pelleted beads were transferred to Handee spin cup columns (Pierce Biotechnology) and washed as described in the kit. Chromatin was eluted from the beads by incubating two times with 250 μL elution buffer. Eluted chromatin was uncrosslinked, subjected to RNase A and proteinase K digestions, and purified using the Qiaquick PCR Purification kit. Cleaned immunoprecipitated DNA was eluted from the column using 50 μL nuclease-free H2O.

Quantitative real-time PCR. Chromatin immunoprecipitation products were tested for the presence of the TRE site found in the matrix metalloproteinase-1 promoter using primers designed to detect the specified region from Applied Biosystems. These custom probes were based on the promoter sequence first described by Angel and colleagues (25) and were confirmed against the human genome sequence. Chromatin immunoprecipitation DNA (4 μL) in triplicate was used for quantitative PCR using Applied Biosystems TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detector (Applied Biosystems). Fold enrichment of the immunoprecipitated fragment was determined using the comparative Ct method using the following equation: 2−(Ct IP − Ct Input). Fold enrichments for each experiment were normalized to the respective control sample. The glyceraldehyde-3-phosphate dehydrogenase promoter was also amplified from input samples and from the RNA polymerase II immunoprecipitation samples for each experiment using normal PCR primers and conditions supplied by the Upstate kit as a control (RNA polymerase data not shown).

Electrophoretic mobility shift assay. Two micrograms of recombinant heterodimers or 5 μg of nuclear proteins were subjected to electrophoretic mobility shift assay (EMSA) using established protocols (2, 18). Nuclear proteins were extracted from treated HaCaT human keratinocytes 12 h after a dose of 250 J/m2 UVB. Nuclear or recombinant proteins were mixed with either sulforaphane, diamide, or vehicle in a final volume of 10 μL and incubated at 37°C for 1 h. Proteins were then mixed with 5× binding buffer [50 mmol/L HEPES (pH 7.9), 250 mmol/L KCl, 0.5 mmol/L EDTA, 12.5 mmol/L DTT, 50% glycerol, 2.5% Triton X-100], 1 μg poly(deoxyinosinic-deoxycytidylic acid), and water to a final volume of 19 μL. This was incubated on ice for 20 min, at which time 1 μL 32P-labeled TRE probe (18) was added and the tubes were incubated at room temperature for 30 min. Products were loaded onto a 6% acrylamide, 0.25× TBE, and 2.4% glycerol nondenaturing gel. Finished gels were dried and exposed to film. All EMSAs displayed only one retention band in addition to the probe front.

Statistical analysis. Primary analyses compared average tumor burden (diameter × height in millimeters) and tumor count (multiplicity) at week 25 in the three treatment groups (acetone, 1 μmol sulforaphane, and 2.5 μmol sulforaphane). These cross-sectional analyses among the three treatment groups used the Kruskal-Wallis test. A nonparametric test for linear trend across treatment groups used Stata's nptrend command. For these primary analyses, statistical significance was assessed at P = 0.05; 2 × 2 analyses used the Wilcoxon rank-sum test. These post hoc multiple comparisons used a Bonferonni corrected P value of 0.025. For the luciferase assay, a two-tailed Student's t test was used to calculate significance between the fold induction of the acetone versus sulforaphane-treated mice. The same analysis was used for comparison of the normalized quantitative real-time PCR data. In both cases, significance was defined as P < 0.05.

Sulforaphane inhibits UVB-induced skin carcinogenesis in SKH-1 mice. Treatment of mouse back skin with sulforaphane at either 1 or 2.5 μmol/mouse produced marked reduction in tumor multiplicity and tumor burden, although there were no differences noted in tumor type between the groups. An overall test for differences in tumor multiplicity among the four experimental groups at week 25 using the Kruskal-Wallis test was borderline significant. (P = 0.06). However, comparison of the acetone group to the 2.5 μmol sulforaphane group indicated a statistically significant difference at week 25 (58% fewer tumors with 2.5 μmol sulforaphane; P = 0.03). A nonparametric test for linear relationship between tumor count and increasing sulforaphane dose for weeks 15 to 25 was also statistically significant (P = 0.007; Fig. 1A). Tumor burden was not different among the experimental groups at week 25. However, there was a statistically significant trend toward lower tumor burden in the groups treated with sulforaphane (P < 0.0001; Fig. 1B). Thus, sulforaphane treatment is effective at inhibiting tumorigenesis in this model, especially when using the higher dose of sulforaphane.

Figure 1.

Sulforaphane inhibits UVB-induced squamous cell carcinoma in mice. SKH-1 mice were pretreated with acetone, 1 μmol sulforaphane (SFN), 2.5 μmol sulforaphane, or nothing 1 h before exposure to UVB three times a week for 25 wk. Tumors were counted and measured weekly. The average tumors per mouse (multiplicity) in the 2.5 μmol group was significantly different than the acetone control at week 25 (P = 0.03), and trend analysis from weeks 15 to 25 showed strong significant inhibition of multiplicity with sulforaphane treatment (A; P = 0.007). Sulforaphane treatment also resulted in a significantly lower trend in the average tumor burden for this group (B; P = 0.0001).

Figure 1.

Sulforaphane inhibits UVB-induced squamous cell carcinoma in mice. SKH-1 mice were pretreated with acetone, 1 μmol sulforaphane (SFN), 2.5 μmol sulforaphane, or nothing 1 h before exposure to UVB three times a week for 25 wk. Tumors were counted and measured weekly. The average tumors per mouse (multiplicity) in the 2.5 μmol group was significantly different than the acetone control at week 25 (P = 0.03), and trend analysis from weeks 15 to 25 showed strong significant inhibition of multiplicity with sulforaphane treatment (A; P = 0.007). Sulforaphane treatment also resulted in a significantly lower trend in the average tumor burden for this group (B; P = 0.0001).

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Sulforaphane inhibits UVB-induced AP-1 luciferase in vivo. Full-thickness skin punch biopsies from the ears of AP-1 luciferase mice confirm significant activation of AP-1 luciferase by UVB (P = 0.0002; data not shown). However, the 24-fold luciferase induction by UVB was significantly reduced to 13-fold with sulforaphane pretreatment (P = 0.01; Fig. 2). Control punches taken 24 h before UVB exposure show luciferase expression levels in both vehicle and sulforaphane-treated ears to be very low overall (data not shown), in accordance with previous studies (21). Thus, sulforaphane is effective at reducing AP-1 activation after UVB in mouse skin but does not appear to influence baseline AP-1 activity. These data are in accordance with previous findings in cultured keratinocytes (18) and are the first to indicate that sulforaphane can inhibit AP-1 activation in vivo.

Figure 2.

Inhibition of UVB-induced AP-1 luciferase by sulforaphane in mouse skin. AP-1 luciferase mouse ears were pretreated with acetone or sulforaphane before acute treatment with UVB. Luciferase assays were done on pre-UV–treated and post-UV–treated ear punches, and fold activation by UVB was calculated for each mouse. *, P = 0.01, statistically significant difference between acetone- and sulforaphane-treated mice after UVB.

Figure 2.

Inhibition of UVB-induced AP-1 luciferase by sulforaphane in mouse skin. AP-1 luciferase mouse ears were pretreated with acetone or sulforaphane before acute treatment with UVB. Luciferase assays were done on pre-UV–treated and post-UV–treated ear punches, and fold activation by UVB was calculated for each mouse. *, P = 0.01, statistically significant difference between acetone- and sulforaphane-treated mice after UVB.

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Sulforaphane inhibits nuclear binding of cFos to the TRE. Sulforaphane is known to influence many factors in the cell that could affect UVB-induced AP-1 luciferase activation. To confirm that the inhibition of AP-1 noted in our previous results was due to inhibition of AP-1 binding to DNA in the cell, we performed chromatin immunoprecipitation assays using keratinocytes in culture. Quantitative PCR to amplify the TRE in the collagenase-1 (matrix metalloproteinase-1) promoter, the same sequence as the promoter in the AP-1 luciferase construct, was done. Our data show ∼2.5-fold activation of cFos DNA binding in cells treated with UVB (P < 0.01; Fig. 3A), in agreement with previous observations using other assays (1). In addition, our data clearly indicate that sulforaphane inhibits cFos from binding to the matrix metalloproteinase-1 TRE after UVB exposure. The level of cFos binding is significantly reduced by sulforaphane treatment when compared with the UVB-alone samples (P < 0.01). These results are supported by low levels of binding in all of the negative controls (no antibody or IgG controls), which are not affected by sulforaphane or UVB treatment. PCR of the glyceraldehyde-3-phosphate dehydrogenase promoter from each Input DNA sample confirms that the initial starting chromatin concentrations were equivalent (Fig. 3B).

Figure 3.

Sulforaphane inhibits cFos binding to the TRE in the nucleus. HaCaT keratinocytes were pretreated and posttreated with acetonitrile or 10 μmol/L sulforaphane before exposure to UVB and harvest for chromatin immunoprecipitation assay. Samples were immunoprecipitated with an antibody against cFos, normal IgG, or no antibody. Real-time quantitative PCR was done using a probe specific for the TRE from the collagenase-1 promoter. Data are pooled from three independent experiments. *, P < 0.001, statistically significant difference between control and UVB alone–treated samples; **, P = 0.0013, UVB alone versus sulforaphane + UVB (A). Input samples were used for amplification of the glyceraldehyde-3-phosphate dehydrogenase promoter to show that each sample started with equivalent amounts of chromatin (B).

Figure 3.

Sulforaphane inhibits cFos binding to the TRE in the nucleus. HaCaT keratinocytes were pretreated and posttreated with acetonitrile or 10 μmol/L sulforaphane before exposure to UVB and harvest for chromatin immunoprecipitation assay. Samples were immunoprecipitated with an antibody against cFos, normal IgG, or no antibody. Real-time quantitative PCR was done using a probe specific for the TRE from the collagenase-1 promoter. Data are pooled from three independent experiments. *, P < 0.001, statistically significant difference between control and UVB alone–treated samples; **, P = 0.0013, UVB alone versus sulforaphane + UVB (A). Input samples were used for amplification of the glyceraldehyde-3-phosphate dehydrogenase promoter to show that each sample started with equivalent amounts of chromatin (B).

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Both sulforaphane and diamide, cysteine-oxidizing agents, effectively block binding of nuclear AP-1 to the TRE in vitro. Nuclear extracts from cells either mock-irradiated or exposed to 250 J/m2 UVB were incubated with sulforaphane or diamide before the addition of TRE probe and EMSA analysis. The dose-dependent inhibition of AP-1 binding due to sulforaphane exposure in vitro (Fig. 4A) is typical of our previous results (18). At a dose of 1 mmol/L sulforaphane, the binding of nuclear AP-1 to the TRE was reduced to baseline levels. Diamide, another cysteine-oxidizing agent, was also very effective at inhibiting the binding of AP-1 under the same conditions (Fig. 4B).

Figure 4.

Sulforaphane and diamide block AP-1 binding to the TRE in vitro. Nuclear extracts from mock- or UVB-treated cells were incubated with sulforaphane (A), diamide (B), or the appropriate vehicle control and then exposed to radiolabeled TRE probe and run on an EMSA gel. Both of these cysteine oxidants reduce the ability of AP-1 to bind to the TRE in a dose-dependent manner.

Figure 4.

Sulforaphane and diamide block AP-1 binding to the TRE in vitro. Nuclear extracts from mock- or UVB-treated cells were incubated with sulforaphane (A), diamide (B), or the appropriate vehicle control and then exposed to radiolabeled TRE probe and run on an EMSA gel. Both of these cysteine oxidants reduce the ability of AP-1 to bind to the TRE in a dose-dependent manner.

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Isolation and characterization of recombinant truncated cFos and cJun. PCR products encoding the β-zip region of cFos and cJun were cloned into bacterial expression vectors encoding a NH2-terminal His tag to create tFos and tJun. These truncated proteins contain only two cysteine residues each: one in the DNA-binding domain (Cys154 for cFos and Cys272 for cJun) and one proximal to the COOH terminus in the leucine zipper domain (Fig. 5A). Recombinant proteins were purified and visualized on a SDS-PAGE gel (Fig. 5B). After dimerization of tFos and tJun subunits to form tAP-1, EMSA analysis showed that these proteins bind tightly to the TRE. Preincubation of tAP-1 with cold wild-type TRE oligos reduced binding to 32P-labeled TRE in a dose-dependent manner, whereas cold mutant oligos did not (Fig. 6A). The tAP-1 dimer therefore specifically binds to the TRE. This binding was inhibited by sulforaphane pretreatment in a dose-dependent fashion, similar to that noted with nuclear extracts (Fig. 6B). Thus, the recombinant tAP-1 protein reacts to the TRE and to sulforaphane in a manner similar to that of its nuclear counterpart.

Figure 5.

Structures and expression of recombinant tFos and tJun. The β-zip domains of cFos and cJun were cloned into bacterial expression vectors. The domain structures of the resulting truncated proteins are shown in comparison with their full-length counterparts (A). Mutated tFos and tJun have the identical amino acid sequence as their wild-type forms, except that the cysteine in the DNA-binding domain is replaced with serine. Recombinant tFos and tJun and their mutated forms were isolated using Ni-NTA resin and were visualized using SDS-PAGE and BioSafe Coomassie blue staining (B).

Figure 5.

Structures and expression of recombinant tFos and tJun. The β-zip domains of cFos and cJun were cloned into bacterial expression vectors. The domain structures of the resulting truncated proteins are shown in comparison with their full-length counterparts (A). Mutated tFos and tJun have the identical amino acid sequence as their wild-type forms, except that the cysteine in the DNA-binding domain is replaced with serine. Recombinant tFos and tJun and their mutated forms were isolated using Ni-NTA resin and were visualized using SDS-PAGE and BioSafe Coomassie blue staining (B).

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Figure 6.

Truncated recombinant AP-1 binds specifically to the TRE and is inhibited by sulforaphane in a manner dependent on the presence of the DNA-binding cysteine. Recombinant tFos and tJun were heterodimerized to form functional tAP-1. The binding specificity of tAP-1 was confirmed through EMSA analysis in which wild-type cold competitor oligos successfully inhibited tAP-1 from binding to the labeled TRE in a dose-dependent fashion, but mutant (mut.) competitors did not (A). tAP-1 was also incubated with sulforaphane for 1 h at 37°C before being mixed with buffer and labeled probe. The ability of sulforaphane (SFN) to inhibit tAP-1 TRE binding in a dose-dependent fashion was therefore confirmed (B). Wild-type tAP-1 was then compared with mutant tAP-1 using EMSAs in which both dimers were pretreated with 1 mmol/L sulforaphane or 7 mmol/L diamide as above. Mutation of the DNA-binding cysteine in tAP-1 results in a loss of activity of both sulforaphane and diamide on the TRE binding response of these proteins (C).

Figure 6.

Truncated recombinant AP-1 binds specifically to the TRE and is inhibited by sulforaphane in a manner dependent on the presence of the DNA-binding cysteine. Recombinant tFos and tJun were heterodimerized to form functional tAP-1. The binding specificity of tAP-1 was confirmed through EMSA analysis in which wild-type cold competitor oligos successfully inhibited tAP-1 from binding to the labeled TRE in a dose-dependent fashion, but mutant (mut.) competitors did not (A). tAP-1 was also incubated with sulforaphane for 1 h at 37°C before being mixed with buffer and labeled probe. The ability of sulforaphane (SFN) to inhibit tAP-1 TRE binding in a dose-dependent fashion was therefore confirmed (B). Wild-type tAP-1 was then compared with mutant tAP-1 using EMSAs in which both dimers were pretreated with 1 mmol/L sulforaphane or 7 mmol/L diamide as above. Mutation of the DNA-binding cysteine in tAP-1 results in a loss of activity of both sulforaphane and diamide on the TRE binding response of these proteins (C).

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DNA-binding cysteine is required for sulforaphane- or diamide-induced inhibition in vitro. To test for direct interaction of sulforaphane with the DNA-binding cysteines in AP-1, we mutated the DNA-binding cysteines in tFos and tJun to serines, leaving the remaining cysteines near the COOH terminus intact. Both wild-type and mutant heterodimers were then treated with sulforaphane or diamide and analyzed for their ability to bind to the TRE via EMSA. As shown in Fig. 6C, mutation of the DNA-binding cysteine resulted in loss of sensitivity to treatment with either of these oxidizing agents. In fact, although binding to the TRE is completely blocked by pretreatment of the wild-type dimer with 7 mmol/L diamide, the mutant dimer is totally immune to this inhibition. The mutant form of tAP-1 is also completely resistant to inhibition by 1 mmol/L sulforaphane.

To our knowledge, this is the first study to report an inhibitory effect of sulforaphane on UVB-induced skin carcinogenesis in mice exposed to both sulforaphane and UVB simultaneously. Other reports have described the effect of sulforaphane using chemically induced mouse skin carcinogenesis (12, 13) or a UVB model using a chemotherapeutic treatment protocol with broccoli extracts (UVB was stopped before the extract was applied; ref. 11). All of these studies described a protective effect of sulforaphane treatment. Therefore, the current data corroborate previous chemopreventive reports and do so using purified sulforaphane and a model of concurrent UVB/agent exposure, which may be more relevant to human outcomes. Sulforaphane does not absorb light in the UV spectrum or produce a sunscreen effect (26). This provides us with a positive framework for using sulforaphane as a topical chemopreventive agent in conjunction with studies to identify molecular mechanisms of sulforaphane chemopreventive effects in the skin.

Many of the molecular studies of sulforaphane have focused on its effects on the Nrf2 transcription factor pathway (12, 27, 28). Nrf2 and its effector proteins help to protect cells from oxidative insults. Although Nrf2 is implicated in protecting the skin against carcinogenic chemicals, its role in UV-induced carcinogenesis is unclear. In cell culture, different doses or wavelengths of UV can induce or reduce Nrf2 levels depending on the cell type (2931). Some have suggested that transient Nrf2 activation in the skin by electrophilic compounds (such as sulforaphane) may be protective against tumorigenesis, but constitutive activation of Nrf2 may lead to malignant conversion (32). Protection from UV-induced carcinogenesis by sulforaphane might also involve modulation of the inflammatory response by Nrf2 (3337). However, a recent report discovered that although Nrf2 knockout mice had increased oxidative DNA damage, inflammation, and sunburn cell formation compared to wild-type mice after acute UVB exposure, chronic UVB treatment revealed no difference in the incidence rate or mean number of tumors between wild-type and Nrf2 knockout mice (38). Therefore, the ability of sulforaphane to inhibit UVB-induced nonmelanoma skin cancer may be due to factors other than Nrf2 stimulation.

The experiments reported here show for the first time the ability of sulforaphane to inhibit AP-1 activity in vivo. Earlier luciferase assays and EMSAs indicated that sulforaphane could regulate the DNA binding of AP-1 in vitro (18). In our transgenic mouse model, we have successfully shown that sulforaphane inhibits UVB-induced AP-1 luciferase activity in the skin. We have also confirmed through chromatin immunoprecipitation analysis that UVB causes increased binding of cFos to the TRE and that this binding is inhibited by sulforaphane. Although others have reported that sulforaphane slightly increases AP-1 luciferase activity at low doses, our results do not indicate increased basal AP-1-luciferase activity with sulforaphane pretreatment (data not shown; ref. 39). These differences may be due to cell type–specific reactions. The data in Figs. 2 and 3 support our hypothesis that inhibition of AP-1 may be a contributing factor to the ability of topical sulforaphane to block UVB-induced squamous cell carcinoma in mice.

Sulforaphane is known to bind to reactive thiol groups, especially cysteines, and may affect protein function through this mechanism (14). Diamide, another thiol oxidative agent, has been used previously to inhibit recombinant AP-1 binding in vitro (7). Treatment of nuclear extracts with either diamide or sulforaphane showed dose-dependent inhibition of AP-1 DNA binding. Because both compounds interact with cysteines, the inhibition of TRE binding is likely a result of cysteine oxidation of the AP-1 transcription factor. We have noted this reaction to sulforaphane previously at the same dose levels (18), which are likely to be physiologically relevant (40). Although the doses needed for inhibition of AP-1 binding in vitro are higher than those used in luciferase assays, sulforaphane is known to accumulate in the cell when added to culture medium. Treatment of mouse hepatoma cells with micromolar concentrations of sulforaphane yielded millimolar concentrations in cellular lysates (40). The data in Fig. 4 suggest a cysteine-specific chemical reaction, because nuclear extracts were exposed to both of these oxidants in a test tube where transcriptional or translational input is minimal.

We next addressed the importance of the specific DNA-binding cysteines of AP-1 (Cys154 in Fos and Cys272 in Jun) in the reaction with sulforaphane and diamide. Mutations of the DNA-binding domain of AP-1 can contribute to the oncogenic nature of AP-1 family members (4143). We therefore purified His-tagged recombinant truncated forms of cFos and cJun consisting of the DNA-binding and leucine zipper (β-zip) domains, modeling them after those used by Abate and colleagues (7). The truncated AP-1 proteins were able to dimerize and bind specifically to the TRE in a manner consistent with that observed using nuclear extracts. The fact that sulforaphane could dose-dependently inhibit truncated AP-1 from binding to the TRE suggests functional similarity between the recombinant form and its endogenous counterpart. However, the complete lack of response to cysteine oxidation by either sulforaphane or diamide when the DNA-binding cysteines are mutated supports our hypothesis: sulforaphane chemically oxidizes Cys154 in Fos and Cys272 in Jun to inhibit binding of AP-1 to the TRE. The cysteine-to-serine mutation creates a “permanently reduced” DNA-binding domain, which is unaffected by sulforaphane and leads to enhanced TRE binding (Fig. 6C).

Sulforaphane and other isothiocyanates are gaining credibility as potential “natural” chemopreventive agents. These natural agents are present in our diet and are easily tolerated by our metabolism. Orally administered broccoli sprout extracts containing sulforaphane have been safely tolerated by volunteers (44). Other groups are testing the efficacy of sulforaphane or related compounds for possible use in humans to prevent hepatocarcinoma, breast cancer, and nonmelanoma skin cancer (2628, 45), although to date there are no published studies regarding oral administration of sulforaphane and the prevention of skin carcinoma. Due to the current focus on sulforaphane-induced Nrf2 activation in chemoprevention, many of these studies justifiably turn to markers of Nrf2 activity to measure the potential efficacy of this compound. Many other molecular targets of sulforaphane have been identified in the cell, including transcription factors such as nuclear factor-κB, which may be subject to a similar form of thiol-mediated redox regulation as AP-1 (46). The results described here suggest that inhibition of AP-1 is also important to consider when studying the properties of sulforaphane in human skin cancer chemoprevention trials.

No potential conflicts of interest were disclosed.

Grant support: NIH grants CA23074, CA27502, R25T CA78447, 1K07CA132956-01A1, and ES06694.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Anne Cione for administrative assistance, Marc Oshiro for chromatin immunoprecipitation assay support, and Dr. James A. Goodrich for providing plasmids.

1
Chen W, Borchers AH, Dong Z, Powell MB, Bowden GT. UVB irradiation-induced activator protein-1 activation correlates with increased c-fos gene expression in a human keratinocyte cell line.
J Biol Chem
1998
;
273
:
32176
–81.
2
Cooper SJ, MacGowan J, Ranger-Moore J, Young MR, Colburn NH, Bowden GT. Expression of dominant negative c-jun inhibits ultraviolet B-induced squamous cell carcinoma number and size in an SKH-1 hairless mouse model.
Mol Cancer Res
2003
;
1
:
848
–54.
3
Barthelman M, Chen W, Gensler HL, Huang C, Dong Z, Bowden GT. Inhibitory effects of perillyl alcohol on UVB-induced murine skin cancer and AP-1 transactivation.
Cancer Res
1998
;
58
:
711
–6.
4
Li JJ, Dong Z, Dawson MI, Colburn NH. Inhibition of tumor promoter-induced transformation by retinoids that transrepress AP-1 without transactivating retinoic acid response element.
Cancer Res
1996
;
56
:
483
–9.
5
Kramer-Stickland K, Edmonds A, Bair WB III, Bowden GT. Inhibitory effects of deferoxamine on UVB-induced AP-1 transactivation.
Carcinogenesis
1999
;
20
:
2137
–42.
6
Bowden GT. Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signalling.
Nat Rev Cancer
2004
;
4
:
23
–35.
7
Abate C, Patel L, Rauscher FJ III, Curran T. Redox regulation of fos and jun DNA-binding activity in vitro.
Science
1990
;
249
:
1157
–61.
8
Xanthoudakis S, Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity.
EMBO J
1992
;
11
:
653
–65.
9
Conaway CC, Wang CX, Pittman B, et al. Phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates inhibit malignant progression of lung adenomas induced by tobacco carcinogens in A/J mice.
Cancer Res
2005
;
65
:
8548
–57.
10
Hu R, Khor TO, Shen G, et al. Cancer chemoprevention of intestinal polyposis in ApcMin/+ mice by sulforaphane, a natural product derived from cruciferous vegetable.
Carcinogenesis
2006
;
27
:
2038
–46.
11
Dinkova-Kostova AT, Jenkins SN, Fahey JW, et al. Protection against UV-light-induced skin carcinogenesis in SKH-1 high-risk mice by sulforaphane-containing broccoli sprout extracts.
Cancer Lett
2006
;
240
:
243
–52.
12
Xu C, Huang MT, Shen G, et al. Inhibition of 7,12-dimethylbenz(a)anthracene-induced skin tumorigenesis in C57BL/6 mice by sulforaphane is mediated by nuclear factor E2-related factor 2.
Cancer Res
2006
;
66
:
8293
–6.
13
Gills JJ, Jeffery EH, Matusheski NV, Moon RC, Lantvit DD, Pezzuto JM. Sulforaphane prevents mouse skin tumorigenesis during the stage of promotion.
Cancer Lett
2006
;
236
:
72
–9.
14
Hong F, Freeman ML, Liebler DC. Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane.
Chem Res Toxicol
2005
;
18
:
1917
–26.
15
Zhang DD, Lo SC, Sun Z, Habib GM, Lieberman MW, Hannink M. Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway.
J Biol Chem
2005
;
280
:
30091
–9.
16
Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress.
Free Radic Res
1999
;
31
:
273
–300.
17
Kim BR, Hu R, Keum YS, et al. Effects of glutathione on antioxidant response element-mediated gene expression and apoptosis elicited by sulforaphane.
Cancer Res
2003
;
63
:
7520
–5.
18
Zhu M, Zhang Y, Cooper S, Sikorski E, Rohwer J, Bowden GT. Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism.
Mol Carcinog
2004
;
41
:
179
–86.
19
Bair WB III, Hart N, Einspahr J, et al. Inhibitory effects of sodium salicylate and acetylsalicylic acid on UVB-induced mouse skin carcinogenesis.
Cancer Epidemiol Biomarkers Prev
2002
;
11
:
1645
–52.
20
Rincon M, Flavell RA. AP-1 transcriptional activity requires both T-cell receptor-mediated and co-stimulatory signals in primary T lymphocytes.
EMBO J
1994
;
13
:
4370
–81.
21
Bachelor MA, Cooper SJ, Sikorski ET, Bowden GT. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model.
Mol Cancer Res
2005
;
3
:
90
–9.
22
Schenk PM, Baumann S, Mattes R, Steinbiss HH. Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs.
Biotechniques
1995
;
19
:
196
–8, 200.
23
Ferguson HA, Goodrich JA. Expression and purification of recombinant human c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro.
Nucleic Acids Res
2001
;
29
:
E98
.
24
Fitzgerald M, Oshiro M, Holtan N, et al. Human pancreatic carcinoma cells activate maspin expression through loss of epigenetic control.
Neoplasia
2003
;
5
:
427
–36.
25
Angel P, Baumann I, Stein B, Delius H, Rahmsdorf HJ, Herrlich P. 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5′-flanking region.
Mol Cell Biol
1987
;
7
:
2256
–66.
26
Talalay P, Fahey JW, Healy ZR, et al. Sulforaphane mobilizes cellular defenses that protect skin against damage by UV radiation.
Proc Natl Acad Sci U S A
2007
;
104
:
17500
–5.
27
Dinkova-Kostova AT, Fahey JW, Wade KL, et al. Induction of the phase 2 response in mouse and human skin by sulforaphane-containing broccoli sprout extracts.
Cancer Epidemiol Biomarkers Prev
2007
;
16
:
847
–51.
28
Cornblatt BS, Ye L, Dinkova-Kostova AT, et al. Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast. Carcinogenesis 
2007
;
28
:
1485
–90.
29
Hirota A, Kawachi Y, Itoh K, et al. Ultraviolet A irradiation induces NF-E2-related factor 2 activation in dermal fibroblasts: protective role in UVA-induced apoptosis.
J Invest Dermatol
2005
;
124
:
825
–32.
30
Durchdewald M, Beyer TA, Johnson DA, Johnson JA, Werner S, auf dem Keller U. Electrophilic chemicals but not UV irradiation or reactive oxygen species activate Nrf2 in keratinocytes in vitro and in vivo.
J Invest Dermatol
2007
;
127
:
646
–53.
31
Marrot L, Jones C, Perez P, Meunier JR. The significance of Nrf2 pathway in (photo)-oxidative stress response in melanocytes and keratinocytes of the human epidermis.
Pigment Cell Melanoma Res
2008
;
21
:
79
–88.
32
Hayes JD, McMahon M. The double-edged sword of Nrf2: subversion of redox homeostasis during the evolution of cancer.
Mol Cell
2006
;
21
:
732
–4.
33
Mochizuki M, Ishii Y, Itoh K, et al. Role of 15-deoxy Δ(12,14) prostaglandin J2 and Nrf2 pathways in protection against acute lung injury.
Am J Respir Crit Care Med
2005
;
171
:
1260
–6.
34
Braun S, Hanselmann C, Gassmann MG, et al. Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound.
Mol Cell Biol
2002
;
22
:
5492
–505.
35
Woo KJ, Kwon TK. Sulforaphane suppresses lipopolysaccharide-induced cyclooxygenase-2 (COX-2) expression through the modulation of multiple targets in COX-2 gene promoter.
Int Immunopharmacol
2007
;
7
:
1776
–83.
36
Lin W, Wu RT, Wu T, Khor TO, Wang H, Kong AN. Sulforaphane suppressed LPS-induced inflammation in mouse peritoneal macrophages through Nrf2 dependent pathway.
Biochem Pharmacol
2008
;
76
:
967
–73.
37
Cheung KL, Khor TO, Kong AN. Synergistic effect of combination of phenethyl isothiocyanate and sulforaphane or curcumin and sulforaphane in the inhibition of inflammation.
Pharm Res
2009
;
26
:
224
–31.
38
Kawachi Y, Xu X, Taguchi S, et al. Attenuation of UVB-induced sunburn reaction and oxidative DNA damage with no alterations in UVB-induced skin carcinogenesis in Nrf2 gene-deficient mice.
J Invest Dermatol
2008
;
128
:
1773
–9.
39
Jeong WS, Kim IW, Hu R, Kong AN. Modulation of AP-1 by natural chemopreventive compounds in human colon HT-29 cancer cell line.
Pharm Res
2004
;
21
:
649
–60.
40
Zhang Y. Role of glutathione in the accumulation of anticarcinogenic isothiocyanates and their glutathione conjugates by murine hepatoma cells.
Carcinogenesis
2000
;
21
:
1175
–82.
41
Okuno H, Akahori A, Sato H, Xanthoudakis S, Curran T, Iba H. Escape from redox regulation enhances the transforming activity of Fos.
Oncogene
1993
;
8
:
695
–701.
42
Chida K, Vogt PK. Nuclear translocation of viral Jun but not of cellular Jun is cell cycle dependent.
Proc Natl Acad Sci U S A
1992
;
89
:
4290
–4.
43
Maki Y, Bos TJ, Davis C, Starbuck M, Vogt PK. Avian sarcoma virus 17 carries the jun oncogene.
Proc Natl Acad Sci U S A
1987
;
84
:
2848
–52.
44
Shapiro TA, Fahey JW, Dinkova-Kostova AT, et al. Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study.
Nutr Cancer
2006
;
55
:
53
–62.
45
Kensler TW, Chen JG, Egner PA, et al. Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a randomized clinical trial in He Zuo township, Qidong, People's Republic of China.
Cancer Epidemiol Biomarkers Prev
2005
;
14
:
2605
–13.
46
Heiss E, Herhaus C, Klimo K, Bartsch H, Gerhauser C. Nuclear factor κB is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms.
J Biol Chem
2001
;
276
:
32008
–15.