TPA-induced Cyclooxygenase-2 Expression Research Articles

LPS promotes invasion through NF-κB pathway dependent repression of PP2Ac in pancreatic cancer cells

Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK–IKKβ.

Piceatannol inhibits phorbol ester-induced expression of COX-2 and iNOS in HR-1 hairless mouse skin by blocking the activation of NF-κB and AP-1.

The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory activity of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) in mouse skin in vivo. Female HR-1 hairless mice were topically treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) with or without piceatannol pretreatment. Epidermal protein expression was assessed by Western blot analysis. The cyclooxygenase-2 (COX-2) expression was detected by immunohistochemistry. The DNA binding of nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) was examined by the electrophoretic mobility gel shift assay. The catalytic activity of IκBα kinase-β (IKKβ) was measured by in vitro kinase assay. Pretreatment with piceatannol attenuated TPA-induced expression of COX-2 and inducible nitric oxide synthase (iNOS) in mouse skin. Piceatannol diminished nuclear translocation and the DNA binding of NF-κB through the blockade of phosphorylation and subsequent degradation of IκBα. Piceatannol attenuated the catalytic activity of IKKβ and inhibited the phosphorylation of mitogen-activated protein (MAP) kinases in TPA-treated mouse skin. In addition, piceatannol decreased TPA-induced expression of c-Fos and the DNA binding of AP-1. Piceatannol inhibits TPA-induced COX-2 and iNOS expression by blocking the activation of NF-κB and AP-1 via suppression of the IKKβ activity and phosphorylation of MAP kinases, which provides a mechanistic basis of its anti-inflammatory effects in mouse skin.

Sulforaphane inhibits phorbol ester-stimulated IKK-NF-κB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-κB activating kinase and ERK

Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK–IKKβ.

Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells

Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

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.

TPA로 유도된 마우스 귀 부종 동물모델에서 소목추출물의 항염증 효과

This study investigated the anti-inflammatory effects of extracts from Caesalpinia sappan L. (CSL) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. Skin inflammation was detected by immunohistochemistry and the protein and mRNA expression levels of cyclooxygenase-2 (COX-2) and cytokines (IL-6, IL-1β and TNF-α) detected by Western blotting and RT-PCR. The activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) were analyzed by Western blotting. CSL extracts markedly inhibited the TPA-induced expression of COX-2 and pro-inflammatory cytokines. Also, CSL extracts significantly reduced the activation of NF-κB and MAPK. These results suggest that CSL extracts may serve as therapeutic agents against skin diseases related to inflammation.

3,3'-Diindolylmethane(DIM)이 Human Mammary Epithelial Cell에서 12-O-tetradecanoylphorbol-13-acetate에 의해 유도된 COX-2 발현에 미치는 영향

3,3"-Diindolylmethane(DIM)은 십자화과 채소에 포함되어 있는 indole-3-carbinol(I3C)이 동물의 산성 위액에서 중합되어 생성된 물질이다. 지금까지 DIM은 유방암, 전립선암 그리고 대장암 세포주에서 항암효과가 있다고 알려져 있으며 그 기작에 대한 연구도 다양하게 진행되고 있다. 그러나 정상세포에서 암화과정 중 암의 촉진과 진행과정의 주요한 항암 표적인 항염증에 대한 연구는 보고된 바 없다. 따라서 본 연구에서는 유방상피세포인 MCF-10A 세포에 12-Ο-tetradecanoylphorbol-13-acetate(TPA)로 염증반응을 유도한 후 DIM이 염증작용에 미치는 영향을 조사하였다. 그 결과 MCF-10A 세포에서 TPA에 의해 유도된 COX-2 단백질 및 mRNA 발현이 DIM 처리에 의해 현저히 감소하였다. 또한 TPA에 의해 유도된 IκB-α의 분해, p65의 핵으로의 이동, NF-κB DNA binding activity 역시 DIM의 처리에 의해 감소하였다. 이는 DIM이 인간의 유방상피세포인 MCF-10A 세포에서 TPA에 의해 유도된 NF-κB 신호전달체계의 활성을 억제함으로써 COX-2의 발현을 억제하여 염증반응을 조절함을 나타낸다. 그러므로 이러한 결과들은 DIM을 염증성 질환 예방제 또는 치료제로 개발할 수 있는 가능성을 시사한다.

Suppression of Transglutaminase-2 is Involved in Anti-Inflammatory Actions of Glucosamine in 12-O-Tetradecanoylphorbol-13-Acetate-Induced Skin Inflammation

Glucosamine (GS) is well known for the treatment of inflam-mation. However, the mechanism and efficacy of GS for skin inflammation are unclear. The aim of this study was to evaluate the effects and mechanism of GS in the mouse 12-O-tetradecanoyl 13-acetate (TPA)-induced ear edema model. TPA-induced ear edema was evoked in ICR or transglutaminase 2 (Tgase-2) (-/-) mice. GS was administered orally (10-100 mg/kg) or topically (0.5-2.0 w/v %) prior to TPA treatment. Orally administered GS at 10 mg/kg showed a 76 or 57% reduction in ear weight or myeloperoxidase, respectively, and a decreased expression of cyclooxy-genase-2 (COX-2), NF-κB and Tgase-2 in TPA-induced ear edema by western blot and immunohistochemistry. Role of Tgase-2 in TPA ear edema is examined using Tgase-2 (-/-) mice and TPA did not induce COX-2 expression in ear of Tgase-2 (-/-) mice. These observations suggested that Tgase-2 is involved in TPA-induced COX-2 expression in the inflamed ear of mice and anti-inflammatory effects of glucosamine is mediated through suppression of Tgase-2 in TPA ear edema.

Phloretin Inhibits Phorbol Ester–Induced Tumor Promotion and Expression of Cyclooxygenase-2 in Mouse Skin: Extracellular Signal-Regulated Kinase and Nuclear Factor-κB as Potential Targets

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.

Diallyl Trisulfide Inhibits Phorbol Ester–Induced Tumor Promotion, Activation of AP-1, and Expression of COX-2 in Mouse Skin by Blocking JNK and Akt Signaling

An inverse relationship exists between the consumption of garlic and the risk of certain cancers. The present study was aimed at investigating the effect of garlic constituent diallyl trisulfide (DATS) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 (COX-2) expression and tumor promotion in mouse skin and to explore the underlying molecular mechanisms. Pretreatment of mouse skin with different garlic-derived allyl sulfides showed DATS to be the most potent in suppressing TPA-induced COX-2 expression. DATS significantly attenuated the DNA binding of activator protein-1 (AP-1), one of the transcription factors that regulate COX-2 expression, in TPA-stimulated mouse skin. DATS also diminished TPA-induced expression of c-Jun and c-Fos, the principal components of AP-1, and blunted the activation of c-Jun NH(2)-terminal kinase (JNK) and Akt. Pharmacologic inhibition of JNK or Akt by SP600125 or LY294002, respectively, resulted in diminished AP-1 DNA binding, reduced levels of c-Jun and c-Fos, and inhibition of COX-2 expression in TPA-treated mouse skin. The JNK or Akt kinase assay, taking c-Jun fusion protein as a substrate, revealed that TPA induced JNK- or Akt-mediated c-Jun phosphorylation in mouse skin, which was significantly attenuated by DATS or respective pharmacologic inhibitors. Evaluation of antitumor-promoting effect of DATS on 7,12-dimethylbenz(a)anthracene-initiated and TPA-promoted mouse skin carcinogenesis showed that pretreatment with DATS significantly reduced the incidence and multiplicity of papillomas. Taken together, the inhibitory effects of DATS on TPA-induced AP-1 activation and COX-2 expression through modulation of JNK or Akt signaling may partly account for its antitumor-promoting effect on mouse skin carcinogenesis.

Piceatannol Inhibits Phorbol Ester-Induced NF-κ B Activation and COX-2 Expression in Cultured Human Mammary Epithelial Cells

There are multiple lines of evidence supporting that inflammation is causally linked to carcinogenesis. Abnormal upregulation of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the prostaglandin biosynthesis, has been implicated in carcinogenesis. Trans-3,4,3′,5′-tetrahydroxystilbene (piceatannol), a naturally occurring hydroxylated stilbene with potent anti-inflammatory and antioxidative activities, has been shown to inhibit the proliferation of several cancer cells by inducing apoptosis or blocking cell cycle progression. In this study, we examined the effect of piceatannol on activation of the nuclear transcription factor NF-κ B, one of the major transcription factors that regulate proinflammatory COX-2 gene transcription, in human mammary epithelial (MCF-10A) cells treated with the tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA). When pretreated to MCF-10A cells, piceatannol markedly inhibited TPA-induced NF-κ B DNA binding to a greater extent than resveratrol and oxyresveratrol, stilbene analogs structurally related to piceatannol. Piceatannol also inhibited TPA-induced phosphorylation and degradation of Iκ Bα as well as nuclear translocation of the phosphorylated form of p65, the functionally active subunit of NF-κ B. Likewise, TPA-induced expression of COX-2 was abrogated by piceatannol pretreatment. The thiol reducing agent dithiothreitol abolished the inhibitory effects of piceatannol on NF-κ B DNA binding activity, suggesting that piceatannol may directly modify NF-κ B or its regulator through reaction with the cysteine thiol(s).

Silibinin prevents TPA-induced MMP-9 expression by down-regulation of COX-2 in human breast cancer cells

Ethnopharmacological relevance The expression of matrix metalloproteinase-9 (MMP-9) and cyclooxygenase-2 (COX-2) are pivotal steps in breast cancer pathogenesis. In a previous study, we reported that silibinin suppresses TPA-induced MMP-9 expression through the Raf/MEK/ERK pathway. Aims of the study Herein we determined the co-relationship between MMP-9 and COX-2, as well as the effect of silibinin on 12- O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and COX-2 expression in the human breast cancer cells, MCF-7 and MDA-MB231. Methods The toxicity of silibinin was evaluated by Quick Cell Proliferation Assay Kit II. MMP-9 and COX-2 expression were analyzed by Zymography and Western blotting, respectively. Adenoviral constitutively active (CA)-MEK was used to activate MEK/ERK pathway. Results The expression of MMP-9 and COX-2 in response to TPA was increased, whereas TPA-induced MMP-9 and COX-2 expression was decreased by silibinin. Our results showed that TPA-induced MMP-9 expression was inhibited by celecoxib in a dose-dependent fashion, but not MMP-1-expression. Both MMP-9 and COX-2 expression were significantly increased by CA-MEK overexpression. In contrast, TPA-induced MMP-9 and COX-2 expression was decreased by UO126 (MEK1/2 inhibitor). Conclusion Silibinin down-regulates TPA-induced MMP-9 expression through inhibition of COX-2 expression in breast cancer cells.

Dietary grape seed proanthocyanidins inhibit 12-O-tetradecanoyl phorbol-13-acetate-caused skin tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated mouse skin, which is associated with the inhibition of inflammatory responses

Grape seed proanthocyanidins (GSPs) possess anticarcinogenic activities. Here, we assessed the effects of dietary GSPs on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. Administration of dietary GSPs (0.2 and 0.5%, wt/wt) supplemented with control AIN76A diet resulted in significant inhibition of TPA-induced skin tumor promotion in C3H/HeN mice. The mice treated with GSPs developed a significantly lower tumor burden in terms of the percentage of mice with tumors (P < 0.05), total number of tumors per group (P < 0.01, n = 20) and total tumor volume per tumor-bearing mouse (P < 0.01-0.001) as compared with the mice that received the control diet. GSPs also delayed the malignant progression of papillomas into carcinomas. As TPA-induced inflammatory responses are used routinely as markers of skin tumor promotion, we assessed the effect of GSPs on biomarkers of TPA-induced inflammation. Immunohistochemical analysis and western blotting revealed that GSPs significantly inhibited expression of cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)) and markers of proliferation (proliferating cell nuclear antigen and cyclin D1) in both the DMBA-initiated/TPA-promoted mouse skin and skin tumors. In short-term experiments in which the mouse skin was treated with acute or multiple TPA applications, we found that dietary GSPs inhibited TPA-induced edema, hyperplasia, leukocytes infiltration, myeloperoxidase, COX-2 expression and PGE(2) production in the mouse skin. The inhibitory effect of GSPs was also observed against other structurally different skin tumor promoter-induced inflammation in the skin. Together, our results show that dietary GSPs inhibit chemical carcinogenesis in mouse skin and that the inhibition of skin tumorigenesis by GSPs is associated with the inhibition of inflammatory responses caused by tumor promoters.

Inhibitory effects of oligonol on phorbol ester-induced tumor promotion and COX-2 expression in mouse skin: NF-κB and C/EBP as potential targets

Plant polyphenols possess anti-oxidant and anti-inflammatory activities and are hence potential candidates for preventing cancer. The present study was aimed at evaluating the anti-inflammatory and anti-tumor promoting activity of oligonol, a formulation of catechin-type oligomers, in mouse skin stimulated with a proto-type tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment of mouse skin with oligonol significantly inhibited TPA-induced expression of cyclooxygenase-2 (COX-2). Oligonol diminished nuclear translocation and DNA binding of nuclear factor-kappaB (NF-κB) via blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Moreover, oligonol suppressed TPA-induced DNA binding of CCAAT/enhancer-binding protein (C/EBP) in mouse skin. Oligonol pretreatment also attenuated the phosphorylation and/or catalytic activities of extracellular signal-regulated protein kinase-1/2 (ERK1/2) and p38 mitogen-activated protein (MAP) kinase. Moreover, p38 MAP kinase inhibitor SB203580, but not the MEK inhibitor U0126, negated TPA-induced DNA binding of C/EBP. In addition, oligonol reduced the incidence and the multiplicity of papillomas and squamous cell carcinomas in 7,12-dimethylbenz[ a]anthracene (DMBA)-initiated and TPA-promoted mouse skin, and prolonged the survival of tumor-bearing mice. Pretreatment with oligonol diminished the levels of proliferating cell nuclear antigen and expression of COX-2 in papillomas and carcinomas, respectively, as compared to DMBA plus TPA treatment alone. Taken together, the above findings suggest that oligonol inhibits TPA-induced COX-2 expression by blocking the activation of NF-κB and C/EBP via modulation of MAP kinases and suppresses chemically induced mouse skin tumorigenesis.

Roles of ERK and p38 mitogen-activated protein kinases in phorbol ester-induced NF-κB activation and COX-2 expression in human breast epithelial cells

Inappropriate up-regulation of cyclooxygenase-2 (COX-2) has been implicated in pathogenesis of various types of human cancer. Thus, COX-2 has been recognized as an important target for the chemoprevention of several human malignancies including breast cancer. COX-2 expression is known to be regulated by the eukaryotic transcription factor NF-κB. In an attempt to link the NF-κB activation and COX-2 induction during mammary carcinogenesis, we have examined the effects of 12- O-tetradecanoylphorbol-13-acetate (TPA), a prototype tumor promoter and a mitogen, on NF-κB activation and COX-2 expression in the immortalized human breast epithelial cell line (MCF10A). Treatment of MCF10A cells with TPA resulted in transient induction of NF-κB DNA binding with maximal activation observed at 30 min. Increased DNA binding of NF-κB was accompanied by enhancement of its transcriptional activity as determined by the luciferase reporter gene assay. Under the same experimental conditions, expression of COX-2 mRNA and its protein product peaked at 2 h and 4 h, respectively. TPA treatment caused an increase in the production of prostaglandin E 2. Treatment of cells with the NF-κB inhibitor pyrrolidine dithiocarbamate resulted in significant suppression of TPA-induced COX-2 expression. TPA induced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) via phosphorylation. PD98059 (ERK inhibitor) and SB203580 (p38 MAPK inhibitor) down-regulated the COX-2 expression induced by TPA. Furthermore, TPA-induced COX-2 induction as well as NF-κB activation was blocked in MCF10A cells transfected with dominant negative mutant ERK1/2 or p38 MAPK. These results suggest that both p38 and ERK MAPKs activates NF-κB signaling, which in turn induces COX-2 expression in TPA-stimulated human mammary epithelial cells.

A Galactolipid Possesses Novel Cancer Chemopreventive Effects by Suppressing Inflammatory Mediators and Mouse B16 Melanoma

Crassocephalum rabens (Asteraceae) is a popular anti-inflammatory folk medicine and food supplement. We investigated the cancer chemopreventive bioactivity of C. rabens phytocompounds in vitro and in vivo using cell- and gene-based bioassays and a mouse B16 melanoma model. The bioactive glyceroglycolipid 1,2-di-O-alpha-linolenoyl-3-O-beta-galactopyranosyl-sn-glycerol (dLGG) that was identified from C. rabens was found in vitro and in vivo to be a potent nitric oxide (NO) scavenger. dLGG treatment inhibited both mRNA and protein expression of inducible NO synthase and cyclooxygenase-2 (COX-2) in murine macrophages and inhibited COX-2 gene transcription in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B16 cells. In immunohistochemical studies, dLGG inhibited TPA-induced expression of COX-2 and nitration of proteins in mouse skin. dLGG could also significantly inhibit lipopolysaccharide-induced prostaglandin E(2) production in murine macrophages. Furthermore, dLGG prevented nuclear translocation of cytoplasmic nuclear factor-kappaB (NF-kappaB) by suppressing IkappaBalpha phosphorylation and degradation. Structure-activity relationship study by electrophoretic mobility shift assay indicated that the dilinolenoylglycerol moiety in dLGG is the essential structural feature preventing NF-kappaB.DNA complex formation. A dLGG-enriched extract from C. rabens (10 mg/kg) markedly suppressed B16 melanoma growth in C57BL/6J mice following i.p. administration, an effect comparable with that of cisplatin, a cancer chemotherapeutic drug. This study shows the detailed molecular mechanism(s) underlying the anti-inflammatory and tumor-suppressive effects of a natural galactolipid.

Humulone inhibits phorbol ester-induced COX-2 expression in mouse skin by blocking activation of NF-κB and AP-1: IκB kinase and c-Jun-N-terminal kinase as respective potential upstream targets

Humulone, a bitter acid derived from hop (Humulus lupulus L.), possesses antioxidative, anti-inflammatory and other biologically active activities. Although humulone has been reported to inhibit chemically induced mouse skin tumor promotion, the underlying mechanisms are yet to be elucidated. Since an inappropriate over-expression of cyclooxygenase-2 (COX-2) is implicated in carcinogenesis, we investigated effects of humulone on COX-2 expression in mouse skin stimulated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical application of humulone (10 mumol) significantly inhibited TPA-induced epidermal COX-2 expression. Humulone also diminished TPA-induced DNA binding of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Pre-treatment with humulone attenuated TPA-induced phosphorylation of p65 and nuclear translocation of NF-kappaB subunit proteins. Humulone blunted TPA-induced activation of inhibitory kappaB (IkappaB) kinase (IKK) in mouse skin, which accounts for its suppression of phosphorylation and subsequent degradation of IkappaBalpha. An in vitro kinase assay revealed that humulone could directly inhibit the catalytic activity of IKKbeta. Humulone suppressed the activation of mitogen-activated protein kinases (MAPKs) in TPA-treated mouse skin. The roles of extracellular signal-regulated protein kinase-1/2 and p38 MAPK in TPA-induced activation of NF-kappaB in mouse skin had been defined in our previous studies. The present study revealed that topical application of SP600125, a pharmacological inhibitor of c-Jun-N-terminal kinase (JNK), abrogated the activation of AP-1 and the expression of COX-2 in TPA-treated mouse skin. Taken together, humulone suppressed TPA-induced activation of NF-kappaB and AP-1 and subsequent expression of COX-2 by blocking upstream kinases IKK and JNK, respectively, which may account for its antitumor-promoting effects on mouse skin carcinogenesis.

KG‐135 Inhibits COX‐2 Expression by Blocking the Activation of JNK and AP‐1 in Phorbol Ester–Stimulated Human Breast Epithelial Cells

Ginsenosides, ingredients of ginseng, have a wide array of pharmacologic effects. Especially, ginsenosides Rk1, Rg3, and Rg5 derived from heat-processed ginseng have been shown to possess substantial anti-tumor-promoting effects. KG-135 is a formulated complex that contains several antitumorigenic ginsenosides, such as Rk1, Rg3, Rg5, Rk2, Rk3, Rs3, Rs4, Rs5, Rs6, Rs7, etc. The present article was aimed at evaluating the chemopreventive as well as anti-inflammatory effects of KG-135 in the human breast epithelial cell line (MCF-10A). 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 this study, we found that KG-135 inhibited COX-2 expression in MCF-10A cells stimulated with a prototype tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the transcription factor activator protein-1 (AP-1) plays a role in tumor promotion and is also known to regulate COX-2 induction, we attempted to determine the effect of KG-135 on TPA-induced activation of AP-1. Cotreatment with KG-135 resulted in a decrease in TPA-induced DNA binding of AP-1. In addition, KG-135 inhibited TPA-induced phosphorylation of c-Jun N-terminal kinases (JNK) that regulates COX-2 expression in MCF-10A cells. The JNK inhibitor SP600125 attenuated COX-2 expression in TPA-treated MCF-10A cells. Taken together, the above findings suggest that KG-135 inhibits TPA-induced COX-2 expression in MCF-10A cells by blocking the JNK/AP-1 signaling pathway.

Inhibitory Effects of Asiatic Acid on 7,12-Dimethylbenz[a]anthracene and 12-O-Tetradecanoylphorbol 13-Acetate-Induced Tumor Promotion in Mice

Asiatic acid, a pentacyclic triterpene, has been reported to induce apoptosis of various human cancer cells. In the present study, we assessed the anti-tumor promoting effect of asiatic acid against 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated skin tumorigenesis in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated ICR mice. Topical application of asiatic acid prior to each application of TPA resulted in a significant reduction in skin tumor formation. We also found that pre-application of asiatic acid alleviated TPA-induced [3H]thymidine incorporation, which is a conventional marker for skin tumor promotion. In addition, asiatic acid inhibited the TPA-induced generation of nitric oxide (NO) and expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are known to play important roles in tumor growth, especially in the promotion stage. In addition, topical application of aminoguanidine (AG), a selective iNOS inhibitor, and N(G)-nitro-L-arginine-methyl ester (NAME), another iNOS inhibitor, 30 min prior to TPA treatment significantly inhibited the TPA-induced COX-2 expression. These results suggest that asiatic acid may exert anti-tumorigenesis through inhibitory actions in NO and COX-2 signals.

Effects of 6-(methylsulfinyl)hexyl isothiocyanate on cyclooxygenase-2 expression induced by lipopolysaccharide, interferon-γ and 12-O-tetradecanoylphorbol-13-acetate

6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) is a bioactive compound extracted from a typical Japanese spice, wasabi (Wasabia japonica (Miq.) Matsumura). In the present study, we found that 6-MITC suppressed the expression of cyclooxygenase-2 (COX-2) induced by lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), but did not suppress that induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), in murine macrophage RAW264. Molecular mechanisms were investigated by targeting the transcriptional factors including activator protein-1 (AP-1), CCAAT/enhancer-binding protein delta (C/EBPdelta), CRE-binding protein (CREB) and nuclear factor kappaB (NF-kappaB), which bind to the core element of COX-2 promoter. LPS induced activation of all of these factors and 6-MITC suppressed LPS-induced activation of AP-1, C/EBPdelta, CREB, but not NF-kappaB. IFN-gamma did not induce any activation of these factors, but 6-MITC suppressed IFN-gamma-induced COX-2 expression, suggesting that the upstream region of the core element is linked for this suppression. Finally, TPA stimulated the activation of CREB and AP-1, but 6-MITC did not block TPA-induced COX-2 expression. These results suggest that LPS, IFN-gamma and TPA regulate COX-2 expression through different mechanisms, and 6-MITC acts as a potent inhibitor of COX-2 expression induced by LPS or IFN-gamma.