Jun-Wan Shin

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

Zerumbone, a sesquiterpene derived from tropical ginger, contains an electrophilic α,β-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, α-humulene and 8-hydroxy-α-humulene, the structural analogues of zerumbone that lack the α,β-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. ©2011 AACR.

Docosahexaenoic Acid Inhibits UVB-Induced Activation of NF-κB and Expression of COX-2 and NOX-4 in HR-1 Hairless Mouse Skin by Blocking MSK1 Signaling

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.

Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo

Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.

Ultraviolet B radiation activates NF-κB and induces iNOS expression in HR-1 hairless mouse skin: Role of IκB kinase-β†

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/m2) 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.

Abstract B67: Curcumin induces stabilization of Nrf2 protein by decreasing the activity of Cullin3-Rbx1 E3 ubiquitin ligase

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.

Abstract 847: Resolvin D1 stimulates efferocytosis through p50/p50-mediated suppression of tumor necrosis factor-α

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Phagocytosis of apoptotic neutrophils by macrophages, called efferocytosis, is critical to resolution of inflammation as this process prevents the exposure of surrounding tissues at the inflammatory site to the toxic contents of lytic cells. Docosahexaenoic acid-derived resolvin D1 (RvD1), endogenously generated during resolution of inflammation, is known to stimulate efferocytosis, but little is known about the mechanism of its action. In the present study, we found that lipopolysaccharide (LPS; 200 μg/ml) suppressed efferocytosis by murine macrophage-like RAW264.7 cells, and RvD1 (50 nM) restored the phagocytic ability of these cells by down-regulating TNF-α expression. The inhibitory effect of RvD1 on LPS-induced TNF-α expression was associated with enhanced nuclear localization of p50/p50 homodimer and concomitant reduction in the nuclear levels of p65/p50 heterodimer. RvD1 triggered extracellular signal-regulated kinase (ERK)- and Akt-mediated nuclear factor κB1 (NF-κB1) p105 degradation and subsequently translocation of p50/p50 homodimer to nucleus in RAW264.7 cells. In contrast, LPS-induced nuclear translocation and DNA binding of p65/p50 heterodimer were attenuated by RvD1 treatment through inhibition of IκBα degradation. siRNA knockdown of NF-κB p50 abolished RvD1-mediated suppression of TNF-α expression and resulted in impaired efferocytosis, suggesting that the replacement of p65/p50 with p50/p50 homodimer is an essential event for RvD1-mediated stimulation of efferocytosis. In a murine peritonitis model, intraperitoneal administration of RvD1 (300 ng) abrogated zymosan A (30 mg/kg)-induced TNF-α production, thereby reactivating efferocytosis. In addition, RvD1 significantly reduced polymorphonuclear leukocyte infiltration while it increased monocyte infiltration. Taken together, these findings indicate that RvD1 expedites resolution of inflammation via p50/p50-mediated repression of TNF-α production. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2011-847

Abstract B72: Resolvin D1, derived from n-3 polyunsaturated fatty acid, stimulates efferocytosis and protects macrophages from oxidative stress-induced apoptosis

Phagocytosis of apoptotic neutrophils by macrophages, called efferocytosis, is critical to resolution of inflammation as this process prevents the exposure of surrounding tissues at the inflammatory site to the toxic contents of lytic cells. Docosahexaenoic acid (DHA)-derived resolvin D1 (RvD1), endogenously generated during resolution of inflammation, is known to stimulate efferocytosis, but little is known about the mechanism of its action. In the present study, we found that lipopolysaccharide (LPS; 200 g/ml) suppressed efferocytosis by murine macrophage-like RAW264.7 cells, and RvD1 (50 nM) restored the phagocytic ability of these cells by down-regulating TNF-α expression. The inhibitory effect of RvD1 on LPS-induced TNF-α expression was associated with enhanced nuclear localization of p50/p50 homodimer and concomitant reduction in the nuclear levels of p65/p50 heterodimer. siRNA knockdown of NF-κB p50 abolished RvD1-mediated suppression of TNF-α expression and resulted in impaired efferocytosis, suggesting that the replacement of p65/p50 with p50/p50 homodimer is an essential event for RvD1-mediated stimulation of efferocytosis. In a murine peritonitis model, intraperitoneal administration of RvD1 (300 ng) abrogated zymosan A (30 mg/kg)-induced TNF-α production, thereby reactivating efferocytosis. In addition, RvD1 protected RAW264.7 cells from oxidative stress-induced apoptosis during efferocytosis. The generation of reactive oxygen species after efferocytosis was markedly blocked in RvD1-exposed macrophages. Moreover, RvD1 upregulated the anti-apoptotic Bcl-2 and Bcl-xL expression and downregulated the proapoptotic Bax and Bad expression. In conclusion, RvD1 expedites resolution of inflammation through stimulating efferocytosis while preserving viability of macrophage during efferocytosis. These findings account, at least in part, for the chemopreventive effects of DHA on inflammation-associated carcinogenesis. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B72.

Abstract 5665: Zerumbone inhibits phorbol ester-induced intracellular ROS accumulation and neoplastic transformation by inducing Nrf2-driven heme oxygenase-1 expression in mouse epidermal cells

Zerumbone, derived from tropical ginger (Zingibers zerumbet, Zingiberaceae), was found to suppress chemically-induced skin tumor formation in mice. However, the molecular mechanisms underlying the chemopreventive effects of zerumbone on mouse skin carcinogenesis are poorly understood. Here, we report that topical application of zerumbone onto dorsal skin of HR-1 hairless mice induced expression of heme oxygenase-1 (HO-1) through activation of Nrf2, a major transcription factor that plays a pivotal role in regulating expression of several antioxidant/detoxifying enzymes. To verify the possible involvement of Nrf2 in zerumbone-derived induction of HO-1, we compared HO-1 protein levels in the zerumbone-treated skin of Nrf2 wild-type (+/+) and Nrf2 knock out (−/−) mice. Nrf2-deficient mice expressed HO-1 in the skin to a much lesser extent than did the wild-type animals following topical application of zerumbone (10 μmol). Moreover, zerumbone (10 μM) inhibited anchorage-independent growth as well as ROS production in mouse epidermal JB6 cells stimulated with 12-0-tetradecanoylphorbol-13-acetate (TPA; 10 ng/ml). Likewise, treatment of JB6 cells with zerumbone resulted in a markedly increased nuclear translocation of Nrf2 as well as the promoter activity of HO-1. In addition, siRNA knock down of nrf2 in JB6 cells diminished the zerumbone-induced expression of HO-1. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that zerumbone induced the direct binding of Nrf2 to antioxidant response element (ARE). We hypothesized that the electrophilic α,β-unsaturated carbonyl moiety present in zerumbone could modify critical cysteine thiols of Keap1, a cytosolic repressor of Nrf2, thereby facilitating its dissociation from Keap1 for nuclear translocation. α-Humulene and 8-hydroxy-α-humulene, structural derivatives of zerumbone that lack the α,β-unsaturated carbonyl group, were unable to activate Nrf2 and induce HO-1 expression in both mouse skin in vivo and cultured mouse epidermal cells. Unlike zerumbone, these non-electrophilic analogs failed to suppress TPA-induced JB6 cell transformation and ROS accumulation. Interestingly, treatment of JB6 cells with 50 μM carbon monoxide-releasing molecule (CORM) markedly reduced ROS accumulation indicating that carbon monoxide formed as a consequence of zerumbone-induced HO-1 expression has an important role in abolishing TPA-induced ROS generation. Taken together, the above findings suggest that up-regulation of HO-1 by zerumbone is mediated via the Nrf2 signaling pathway, which provides a mechanistic basis for the anti-tumor promoting effects of this sesquiterpene on mouse skin carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5665.

Abstract 5663: Docosahexaenoic acid protects against UV-induced oxidative stress through upregulation of heme oxygenase-1 and NAD(P)H: Quinone oxidoreductase in mouse epidermal cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC UV radiation is an important environmental factor in the pathogenesis of skin aging and cancer. Exposure of mammalian skin to UV results in oxidative damage, caused by increased cellular levels of reactive oxygen species (ROS). These include DNA breaks, protein inactivation, altered gene expression, loss of membrane lipid-bound essential polyunsaturated fatty acids and apoptosis. Omega-3 fatty acids, which occur at high levels in some fish oils, are known to have ROS scavenging activity and exert protective effects against photoaging and cancer. Their chemopreventive effects against UV-induced carcinogenesis are thought to be mediated through induction of antioxidant / phase 2 detoxifying enzymes, which are involved in elimination of ROS. In our present study, docosahexaenoic acid (DHA), a representative ω-3 fatty acid, significantly up-regulated the expression of two representative antioxidant enzymes, heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase (NQO1) in a time-dependent manner with maximal induction observed at 12 hr and 24 hr, respectively in mouse epidermal JB6 cells. Nuclear factor E2-related factor 2 is a principal transcription factor that plays a pivotal role in the induction of many genes encoding antioxidant and other cytoprotective proteins. DHA (50 μM) treatment enhanced nuclear translocation of Nrf2 and its subsequent binding to antioxidant response element (ARE). DHA treatment increased the activity of mouse HO-1 promoter in JB6 cells as as assessed by the luciferase reporter assay. siRNA knock down of Nrf2 abrogated DHA-induced expression of HO-1 and NQO1. Moreover, DHA pretreatment significantly suppressed intracellular ROS formation in response to UVB. Over expression of HO-1 vector in JB6 cell abolished the ROS generation induced by UVB. These findings, taken together, suggest that DHA exerts protective effects against UVB-induced oxidative stress in mouse epidermal cells through Nrf2-induced expression of HO-1 and NQO1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5663.