Zerumbone, a sesquiterpene derived from tropical ginger, contains an electrophilic a,b-unsaturated carbonyl moiety and was found to suppress chemically induced papilloma formation in mouse skin. Here, we report that topical application of zerumbone onto dorsal skin of hairless mice induces activation of NF-E2-related factor 2 (Nrf2) and expression of heme oxygenase-1 (HO-1). We compared the levels of HO-1 protein in the skin of zerumbone-treated Nrf2 wild-type and Nrf2 knockout mice, and nrf2-deficient mice expressed HO-1 protein to a much lesser extent than the wild-type animals following topical application of zerumbone. Treatment of mouse epidermal JB6 cells with zerumbone caused a marked increase of Nrf2 nuclear translocation followed by the promoter activity of HO-1, and also enhanced direct binding of Nrf2 to the antioxidant response element. Moreover, knockdown of Nrf2 in JB6 cells diminished the zerumbone-induced upregulation of HO-1. Notably, a-humulene and 8-hydroxy-a-humulene, the structural analogues of zerumbone that lack the a,b-unsaturated carbonyl group, failed to activate Nrf2 and were unable to increase HO-1 expression. Unlike zerumbone, these nonelectrophilic analogues could not suppress the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced JB6 cell transformation and the intracellular accumulation of reactive oxygen species (ROS). Interestingly, when JB6 cells were treated with carbon monoxide-releasing molecule that mimics the HO-1 activity, the TPA-induced ROS production was markedly reduced. Taken together, these findings suggest that upregulation of HO-1 expression by zerumbone is mediated through activation of Nrf2 signaling, which provides a mechanistic basis for the chemopreventive effects of this sesquiterpene on mouse skin carcinogenesis. Cancer Prev Res; 4(6); 860-70. Ó2011 AACR.

Conjugated linoleic acid (CLA) has been reported to inhibit mouse skin carcinogenesis, particularly in the promotion stage, but underlying molecular mechanisms remain poorly understood. Since persistent induction of cyclooxygenase-2 (COX-2) is frequently implicated in carcinogenesis, we investigated the effect of cis-9,trans-11-CLA (9Z,11E-CLA) on the tumor promoter-induced COX-2 expression in HR-1 hairless mouse skin in vivo. Topical application of 9Z,11E-CLA caused significant inhibition of COX-2 expression at 6 h induced by 10 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) in HR-1 mouse skin. Since NF-kappaB is known to regulate COX-2 gene expression, we determined the effect of 9Z,11E-CLA on TPA-induced activation of this transcription factor. Treatment of mouse skin with 9Z,11E-CLA reduced TPA-induced DNA binding as well as nuclear translocation of NF-kappaB by blocking phosphorylation and subsequent degradation of IkappaBalpha. In addition, 9Z,11E-CLA attenuated TPA-induced phosphorylation of extracellular signal-regulated protein kinase, p38 mitogen-activated protein kinase and Akt. To further elucidate the molecular mechanism underlying the inactivation of NF-kappaB by 9Z,11E-CLA, we investigated its effect on TPA-induced activation of IkappaB kinase (IKK), an upstream kinase that regulates NF-kappaB via phosphorylation and degradation of IkappaBalpha. 9Z,11E-CLA treatment down-regulated phosphorylation and catalytic activities of IKKalpha/beta in TPA-treated mouse skin. Co-treatment of mouse skin with the IKKbeta-specific inhibitor SC-514 (1 micromol) attenuated TPA-induced activation of Akt and NF-kappaB, and also the expression of COX-2 in hairless mouse skin. Taken together, 9Z,11E-CLA inhibits NF-kappaB driven-COX-2 expression by blocking the IKK and PI3K-Akt signaling in TPA-treated hairless mouse skin in vivo, which may account for its previously reported anti-tumor promoting effects.

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Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Docosahexaenoic acid (DHA), a representative ω-3 polyunsaturated fatty acid, has been reported to possess anti-inflammatory and chemopreventive properties. In the present study, we investigated the molecular mechanisms underlying the inhibitory effects of DHA on UVB-induced inflammation in mouse skin. Our study revealed that topical application of DHA prior to UVB irradiation attenuated the expression of cyclooxygenase-2 (COX-2) and NAD(P)H:oxidase-4 (NOX-4) in hairless mouse skin. DHA pretreatment also attenuated UVB-induced DNA binding of nuclear factor-kappaB (NF-κB) through the inhibition of phosphorylation of IκB kinase-α/β, phosphorylation and degradation of IκBα and nuclear translocation of p50 and p65. In addition, UVB-induced phosphorylation of p65 at the serine 276 residue was significantly inhibited by topical application of DHA. Irradiation with UVB induced phosphorylation of mitogen and stress-activated kinase-1 (MSK1), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, and all these events were attenuated by pretreatment with DHA. Blocking ERK and p38 MAP kinase signaling by U0126 and SB203580, respectively, diminished MSK1 phosphorylation in UVB-irradiated mouse skin. Pretreatment with H-89, a pharmacological inhibitor of MSK1, abrogated UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 in mouse skin. In conclusion, topically applied DHA inhibits the UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 by blocking the phosphorylation of MSK1, a kinase downstream of ERK and p38 MAP kinase, in hairless mouse skin.

Exposure to ultraviolet B (UVB) radiation is known to cause inflammatory tissue damage and skin cancer. One of the molecular links between inflammation and cancer is the eukaryotic transcription factor nuclear factor-kappaB (NF-κB), which is known to regulate expression of various pro-inflammatory genes including inducible nitric oxide synthase (iNOS). The present study was aimed at elucidating the molecular mechanisms underlying UVB-induced NF-κB activation and iNOS expression in hairless mouse skin. Irradiation of male HR-1 hairless mouse skin with UVB (5 kJ/m(2) ) resulted in increased degradation of IκBα, nuclear translocation of p65 and p50, and the DNA binding of NF-κB. Exposure to UVB radiation induced the phosphorylation and the catalytic activity of an upstream kinase IκB kinase-β (IKKβ). Pharmacological inhibition of IKKβ attenuated UVB-induced NF-κB activation in mouse skin. Irradiation of mouse skin with UVB also increased phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Pretreatment with SC-514, a specific inhibitor of IKKβ, attenuated UVB-induced phosphorylation of ERK and p38 MAP kinase. A kinetic study showed that UVB significantly increased the expression of iNOS in mouse skin at 6 h postirradiation, which was abrogated by pretreatment with SC-514. In conclusion, the upstream kinase IKKβ is involved in UVB-induced activation of MAP kinases and NF-κB, and expression of iNOS in mouse skin.

The present study investigated the effect of phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cyclooxygenase-2 (COX-2) expression and tumor promotion in mouse skin and explored the underlying molecular mechanisms. Topical application of phloretin significantly inhibited 7,12-dimethylbenz[a]anthracene-initiated and TPA-promoted mouse skin carcinogenesis. Pretreatment with phloretin on the dorsal skin of mice inhibited TPA-induced COX-2 expression in a dose-dependent manner. To elucidate the molecular mechanism underlying COX-2 inhibition by phloretin, we examined its effect on TPA-induced activation of nuclear factor-κB (NF-κB), a ubiquitous transcription factor responsible for TPA-induced COX-2 expression in mouse skin. Topically applied phloretin decreased the TPA-induced DNA binding of NF-κB. In addition, phloretin inhibited the phosphorylation as well as the catalytic activity of extracellular signal-regulated kinase (ERK), which was previously found to activate NF-κB and induce COX-2 expression in TPA-treated mouse skin. Taken together, the inhibitory effects of phloretin on TPA-induced NF-κB activation and COX-2 expression through the modulation of ERK signaling may partly account for its antitumor-promoting effect on mouse skin carcinogenesis.

Curcumin, the yellow pigment of tumeric (Curcuma longa L., Zingiberaceae), is one of the most widely used spices in the East Asia. Curcumin has been shown to possess anti-inflammatory, antioxidative and chemopreventive effects. Although a growing body of evidence suggests that the chemopreventive potential of curcumin depends partly on its ability to induce cytoprotective proteins through the activation of nuclear factor erythroid-related factor-2 (Nrf2), the molecular mechanisms remain elusive. The present study was aimed to elucidate the mechanisms underlying the activation of Nrf2 and induction of cytoprotective gene expression by curcumin. Incubation of mouse epidermal (JB6) cells with curcumin resulted in the induction of heme oxygenase-1 (HO-1) and NAD(P)H oxidoreductase 1 (NQO1) at both mRNA and protein levels. Curcumin-induced expression of HO-1 and NQO1 was abrogated in cells transiently transfected with Nrf2 siRNA. Furthermore, embryo fibroblasts from Nrf2 knock-out mice were not responsive to curcumin, compared with those from wild-type animals, in terms of inducing HO-1 and NQO1 expression. While curcumin treatment increased protein expression of Nrf2, it failed to alter the steady-state level of the Nrf2 mRNA transcript, suggesting that protein stabilization might be involved in curcumin-induced Nrf2 accumulation. Treatment of JB6 cells with curcumin did stabilize Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of Nrf2. Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor of Cullin3-Rbx1 E3 ubiquitin ligase complex, has several reactive cysteine residues, and modification of these cysteine residues has been proposed as a molecular mechanism underlying oxidative and electrophilic stress-induced activation of Nrf2. The thiol-reducing agent dithiothreitol abrogated curcumin-induced accumulation of Nrf2 and expression of cytoprotective proteins. In addition, tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,β-unsaturated carbonyl group, failed to induce cytoprotective protein expression as well as Nrf2 nuclear translocation, indicative of a pivotal role of cysteine residues of Keap1 in curcumin-induced Nrf2 activation. Cells transfected with a mutant Keap1 protein in which cysteine 151 is replaced by serine exhibited reduction in curcumin-induced Nrf2 activation. Although curcumin did not cause dissociation of Cullin3-Rbx1 E3 ubiquitin ligase complex components, it increased interaction of Nrf2 with the Keap1. Thus, it is likely that curcumin inhibits the ability of the Cullin3-Rbx1 E3 ubiquitin ligase to target Nrf2 for ubiquitination by modifying Keap1 Cys 151 residue. This may change the conformation of the complex and saturates the binding capacity of Keap1 to Nrf2, facilitating nuclear translocation of Nrf2. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B67.

Supplementary Figure S1 from Zerumbone Induces Heme Oxygenase-1 Expression in Mouse Skin and Cultured Murine Epidermal Cells through Activation of Nrf2