Original Paper | Published:

[6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-κB in phorbol ester-stimulated mouse skin

Oncogenevolume 24pages25582567 (2005) | Download Citation

Subjects

Abstract

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. The present study was aimed at unraveling the molecular mechanisms underlying previously reported antitumor promoting effects of [6]-gingerol in mouse skin in vivo. One of the well-recognized molecular targets for chemoprevention is cyclooxygenase-2 (COX-2) that is abnormally upregulated in many premalignant and malignant tissues and cells. In our present study, topical application of [6]-gingerol inhibited COX-2 expression in mouse skin stimulated with a prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the transcription factor nuclear factor-kappaB (NF-κB) is known to regulate COX-2 induction, we attempted to determine the effect of [6]-gingerol on TPA-induced activation of NF-κB. Pretreatment with [6]-gingerol resulted in a decrease in both TPA-induced DNA binding and transcriptional activities of NF-κB through suppression of IκBα degradation and p65 nuclear translocation. Phosphorylation of both IκBα and p65 was substantially blocked by [6]-gingerol. In addition, [6]-gingerol inhibited TPA-stimulated interaction of phospho-p65-(Ser-536) with cAMP response element binding protein-binding protein, a transcriptional coactivator of NF-κB. Moreover, [6]-gingerol prevented TPA-induced phosphorylation and catalytic activity of p38 mitogen-activated protein (MAP) kinase that regulates COX-2 expression in mouse skin. The p38 MAP kinase inhibitor SB203580 attenuated NF-κB activation and subsequent COX-2 induction in TPA-treated mouse skin. Taken together, our data suggest that [6]-gingerol inhibits TPA-induced COX-2 expression in mouse skin in vivo by blocking the p38 MAP kinase-NF-κB signaling pathway.

Introduction

Multiple lines of evidence suggest that inflammation is causally linked to carcinogenesis (Balkwill and Coussens, 2004; Greten and Karin, 2004; Pikarsky et al., 2004). Inappropriate upregulation of cyclooxygenase (COX)-2, a rate-limiting enzyme involved in prostaglandin (PG) biosynthesis and inflammation, has been frequently observed in various premalignant and malignant tissues (Williams et al., 1999; Mohan and Epstein, 2003). Further evidence for the implication of COX-2 in tumorigenesis has been corroborated by increased susceptibility of COX-2 overexpressing transgenic mice (Muller-Decker et al., 2002) and relative resistance of COX-2 knockout animals (Tiano et al., 2002) to spontaneous or experimentally-induced carcinogenesis. Therefore, targeted inhibition of COX-2 is now regarded as a promising and rational strategy to prevent cancer (Subbaramaiah and Dannenberg, 2003). Numerous dietary phytochemicals have been shown to inhibit COX-2 induction both in in vitro and in vivo models of carcinogenesis (Bode and Dong, 2004; Surh, 2002).

Recent progress in our understanding of cellular and molecular biology led us to have a deep insight into the complex network of intracellular signaling molecules or events involved in multistage carcinogenesis. Like other early-response gene products, COX-2 can be induced rapidly and transiently by proinflammatory mediators and mitogenic stimuli including cytokines, endotoxins, growth factors, oncogenes, and phorbol ester (Cao and Prescott, 2002). It has been reported that topical application of a prototype tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA) induces expression of COX-2 and its mRNA transcript in mouse skin by activating eucaryotic transcription factors, such as nuclear factor kappaB (NF-κB) and activator protein-1 (AP-1), which are, in turn, regulated by a series of upstream kinases collectively known as mitogen-activated protein (MAP) kinases (Chun et al., 2003, 2004). Modulation of abnormal upregulation of such intracellular signaling molecules is now recognized as the molecular basis of chemoprevention by structurally diverse dietary phytochemicals.

Ginger (Zingiber officinale Roscoe, Zingiberaceae) has been used as a condiment throughout the world for more than 2500 years. Besides its extensive use as a spice, the rhizome of ginger has been utilized in traditional oriental medicine to ameliorate such symptoms as inflammation, rheumatic disorders and gastrointestinal discomforts (Newall et al., 1996). Among the major pungent principles of ginger, [6]-gingerol (1-[4’-hydroxy-3’-methyoxyphenyl]-5-hydroxy-3-decanone; Figure 1) exhibits diverse pharmacological effects including antioxidant and anti-inflammatory activities (Surh, 1999). [6]-Gingerol has also been reported to inhibit TPA-mediated tumor promotion, induction of ornithine decarboxylase activity and tumor necrosis factor-alpha (TNF-α) production in mouse skin (Park et al., 1998). More recently, [6]-gingerol has been found to inhibit epidermal growth factor-induced AP-1 activation and neoplastic transformation in mouse epidermal JB6 cells (Bode et al., 2001). However, the exact molecular mechanisms underlying the chemopreventive effect of [6]-gingerol remain largely unresolved.

Figure 1
Figure 1

Chemical structure of [6]-gingerol

In the present study, we attempted to investigate the effect of [6]-gingerol on TPA-induced COX-2 expression in mouse skin in vivo and to explore underlying molecular mechanisms. Our study revealed that topical application of [6]-gingerol significantly inhibited TPA-induced COX-2 expression by blocking the activation of p38 MAP kinase and NF-κB.

Results

Inhibitory effects of [6]-gingerol on TPA-induced COX-2 expression in mouse skin

The prototype tumor promoter TPA is one of the potent inducers of COX-2 expression in various cell lines (Lee et al., 2000; Grab et al., 2004) as well as in mouse skin in vivo (Chun et al., 2003; Kundu et al., 2003). Previous studies from this laboratory have demonstrated that topical application of TPA (10 nmol) onto shaven back of female ICR mice induces the expression of COX-2 protein maximally at 4 h (Chun et al., 2003). In the present study, topical application of [6]-gingerol (30 μmol) 30 min prior to TPA resulted in a statistically significant (P<0.001) decrease in the level of COX-2 protein in mouse skin 4 h after TPA treatment (Figure 2a), while the expression of the house-keeping enzyme COX-1 remained almost unchanged. The inhibition of TPA-stimulated COX-2 expression by [6]-gingerol (30 μmol) was comparable to that achieved with the reference compound curcumin (10 μmol). In addition, immunohistochemical analysis verified the TPA-induced expression of COX-2, predominantly localized in the epidermal layer, which was significantly (P<0.001) abolished by pretreatment with [6]-gingerol (Figure 2b and c).

Figure 2
Figure 2

Inhibitory effects of [6]-gingerol on phorbol ester-induced COX-2 expression in mouse skin. Female ICR mice were treated topically with either [6]-gingerol (5 or 30 μmol) or curcumin (10 μmol) dissolved in 0.2 ml acetone. After 30 min, mice were treated topically with 10 nmol TPA in 0.2 ml acetone and killed 4 h later. Control animals were treated with acetone in lieu of TPA. (a) Total cell lysates were analysed for COX-1 and COX-2 expression by immunoblotting. Quantification of COX-2 immunoblot was normalized to that of actin followed by statistical analysis of relative image density in comparison to control (*P<0.001). (b) Mice treated as mentioned above were killed after 4 h of TPA application. Skin samples from mice treated with acetone, TPA alone and [6]-gingerol (30 μmol) plus TPA were subjected to immunohistochemical analysis by using COX-2 antibody as described in Materials and methods. Positive COX-2 staining yielded a brown-colored product (arrow). (c) Percent of COX-2 positivity in epidermal layer was determined by counting the number of total and COX-2-positive cells from 10 equal sections of immunostained tissues from each animal (*P<0.001)

Inhibition of TPA-stimulated activation of NF-κB by [6]-gingerol in mouse skin

Since the induction of COX-2 is frequently regulated by NF-κB (Nakao et al., 2000; Lim et al., 2001; Surh et al., 2001), we attempted to examine the effects of [6]-gingerol on TPA-stimulated DNA binding and transcriptional activity of NF-κB in mouse skin. For this purpose, nuclear extracts from TPA-treated mouse skin, with or without [6]-gingerol pretreatment, were analysed by EMSA using the oligonucleotide harboring the NF-κB binding sequence present in the mouse COX-2 promoter region. As shown in Figure 3a, [6]-gingerol caused significant inhibition of TPA-induced DNA binding of NF-κB. Since NF-κB exists predominantly as a heterodimer of p50 and p65/RelA proteins and its binding to DNA in response to mitogenic stimuli depends on the nuclear translocation of these subunits (Baldwin, 1996; May and Ghosh, 1997), we examined the effect of [6]-gingerol on TPA-stimulated nuclear translocation of both p50 and p65/RelA. In agreement with its inhibitory effect on NF-κB DNA binding, [6]-gingerol reduced the levels of p50 and p65/RelA in nuclear fractions prepared from TPA-treated mouse skin (Figure 3b). Immunohistochemical analysis revealed that topical application of 10 nmol TPA increased both positivity and nuclear translocation of p65, a major functionally active subunit of NF-κB, in the epidermal layer (Figure 3c). Pretreatment with [6]-gingerol prevented TPA-induced positivity as well as nuclear accumulation of p65, which further supports the inhibitory effect of [6]-gingerol on the NF-κB signaling in TPA-stimulated mouse skin (Figure 3c). In addition, we investigated the effect of [6]-gingerol on the transcriptional activity of NF-κB in transgenic mice harboring the NF-κB luciferase reporter gene. Our study revealed that topical application of TPA (10 nmol) induced NF-κB transactivation as determined by the luciferase reporter gene assay. Pretreatment with [6]-gingerol led to the attenuation of the TPA-stimulated NF-κB luciferase activity (Figure 3d).

Figure 3
Figure 3

Inhibitory effects of [6]-gingerol on phorbol ester-induced activation of NF-κB in mouse skin in vivo. Shaven backs of female ICR mice were treated either with acetone or [6]-gingerol (5 or 30 μmol) 30 min prior to TPA (10 nmol) except the control animal, which was treated with acetone only. At 1 h after the treatment, the epidermal nuclear extracts were prepared. (a) Nuclear protein (10 μg) was incubated with the radiolabeled oligonucleotide containing the NF-κB consensus sequence for analysis by EMSA. Lane 1, acetone control; lane 2, TPA alone; lane 3, [6]-gingerol (5 μmol)+TPA; lane 4, [6]-gingerol (30 μmol)+TPA; lane 5, free probe alone (no nuclear extracts); lane 6, TPA-treated sample+100-fold excess unlabeled oligonucleotide. (b) Nuclear protein (40 μg) was separated by 12% SDS–polyacrylamide gel and immunoblot was performed by using a primary antibody specific to detect p50 or p65. (c) Mice treated as mentioned above were killed after 1 h of TPA application. Skin samples from mice treated with acetone, TPA alone and [6]-gingerol (30 μmol) plus TPA were subjected to immunohistochemical analysis by using anti-p65 antibody as described in Materials and methods. Positive p65 staining yielded a partial brown-colored product, while cells showing complete red staining indicated nuclear translocation of p65. Images in the inset represent respective areas of the immunostained epidermis showing positivity and nuclear translocation of p65. (d) Transgenic mice harboring NF-κB luciferase gene were treated topically with either [6]-gingerol (30 μmol) or acetone 30 min prior to TPA, while control animals were treated with acetone only. Skin tissue lysates, prepared after 4 h of TPA treatment, were subjected to the luciferase reporter gene assay according to the protocol described in Materials and methods (*P<0.001)

Inhibition of TPA-induced phosphorylation of IκBα and p65 by [6]-gingerol

The nuclear translocation of NF-κB, which remains sequestered in the cytoplasm as an inactive complex with an inhibitory protein such as IκBα, is dependent on the release of p65–p50 heterodimer as a consequence of phosphorylation and subsequent degradation of its inhibitory counterpart (Karin, 1999). We, therefore, examined whether [6]-gingerol could block TPA-induced phosphorylation of IκBα as a mechanism of its inhibitory effect on p65/RelA nuclear translocation. As shown in Figure 4a, TPA-induced phosphorylation of IκBα at serine-32 and -36 residues was inhibited by [6]-gingerol.

Figure 4
Figure 4

Effects of [6]-gingerol on the phosphorylation of IκBα and p65. (a) Mice were treated topically with acetone or [6]-gingerol (5 or 30 μmol) 30 min prior to TPA and killed 1 h later. Cytosolic extract was prepared as described in Materials and methods. The expression of phosphorylated IκBα was measured by immunoblot analysis of cytosolic proteins (50 μg) after separation over 10% SDS–polyacrylamide gel. Data presented are representative of two independent experiments showing similar trend. (b) Shaved back of female ICR mice were treated either with acetone or TPA (10 nmol) for indicated periods. Total tissue lysates (60 μg) were separated by 10% SDS–polyacrylamide gel, transferred to PVDF membrane, and immunobloted by using phospho-p65-(Ser-536) antibody. The immunoblot is representative of two independent experiments eliciting similar pattern. (c) Whole-tissue extracts (50 μg) prepared from mouse skin treated with either acetone or [6]-gingerol followed by TPA treatment were subjected to immunoblot analysis to determine the level of phospho-p65-(Ser-536). Control animals were treated with acetone in lieu of TPA. (d) Inhibitory effect of [6]-gingerol on the interaction between CBP and phospho-p65. Mice were treated as described in Figure 3. Nuclear extracts (200 μg) were immunoprecipitated by using CBP antibody. The immunoprecipitate was separated by running through 10% SDS–polyacrylamide gel and immunobloted with a specific antibody against phospho-p65-(Ser-536). IP, immunoprecipitation

The transcriptional activation of NF-κB-regulated genes requires not only the increased binding of NF-κB to their promoter regions, but also the phosphorylation of p65/RelA, which is the functionally active subunit of NF-κB. The phosphorylation of p65/RelA at serine 536 residue, located on its transactivation domain (TAD), has been reported as a critical event for transcriptional activation of target genes (Jiang et al., 2003). A kinetic study showed that TPA induced a gradual increase in p65/RelA phosphorylation at serine 536 residue (Figure 4b). [6]-Gingerol pretreatment significantly inhibited phosphorylation of p65-(Ser-536) induced by TPA (Figure 4c).

Inhibitory effect of [6]-gingerol on the interaction of phospho-p65-(Ser-536) with CBP

It has been demonstrated that CBP, by dint of its intrinsic histone acetyl transferase activity, acts as a transcriptional coactivator and that the interaction of CBP with phospho-p65/RelA is a critical event in recruiting other key components of the transcriptional machinery to form a transcription initiation complex (Zhong et al., 1998). To explore the mechanism underlying the inhibition of NF-κB transcriptional activity by [6]-gingerol, we examined the effect of this compound on CBP interaction with p65/RelA phosphorylated at serine 536 residue. Nuclear extracts from TPA-treated mouse skin with or without [6]-gingerol pretreatment was immunoprecipitated with anti-CBP antibody, followed by Western blotting using a specific antibody against phospho-p65-(Ser-536). Topical application of TPA resulted in dramatic increase in the interaction of CBP with phospho-p65-(Ser-536) in mouse skin, which was strongly inhibited by [6]-gingerol pretreatment (Figure 4d).

Effects of [6]-gingerol on activation of MAP kinases in TPA-treated mouse skin

MAPKs were reported to regulate, at least in part, the TPA-induced COX-2 expression in mouse skin (Chun et al., 2003). Therefore, we determined the effect of [6]-gingerol on TPA-induced activation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 MAP kinase and c-Jun-N-terminal kinase (JNK), which are representative MAP kinases involved in a wide array of cellular signaling cascades (Surh, 2003). According to Chun et al. (2003), the activation of ERK1/2 and p38 MAP kinase via phosphorylation peaked at 1 h and sustained up to 4 h after topical application of TPA in mouse skin. Western blot analysis revealed that [6]-gingerol suppressed TPA-induced phosphorylation of p38 MAP kinase, but had no inhibitory effect on ERK1/2 phosphorylation (Figure 5a). Nonradioactive kinase assay revealed that [6]-gingerol, in addition to its inhibitory effect on phosphorylation of p38, inhibited the catalytic activity of the enzyme as indicated by reduced phosphorylation of its substrate protein ATF-2 (Figure 5b). JNK is another representative MAP kinase that plays an important role in regulating cell proliferation and tumor promotion (Huang et al., 1999; Chen et al., 2001). JNK is more likely to contribute to the activation of AP-1 rather than that of NF-κB (Karin and Delhase, 1998). When topically applied, TPA induced JNK phosphorylation and AP-1 DNA binding (data not shown).

Figure 5
Figure 5

Effects of [6]-gingerol on p38 MAP kinase-mediated regulation of NF-κB activation. Shaven back of female ICR mice were treated with [6]-gingerol (5 or 30 μmol) 30 min before TPA treatment. Control animals were treated with acetone instead of TPA. Animals were killed at either 1 or 4 h after TPA treatment and total protein was isolated from the dorsal skin and quantified. (a) Protein isolated after 4 h treatment was analysed for pERK1/2, phospho-p38 and phospho-JNK by immunoblotting. Blots are representative of three independent experiments. (b) For the measurement of p38 MAP kinase activity, cell lysates (200 μg) obtained after 1 h treatment were subjected to immunoprecipitation by using an immobilized phospho-p38 monoclonal antibody. The resulting immunoprecipitate was then incubated with ATF-2 fusion protein in the presence of 100 mM ATP. ATF-2 phosphorylation, as a measure of the p38 MAP kinase activity, was determined by a nonradioactive method using phospho-ATF-2 antibody. Data are representative of two independent experiments, which gave rise to a similar trend. (c) Mice were treated on the shaved back area with acetone or SB203580 (4 or 8 μmol) dissolved in 0.2 ml acetone. After 30 min, TPA (10 nmol) was applied to all except the control animals, which were treated with acetone instead of TPA. The expression of pIκBα was measured by Western blot analysis of whole-tissue lysates. Data are representative of two individual experiments. (d) Animals were treated as described in Figure 5c. Western blot analysis of whole-cell extract was performed to determine the effect of SB203580 on the phosphorylation of p-65-(Ser-536) using phospho-specific antibody

Effects of the p38 MAP kinase inhibitor on TPA-induced phosphorylation of IκBα and p65

To delineate the inhibitory effect of [6]-gingerol on TPA-induced p38 MAP kinase activation and its blockade of p65/RelA nuclear translocation, we examined the effect of SB203580, a specific inhibitor of p38 MAP kinase, on TPA-induced NF-κB activation. Topical application of SB203580 resulted in the inhibition of TPA-induced phosphorylation of IκBα (Figure 5c). It was evident from our study that pharmacological inhibition of p38 MAP kinase attenuated TPA-induced p65/RelA phosphorylation at serine 536 residue in a similar way to that of [6]-gingerol (Figure 5d). These findings suggest that TPA-induced activation of p38 MAP kinase may regulate, at least in part, the activation of NF-κB in mouse skin in vivo.

Discussion

Multistage carcinogenesis involves aberrant alterations in intracellular signal transduction pathways resulting in neoplastic conversion of cells. Abnormal upregulation or silencing of many of these intracellular signaling cascades is a reversible processes and may be subjected to intervention with dietary phytochemicals, which may block or reverse deleterious changes in cellular signaling thereby preventing cancer (Bode and Dong, 2000; Nomura et al., 2000; Surh, 2003). As a result of a causal relationship between inflammation and cancer, COX-2 has been recognized as one of the potential molecular targets for chemoprevention (Surh, 2003). Multiple lines of evidence arising from both population-based and laboratory studies suggest that the targeted inhibition of inappropriate overexpression or activity of COX-2 by agents capable of specifically inhibiting this enzyme is effective in preventing certain malignancies (Asano and McLeod, 2004; Chun and Surh, 2004; Gupta et al., 2004; Kismet et al., 2004). The induction of COX-2 in mouse skin is regulated, at least in part, by an eucaryotic transcription factor NF-κB (Chun et al., 2003; Surh, 2003), which makes a bridge between inflammation and tumor promotion (Greten and Karin, 2004). In our study, [6]-gingerol was found to inhibit TPA-induced DNA binding activity of NF-κB by suppressing phosphorylation of IκBα and subsequent nuclear translocation of p50 and p65/RelA subunits of NF-κB.

Although the nuclear translocation of NF-κB has always been highly regarded as the critical event required for the induction of NF-κB-dependent gene expression (Baldwin, 1996; May and Ghosh, 1997), an increasing body of data suggest that the down-regulation of NF-κB DNA-binding activity is not necessarily associated with its reduced transcriptional activity (Harnish et al., 2000; Takahashi et al., 2002). It has been reported that inhibitors of several upstream kinases, such as phosphatidylionositol-3 kinase/Akt, p38 MAP kinase and protein kinase A (PKA), may block the transcriptional activity of NF-κB without affecting its nuclear translocation (Wesselborg et al., 1997; Zhong et al., 1998; Sizemore et al., 1999; Madrid et al., 2001). The physiological importance of transactivation potential of NF-κB was evident in genetically engineered mice lacking glycogen synthase kinase 3β or T2K (TBK1). Cells generated from these knockout animals are still capable of inducing nuclear translocation, but are unable to stimulate transcriptional activity of NF-κB (Bonnard et al., 2000; Hoeflich et al., 2000). Moreover, the efficient transcriptional activation of NF-κB depends on the phosphorylation of its active subunit p65/RelA (Ghosh and Karin, 2002). The proinflammatory cytokines, TNF-α and interleukin-1 have been shown to stimulate p65/RelA phosphorylation and subsequent NF-κB transactivation via mechanisms distinct from those that involve the IκBα phosphorylation and subsequent nuclear translocation of NF-κB (Baldwin, 1996; Bird et al., 1997; Wang and Baldwin, 1998; Sizemore et al., 1999).

Since the activation of MAP kinases has been known to regulate phorbol ester-induced COX-2 expression in mouse skin (Chun et al., 2003, 2004), the inhibition of both phosphorylation and catalytic activity of p38 MAP kinase by [6]-gingerol suggests that its inhibitory effect on COX-2 expression is likely to be mediated via the p38 MAP kinase-mediated signal transduction pathways. The phosphorylation of p38 MAP kinase may, in turn, lead to phosphorylation-dependent activation of diverse transcription factors including AP-1, cAMP response element binding (CREB) protein and NF-κB (Bhat et al., 2002; Saccani et al., 2002; Wilms et al., 2003), which are known to differentially regulate the induction of COX-2 depending on the tissues/cells and stimuli. According to Chun et al. (2004), the activation of the transcription factor AP-1, which results in the upregulation of COX-2 in TPA-treated mouse skin, is regulated by p38 MAP kinase. Since [6]-gingerol showed an inhibitory effect on TPA-induced activation of p38 MAP kinase, it may also inhibit DNA binding of AP-1 in mouse skin stimulated with TPA. Several lines of evidence suggest that p38 MAP kinase may regulate transcriptional activity of NF-κB (Madrid et al., 2001; Saccani et al., 2002; Wilms et al., 2003). To verify the role of p38 MAP kinase as a putative upstream kinase in regulating the activation of NF-κB, the effect of p38 MAP kinase inhibitor SB203580 on the phosphorylation of both IκBα and p65-(Ser-536) was investigated. Topical application of SB203580 suppressed TPA-induced phosphorylation of IκBα, which was in agreement with a recent study by Liao et al. (2004) who demonstrated the inhibition of IκBα phosphorylation by SB203580 in LPS-treated Raw 264.7 cells. We found that SB203580 suppressed TPA-induced phosphorylation of p65 at serine 536, suggesting that p38 MAP kinase regulates nuclear translocation and subsequent transcriptional activation of NF-κB in mouse skin in vivo.

The activity of many inducible transcription factors is regulated through their interaction with transcriptional coactivators such as CBP/p300, which is believed to link enhancer-bound transcription factors with general transcription machinery (Zhong et al., 1998). CBP/p300 has an intrinsic acetyltransferase activity that regulates gene expression, in part, through acetylation of the N-terminal tails of histones. Acetylated histones are associated with transcriptionally active segments of chromatin, whereas deacetylated histones accumulate in transcriptionally repressed regions (Imhof and Wolffe, 1998; Kuo and Allis, 1998). It has been reported that cotransfection of cells with CBP/p300 enhances NF-κB-dependent transcription (Gerritsen et al., 1997). Zhong et al. (1998) have demonstrated that the association of NF-κB with CBP/p300 occurs either by a phosphorylation-independent mechanism or through PKA-dependent phosphorylation of p65/RelA. In the present study, we note that [6]-gingerol abrogated the interaction between CBP and phospho-p65-(Ser-536). Thus, the inhibitory effect of [6]-gingerol on the TPA-stimulated NF-κB transcriptional activity appears to be attributable to its interference with interaction between CBP and phospho-p65-(Ser-536). These findings further support the possibility that the inhibitory effect of [6]-gingerol on COX-2 expression is mediated through suppression of NF-κB transactivation.

Recently, it has been demonstrated that the phosphorylation of the serine residue at 276 located on the Rel homology domain (RHD) of p65/RelA facilitates the interaction of CBP with p65/RelA (Zhong et al., 1998). Therefore, the possible inhibitory effect of [6]-gingerol on phosphorylation of p65/RelA not only at serine 536 but also at serine 276 on RHD cannot be excluded.

The phosphorylation of both IκB and NF-κB is attributed to an upstream regulator IκB kinase (IKK) complex (Sakurai et al., 1999; Yang et al., 2003). A recent study suggests that within the IKK complex, IKKα is solely responsible for p65 phosphorylation, whereas IKKβ is capable of phosphorylating both IκBα and p65 (Sizemore et al., 2002). However, the present study clearly demonstrates that TPA-induced phosphorylation of both IκBα and p65-(Ser-536) is regulated by p38 MAP kinase. Although [6]-gingerol prevented phosphorylation of both IκBα and p65 by targeting p38 MAP kinase, a simultaneous modulation of IKK activity may also comprise part of the molecular mechanism underlying the inhibition of NF-κB transactivation by [6]-gingerol in TPA-stimulated mouse skin.

In conclusion, [6]-gingerol inhibited TPA-induced COX-2 expression via the p38 MAP kinase-NF-κB signaling cascade (Figure 6) in mouse skin in vivo, which provides a mechanistic basis of anti-inflammatory activity of [6]-gingerol. There has been increasing evidence for a causal relationship between inflammation and cancer (Clevers, 2004). Current progress in searching for the molecular targets linking inflammation and cancer has identified NF-κB as a prime proinflammatory mediator, which by promoting transcription of proinflammatory genes, including COX-2, makes the microenvironment of an inflamed tissue as a favorable ground for malignant transformation (Aggarwal, 2004; Balkwill and Coussens, 2004; Pikarsky et al., 2004). Therefore, the inhibition of TPA-induced COX-2 expression through blockade of p38 MAP kinase-NF-κB signaling pathway by [6]-gingerol provides the molecular basis for its previously reported antitumor-promoting effects.

Figure 6
Figure 6

Proposed signaling pathways blocked by [6]-gingerol in its suppression of TPA-induced NF-κB activation and COX-2 expression in mouse skin. Topical application of TPA activates p38 MAP kinase, which, in turn, phosphorylates both IκBα and p65 thereby contributing to the activation of NF-κB and subsequent induction of COX-2. Phosphorylation of p65 at Ser-536 may facilitate the nuclear translocation of NF-κB and its interaction with other components of the transcription initiation complex, particularly CBP/p300. Pretreatment with [6]-gingerol inhibits TPA-induced COX-2 expression by targeting p38 MAP kinase and its downstream signaling events leading to the inactivation of NF-κB

Materials and methods

Materials

[6]-Gingerol (purity >98%) was purchased from Wako Pure Chemicals (Osaka, Japan). TPA was supplied from Alexis Biochemicals (San Diego, CA, USA). Rabbit polyclonal COX-1 and COX-2 antibodies were products of Cayman Chemical Co. (Ann Arbor, MI, USA). Primary antibodies for ERK, pERK, p38, pp38, JNK, pJNK, p50 and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-IκBα and anti-phospho-p65-(Ser-536) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-rabbit, anti-goat and anti-mouse horseradish peroxidase-conjugated secondary antibodies were products of Zymed Laboratories (San Francisco, CA, USA). An oligonucleotide probe containing the NF-κB consensus sequence in mouse COX-2 promoter region was obtained from Bionics (Seoul, Korea). Enhanced chemiluminescence (ECL) detection kit and [γ-32P]ATP were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Animal treatment

Female ICR mice (6–7 weeks of age) were supplied from the Dae-Han/Biolink Experimental Animal Center (Daejeon, Korea). NK-κB luciferase transgenic mice (6–7 weeks of age) were initially provided by Dr Nancy Colburn of National Cancer Institute, USA, and breeded in College of Veterinary Medicine (Seoul National University, Seoul, Korea). The animals were housed in climate-controlled quarters (24°C at 50% humidity) with a 12 h light/12 h dark cycle. The dorsal side of the skin was shaved using an electric clipper, and only those animals in the resting phase of the hair cycle were used in all experiments. [6]-Gingerol and TPA were dissolved in 200 μl of acetone and applied topically to the dorsal shaved area.

Western blot analysis

The female ICR mice were topically treated on shaved backs with indicated doses of [6]-gingerol 30 min before TPA treatment and were killed by the cervical dislocation either 1 or 4 h later. Total protein was isolated and quantified by using BCA protein assay kit. Collected tissues were homogenized in 800 μl of ice-cold lysis buffer. [150 mM NaCl, 0.5% Triton-X 100, 50 mM Tris–HCl (pH 7.4), 20 mM EGTA, 1 mM DTT, 1 mM Na3VO4 and protease inhibitor cocktail tablet]. Lysates were centrifuged at 14 800 g for 30 min, and aliquots of supernatant containing 30 μg protein were boiled in sodium dodecylsulphate (SDS) sample buffer for 5 min before electrophoresis on 12% SDS–polyacrylamide gel. After transfer to PVDF membrane, the blots were blocked with 5% fat-free dry milk–PBST buffer (phosphate-buffered saline containing 0.1% Tween 20) for 1 h at room temperature and then washed in PBST buffer. The membranes were incubated for 4 h at room temperature with 1 : 1000 dilutions of primary antibodies for COX-1, COX-2, ERK, pERK, p38 and pp38, and for 12 h at 4°C with 1 : 500 dilutions of primary antibodies for JNK, pJNK, pIκBα, p50, p65 and phospho-p65-(Ser-536). Blots were washed three times with PBST at 5 min intervals followed by incubation with 1 : 5000 dilution of respective horseradish peroxidase-conjugated secondary antibodies (rabbit, goat or mouse) for 1 h and again washed in PBST for three times. The transferred proteins were visualized with an ECL detection kit according to the manufacturer's instructions.

Immunohistochemical staining of p65 and COX-2

The dissected skin was prepared for immunohistochemical analysis of the expression pattern of COX-2 and p65 in mouse skin treated with TPA in presence or absence of [6]-gingerol. In all samples, 4 μm sections of 10% formalin-fixed, paraffin-embedded tissues were cut on silanized glass slides and deparaffinized three times with xylene and rehydrated through graded alcohol bath. The deparaffinized sections were heated and boiled twice for 6 min in 10 mM citrate buffer, pH 6.0 for antigen retrieval. To diminish nonspecific staining, each section was treated with 3% hydrogen peroxide and 4% peptone casein blocking solution for 15 min. For the detection of COX-2 and p65, slides were incubated with affinity-purified rabbit polyclonal anti-COX-2 antibody (Cayman Chemical Co., Ann Arbor, MI, USA) and mouse monoclonal anti-p65 antibody (Santa Cruz, CA, USA) at room temperature for 40 min in Tris-buffered saline containing 0.05% Tween-20 and then developed using anti-rabbit and anti-mouse HPR EnVisionTM System (Dako, Glostrup, Denmark), respectively. The peroxidase binding sites were detected by staining with 3,3′-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark). Finally, counterstaining was performed using Mayer's hematoxylin.

MAP kinase assay (nonradioactive)

Enzyme assays for determining the catalytic activities of p38 were carried out by using a nonradioactive MAP kinase assay kit (Cell Signaling Technology, Beverly, MA, USA) as described in the protocol provided by the manufacturer. Collected tissues were lysed in 200 μl of lysis buffer per sample. The lysates were centrifuged, and the supernatant (200 μg) was incubated with specific immobilized phospho-p38 monoclonal antibody with gentle rocking for overnight at 4°C. The beads were washed twice each with 500 μl of lysis buffer and the same volume of kinase buffer. The kinase reactions were carried out in the presence of 100 mM ATP and 2 μg of ATF-2 fusion protein at 30°C for 30 min. Phosphorylation of ATF-2 at threonine 71 residue was selectively measured by immunoblotting with a specific antibody.

Preparation of nuclear extracts from mouse skin

The nuclear extract from mouse skin was prepared as described previously (Chun et al., 2003). In brief, scraped dorsal skin of mice was homogenized in 800 μl of hypotonic buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF)]. To the homogenates was added 80 μl of 10% Nonidet P-40 (NP-40) solution, and the mixture was then centrifuged for 2 min at 14 000 g. The supernatant was collected as cytosolic fraction. The precipitated nuclei were washed once with 500 μl of buffer A plus 40 μl of 10% NP-40, centrifuged, resuspended in 200 μl of buffer C [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 20% glycerol] and centrifuged for 5 min at 14 800 g. The supernatant containing nuclear proteins was collected and stored at −70°C after determination of protein concentrations.

Electrophoretic mobility shift assay

The electrophoretic mobility shift assay (EMSA) for NF-κB DNA binding was performed using a DNA–protein binding detection kit, according to the manufacturer's protocol (GIBCO BRL, Grand Island, NY, USA). Briefly, the NF-κB oligonucleotide probe 5′-GAG GGG ATT CCC TTA-3′ (NF-κB binding site in murine COX-2 promoter region underlined) was labeled with [γ-32P]ATP by T4 polynucleotide kinase and purified on a Nick column (Amersham Pharmacia Biotech, Buckinghamshire, UK). The binding reaction was carried out in 25 μl of the mixture containing 5 μl of incubation buffer [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% glycerol, and 0.1 mg/ml sonicated salmon sperm DNA], 10 μg of nuclear extracts, and 100 000 c.p.m. of [γ-32P]ATP-end labeled oligonucleotide. After 50 min incubation at room temperature, 2 μl of 0.1% bromophenol blue was added, and samples were electrophoresed through 6% nondenaturing polyacrylamide gel at 150 V in a cold room for 2 h. Finally, the gel was dried and exposed to X-ray film.

Measurement of NF-κB luciferase activity in mouse skin

The NF-κB luciferase transgenic mice were identified, grouped and housed in accordance with the Animal treatment protocol of Seoul National University. At 2 weeks after grouping, the basal level of NF-κB luciferase activity was measured with skin punch biopsy. NF-κB luciferase reporter gene-bearing mice were randomly divided into three groups. At 2 weeks after the punch biopsy, mice were treated topically with TPA (10 nmol) in the presence or absence of [6]-gingerol onto shaven backs of mice. Control animals were treated with acetone in lieu of TPA. Skin biopsies and tissues were digested in 100 μl of lysis buffer [0.1 M potassium phosphate buffer (pH 7.8), 1% Triton X-100, 1 mM DTT, and 2 mM EDTA] for the measurement of luciferase activity. The NF-κB-dependent luciferase activity in the tissue extract was determined using the luciferase assay reagent from Promega (Madison, WI, USA). The luciferase assay reaction was verified to measure the linear range. The results were adjusted to activity/μg protein and expressed as relative NF-κB luciferase activity.

Immunoprecipitation assay

Tissue homogenates were prepared from mouse skin treated with TPA either in presence or absence of [6]-gingerol. Cellular proteins (200 μg) were subjected to immunoprecipitation by shaking with CBP primary antibody (Santa Cruz Biotechnology) at 4°C for 12 h followed by the addition of 50% protein G-agarose suspension (20 μl) and additional shaking for 2 h at the same condition. After centrifugation at 14 800 g for 2 min, immunoprecipitated beads were collected by discarding the supernatant and washing with cell lysis buffer. After final wash, immunoprecipitate was resuspended in 40 μl of 2 × SDS electrophoresis sample buffer and boiled for 5 min. Supernatant (20 μl) from each sample was collected after centrifugation and loaded on SDS–polyacrylamide gel (0.75 mm thickness). Following Western blot protocol described earlier, separated proteins were transferred from gel to a PVDF membrane, which was then immunoblotted with phospho-p65-(Ser-536) antibody (Cell Signaling Technology, Beverly, MA, USA) to detect the interaction of phospho-p65-(Ser-536) with CBP.

Statistical evaluation

Values were expressed as the mean±s.e.m. of at least three independent experiments. Statistical significance was determined by Student's t-test and a P-value of less than 0.01 was considered to be statistically significant.

References

  1. Aggarwal BB . (2004). Cancer Cell, 6, 203–208.

  2. Asano TK and McLeod RS . (2004). Dis. Colon Rectum, 47, 665–673.

  3. Baldwin Jr AS . (1996). Annu. Rev. Immunol., 14, 649–683.

  4. Balkwill F and Coussens LM . (2004). Nature, 431, 405–406.

  5. Bhat NR, Feinstein DL, Shen Q and Bhat AN . (2002). J. Biol. Chem., 277, 29584–29592.

  6. Bird TA, Schooley K, Dower SK, Hagen H and Virca GD . (1997). J. Biol. Chem., 272, 32606–32612.

  7. Bode AM and Dong Z . (2000). Lancet Oncol., 1, 181–188.

  8. Bode AM and Dong Z . (2004). Mutat. Res., 555, 33–51.

  9. Bode AM, Ma WY, Surh Y-J and Dong Z . (2001). Cancer Res., 61, 850–853.

  10. Bonnard M, Mirtsos C, Suzuki S, Graham K, Huang J, Ng M, Itie A, Wakeham A, Shahinian A, Henzel WJ, Elia AJ, Shillinglaw W, Mak TW, Cao Z and Yeh WC . (2000). EMBO J., 19, 4976–4985.

  11. Cao Y and Prescott SM . (2002). J. Cell Physiol., 190, 279–286.

  12. Chen N, Nomura M, She QB, Ma WY, Bode AM, Wang L, Flavell RA and Dong Z . (2001). Cancer Res., 61, 3908–3912.

  13. Chun K-S, Keum Y-S, Han S-S, Song Y-S, Kim S-H and Surh Y-J . (2003). Carcinogenesis, 24, 1515–1524.

  14. Chun K-S, Kim S-H, Song Y-S and Surh Y-J . (2004). Carcinogenesis, 25, 713–722.

  15. Chun K-S and Surh Y-J . (2004). Biochem. Pharmacol., 68, 1089–1100.

  16. Clevers H . (2004). Cell, 118, 671–674.

  17. Gerritsen ME, Williams AJ, Neish AS, Moore S, Shi Y and Collins T . (1997). Proc. Natl. Acad. Sci. USA, 94, 2927–2932.

  18. Ghosh S and Karin M . (2002). Cell, 109, S81–S96.

  19. Grab LT, Kearns MW, Morris AJ and Daniel LW . (2004). Biochim. Biophys. Acta, 1636, 29–39.

  20. Greten FR and Karin M . (2004). Cancer Lett., 206, 193–199.

  21. Gupta S, Adhami VM, Subbarayan M, MacLennan GT, Lewin JS, Hafeli UO, Fu P and Mukhtar H . (2004). Cancer Res., 64, 3334–3343.

  22. Harnish DC, Scicchitano MS, Adelman SJ, Lyttle CR and Karathanasis SK . (2000). Endocrinology, 141, 3403–3411.

  23. Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O and Woodgett JR . (2000). Nature, 406, 86–90.

  24. Huang C, Li J, Ma WY and Dong Z . (1999). J. Biol. Chem., 274, 29672–29676.

  25. Imhof A and Wolffe AP . (1998). Curr. Biol., 8, R422–R424.

  26. Jiang X, Takahashi N, Ando K, Otsuka T, Tetsuka T and Okamoto T . (2003). Biochem. Biophys. Res. Commun., 301, 583–590.

  27. Karin M . (1999). Oncogene, 18, 6867–6874.

  28. Karin M and Delhase M . (1998). Proc. Natl. Acad. Sci. USA, 95, 9067–9069.

  29. Kismet K, Akay MT, Abbasoglu O and Ercan A . (2004). Cancer Detect. Prev., 28, 127–142.

  30. Kundu JK, Na H-K, Chun K-S, Kim Y-K, Lee S-J, Lee S-S, Lee O-S, Sim Y-C and Surh Y-J . (2003). J. Nutr., 133, 3805S–3810S.

  31. Kuo MH and Allis CD . (1998). Bioessays, 20, 615–626.

  32. Lee MW, Kim JH, Jeong DW, Ahn KH, Toh SH and Surh YJ . (2000). Biol. Pharm. Bull., 23, 517–518.

  33. Liao CH, Sang S, Liang YC, Ho CT and Lin JK . (2004). Mol. Carcinog., 41, 140–149.

  34. Lim JW, Kim H and Kim KH . (2001). Lab. Invest., 81, 349–360.

  35. Madrid LV, Mayo MW, Reuther JY and Baldwin Jr AS . (2001). J. Biol. Chem., 276, 18934–18940.

  36. May MJ and Ghosh S . (1997). Semin. Cancer Biol., 8, 63–73.

  37. Mohan S and Epstein JB . (2003). Oral Oncol., 39, 537–546.

  38. Muller-Decker K, Neufang G, Berger I, Neumann M, Marks F and Furstenberger G . (2002). Proc. Natl. Acad. Sci. USA, 99, 12483–12488.

  39. Nakao S, Ogata Y, Shimizu-Sasaki E, Yamazaki M, Furuyama S and Sugiya H . (2000). Mol. Cell Biochem., 209, 113–118.

  40. Newall CA, Anderson LA and Phillipson JD . (1996). Herbal Medicines: A Guide For Health-Care Professionals Vol. 4. Pharmaceutical Press: London, pp. 296.

  41. Nomura M, Ma W, Chen N, Bode AM and Dong Z . (2000). Carcinogenesis, 21, 1885–1890.

  42. Park K-K, Chun K-S, Lee J-M, Lee S-S and Surh Y-J . (1998). Cancer Lett., 129, 139–144.

  43. Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E and Ben-Neriah Y . (2004). Nature, 431, 461–466.

  44. Saccani S, Pantano S and Natoli G . (2002). Nat. Immunol., 3, 69–75.

  45. Sakurai H, Chiba H, Miyoshi H, Sugita T and Toriumi W . (1999). J. Biol. Chem., 274, 30353–30356.

  46. Sizemore N, Lerner N, Dombrowski N, Sakurai H and Stark GR . (2002). J. Biol. Chem., 277, 3863–3869.

  47. Sizemore N, Leung S and Stark GR . (1999). Mol. Cell. Biol., 19, 4798–4805.

  48. Subbaramaiah K and Dannenberg AJ . (2003). Trends Pharmacol. Sci., 24, 96–102.

  49. Surh Y-J . (1999). Mutat. Res., 428, 305–327.

  50. Surh Y-J . (2002). Food Chem. Toxicol., 40, 1091–1097.

  51. Surh Y-J . (2003). Nat. Rev. Cancer, 3, 768–780.

  52. Surh Y-J, Chun K-S, Cha H-H, Han S-S, Keum Y-S, Park K-K and Lee S-S . (2001). Mutat. Res., 480–481, 243–268.

  53. Takahashi N, Tetsuka T, Uranishi H and Okamoto T . (2002). Eur. J. Biochem., 269, 4559–4565.

  54. Tiano HF, Loftin CD, Akunda J, Lee CA, Spalding J, Sessoms A, Dunson DB, Rogan EG, Morham SG, Smart RC and Langenbach R . (2002). Cancer Res., 62, 3395–3401.

  55. Wang D and Baldwin Jr AS . (1998). J. Biol. Chem., 273, 29411–29416.

  56. Wesselborg S, Bauer MK, Vogt M, Schmitz ML and Schulze-Osthoff K . (1997). J. Biol. Chem., 272, 12422–12429.

  57. Williams CS, Mann M and DuBois RN . (1999). Oncogene, 18, 7908–7916.

  58. Wilms H, Rosenstiel P, Sievers J, Deuschl G, Zecca L and Lucius R . (2003). FASEB J., 17, 500–502.

  59. Yang F, Tang E, Guan K and Wang CY . (2003). J. Immunol., 170, 5630–5635.

  60. Zhong H, Voll RE and Ghosh S . (1998). Mol. Cell, 1, 661–671.

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Acknowledgements

This work was supported by the National Research Laboratory Fund (allocated to Y-J Surh) from the Ministry of Science and Technology, Republic of Korea. We acknowledge the technical support by Professor Yong-Sang Song of Seoul National University Hospital for immunohistochemical analyses.

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Author notes

  1. Sue Ok Kim and Joydeb Kumar Kundu: These two authors contributed equally to this work

Affiliations

  1. College of Pharmacy, Seoul National University, Seoul, 151-742, South Korea

    • Sue Ok Kim
    • , Joydeb Kumar Kundu
    • , Young Kee Shin
    •  & Young-Joon Surh
  2. College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul, 151-742, South Korea

    • Jin-Hong Park
    •  & Myung-Haing Cho
  3. College of Medicine, The Catholic University of Korea, Seoul, 150-010, South Korea

    • Tae-Yoon Kim

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