Regulation Of Cyclooxygenase-2 Expression Research Articles

Vitamin C down‐regulates VEGF production in B16F10 murine melanoma cells via the suppression of p42/44 MAPK activation

It is known that vitamin C induces apoptosis in several kinds of tumor cells, but its effect on the regulation of the angiogenic process of tumors is not completely studied. Vascular endothelial growth factor (VEGF) is the most well-known angiogenic factor, and it has a potent function as a stimulator of endothelial survival, migration, as well as vascular permeability. Therefore, we have investigated whether vitamin C can regulate the angiogenic process through the modulation of VEGF production from B16F10 melanoma cells. VEGF mRNA expression and VEGF production at protein levels were suppressed by vitamin C. In addition, we found that vitamin C suppressed the expression of cyclooxygenase (COX)-2 and that decreased VEGF production by vitamin C was also restored by the administration of prostaglandin E2 which is a product of COX-2. These results suggest that vitamin C suppresses VEGF expression via the regulation of COX-2 expression. Mitogen-activated protein kinases are generally known as key mediators in the signaling pathway for VEGF production. In the presence of vitamin C, the activation of p42/44 MAPK was completely inhibited. Taken together, our data suggest that vitamin C can down-regulate VEGF production via the modulation of COX-2 expression and that p42/44 MAPK acts as an important signaling mediator in this process.

Macrophages induce COX-2 expression in breast cancer cells: role of IL-1β autoamplification

Tumor-associated macrophages and high levels of cyclooxygenase-2 (COX-2) are associated with poor prognosis in breast cancer patients, but their potential interdependence has not been evaluated. The objective of this study was to determine whether macrophages regulate COX-2 expression in breast cancer cells. For this purpose, THP-1 cells were cocultured with HCC1954 breast cancer cells. Coculture led to increased COX-2 expression in the HCC1954 cells and elevated prostaglandin E(2) levels in conditioned media. Similar results were observed when THP-1 cells were incubated with HCC1937 breast cancer cells or when human monocyte-derived macrophages were cocultured with HCC1954 cells. Coculture triggered production of reactive oxygen species (ROS) in HCC1954 cells. COX-2 induction was blocked in cells preincubated with an reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or by silencing p67PHOX, a subunit of NADPH oxidase. ROS production triggered activation of Src and mitogen-activated protein kinases (MAPKs). Blocking Src or MAPK activities or antagonizing the activator protein-1 (AP-1) transcription factor attenuated COX-2 induction in HCC1954 cells. Coculture caused rapid induction of interleukin-1β (IL-1β) in both breast cancer cells and macrophages. Increased IL-1β expression was blocked by an interleukin-1 receptor antagonist (IL-1Ra), suggesting autocrine and paracrine effects. Importantly, macrophage-induced COX-2 expression was blocked in HCC1954 cells preincubated with IL-1Ra or anti-IL-1β IgG. Together, these results indicate that macrophage-mediated induction of COX-2 in breast cancer cells is a consequence of IL-1β-mediated stimulation of ROS→Src→MAPK→AP-1 signaling. IL-1β-dependent induction of COX-2 in breast cancer cells provides a mechanism whereby macrophages contribute to tumor progression and potential therapeutic targets in breast cancer.

TNF-α potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts

The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Here, we show that treatment of 18Co cells, a model of human colonic MFBs, with TNF-α and lysophosphatidic acid (LPA) induced striking synergistic cyclooxygenase-2 (COX-2) protein expression and production of PGE(2). This effect was prevented by the LPA(1) receptor antagonist Ki16425, the G(iα)-specific inhibitor pertussis toxin, and by the preferential protein kinase (PK) C inhibitors GF109203X and Go6983. As a known downstream target of LPA and PKC, we tested whether PKD, recently implicated in the regulation of COX-2 expression in MFB, was involved in this response. TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation stimulated by LPA, as measured by PKD autophosphorylation at Ser(910). LPA-induced PKD activation was also inhibited by Ki16425, pertussis toxin, GF109203X, and Go6983. Transfection of 18Co cells with short interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein, demonstrating a critical role of PKD in this response. Our results imply that cross talk between TNF-α and LPA results in the amplification of COX-2 protein expression via a conserved PKD-dependent signaling pathway that appears to involve the LPA(1) receptor and the G protein G(iα). PKD plays a critical role in the expression of COX-2 in human colonic MFBs and may contribute to an inflammatory microenvironment that promotes tumor growth.

Upregulation of the EP1 receptor for prostaglandin E2 promotes skin tumor progression

Prostaglandin E(2) (PGE(2) ) has been shown to promote the development of murine skin tumors. EP1 is 1 of the 4 PGE(2) G-protein-coupled membrane receptors expressed by murine keratinocytes. EP1 mRNA levels were increased ∼2-fold after topical treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or exposure to ultraviolet (UV) light, as well as increased ∼3- to 12-fold in tumors induced by 7,12-dimethyl-benz[a]anthracene (DMBA) initiation/TPA promotion or by UV exposure. To determine the effect of EP1 levels on tumor development, we generated BK5.EP1 transgenic mice that overexpress EP1 in the basal layer of the epidermis. Skins of these mice were histologically indistinguishable from wild type (WT) mice and had similar levels of proliferation after TPA treatment. Using a DMBA/TPA carcinogenesis protocol, BK5.EP1 mice had a reduced tumor multiplicity compared to WT mice, likely due to the observed down-regulation of protein kinase C (PKC). However, the BK5.EP1 mice had an ∼8-fold higher papilloma to carcinoma conversion rate. When DMBA/anthralin was used, BK5.EP1 mice produced more tumors than WT mice, as well as a ninefold increase in carcinomas, indicating that the tumor response is dependent on the type of tumor promoter agent used. Additionally, although almost undetectable in WT mice, cyclooxygenase-2 (COX-2) was expressed in the untreated epidermis of BK5.EP1 mice. While TPA highly induced COX-2 in WT mice, COX-2 expression in the BK5.EP1 mice did not change after TPA treatment; PGE(2) levels were likewise affected. These data indicate that EP1 is more important in tumor progression than in tumor promotion and that it indirectly regulates COX-2 expression.

Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells

Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway.

Promoter hypermethylation of cyclooxygenase-2 gene in esophageal squamous cell carcinoma

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies including esophageal squamous cell carcinoma (ESCC). However, a subset of ESCC either do not express COX-2 or show low level of expression. It is well established that promoter methylation is a major mechanism that mediates transcriptional silencing of COX-2 in gastric and colorectal cancer, but the data on ESCC are very limited. In this study, we attempted to determine whether COX-2 expression was also regulated by promoter methylation in human ESCC cell lines. We examined the methylation status of the COX-2 promoter in five human ESCC cell lines (EC109, EC9706, KYSE 410, KYSE 150, TE-1) using bisulfite sequencing analysis. Western blot analysis was used to determine COX-2 expression. Quantitative real-time polymerase chain reaction was used to determine COX-2 mRNA level. Prostaglandin (PG) E(2) was detected by ELISA. The promoter was densely methylated in TE-1 and KYSE 150, which had a low level of COX-2 expression and less methylated in other three cell lines (EC109, EC9706, KYSE 410), with high level of COX-2 expression. Treatment with 5-aza-deoxycytidine (5-aza-DC), a DNA methyltransferase inhibitor, demethylated the promoter and upregulated COX-2 expression, as well as PGE(2) production in TE-1 and KYSE 150. However, no such effects were observed in EC109. COX-2 protein was negative, but mRNA was positive in TE-1. After treatment with 5-aza-DC, both COX-2 mRNA and protein level had increased. These findings suggest that the promoter methylation may be one of the mechanisms that regulate COX-2 expression in ESCC.

Association between expression of embryonic lethal abnormal vision‐like protein HuR and cyclooxygenase‐2 in oral squamous cell carcinoma

Increased cytoplasmic HuR expression has been noted in several cancer types, where it may contribute to the increased cyclooxygenase-2 (COX-2) expression observed during tumorigenesis and metastasis. To assess the correlation between COX-2 and HuR in oral squamous cell carcinoma (OSCC), the expression patterns of HuR and COX-2 were assessed via immunohistochemistry analyses of 103 OSCC samples. Cytoplasmic HuR expression was significantly associated with COX-2 expression (p < .025) and lymph node metastasis (p < .050) and distant metastasis (p < .025). In multivariate analysis, cytoplasmic HuR expression was identified as an independent prognostic parameter for reduced overall survival. The inhibition of HuR expression by siRNA or leptomycin B (LMB) caused a reduction in the inducibility of COX-2 in oral cancer cells. Our results indicate that the cytoplasmic expression of HuR is associated with COX-2 expression in OSCCs and HuR can regulate COX-2 expression in oral cancer cell lines.

185 ROLE OF C-TYPE NATRIURETIC PEPTIDE (CNP) IN OSTEOGENESIS AND TREATMENT OF OSTEOPOROSIS

miRNA sequences. Inhibition of NF-κB and p38-MAPK activation with specific inhibitors down-regulated the expression of COX-2 but up-regulated the expression of miR-101_3, miR-199a* in human OA chondrocytes suggesting negative regulation of miR-101_3, miR-199a* by the activation of these pathways. Conclusions: Our data implicate miR-199a* in the post transcriptional regulation of COX-2 expression in human OA chondrocytes. These results also identify miR-199a* as a novel therapeutic target for the treatment of OA.

Identification of PARP-1 as one of the transcription factors binding to the repressor element in the promoter region of COX-2

Cyclooxygenase-2 (COX-2) plays important roles in the development of many disease conditions, including pancreatic β-cell dysfunction. Although the processes involved in the transcriptional regulation of COX-2 are well documented, some key elements, especially inhibitory elements, are still unknown. In our previous study, we identified a novel repressor element located in promoter region of mouse COX-2. In this study, we isolated several DNA-binding proteins from NIT-1 cells via DNA affinity chromatography; the most prominent among these proteins was poly (ADP-ribose) polymerase-1 (PARP-1). In this study, gel-supershift assays and chromatin immunoprecipitation assays showed that PARP-1 can bind to the inhibitory element −655/−632 in the promoter region of mouse COX-2 both in vitro and in vivo. Furthermore, overexpression of PARP-1 significantly inhibited promoter activity and decreased COX-2 expression. Conversely, repression of PARP-1 by RNAi upregulated COX-2 expression. These data suggest that PARP-1 plays an important role in the regulation of COX-2 expression via binding to the inhibitory element. Collectively, our findings provide new important information on the transcriptional regulation of COX-2 in pancreatic β-cells.

Protease-activated receptor-2 regulates cyclooxygenase-2 expression in human bile duct cancer via the pathways of mitogen-activated protein kinases and nuclear factor kappa B

Recent studies have suggested that protease-activated receptor-2 (PAR-2) activity correlates with cell proliferation and tumor growth, and its activation induces expression of cyclooxygenase-2 (COX-2). However, no previous reports have investigated PAR-2 signaling pathways in bile duct cancer. The aim of this study was to determine whether PAR-2 activation can regulate COX-2 expression via mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) in human bile duct cancer cells. We immunohistochemically examined PAR-2 and COX-2 expression in 104 resected human specimens of extrahepatic bile duct cancer. We then determined how inhibitors of MAPKs and NF-κB signaling pathways influence COX-2 expression under PAR-2 activation in HuCCT1 and TKKK, human bile duct cancer cell lines. PAR-2 and COX-2 proteins were immunohistochemically recognized in 63 and 57% of specimens and were significantly correlated. PAR-2 agonist peptide activated mRNA and protein expression of COX-2 in HuCCT1 and TKKK. Pharmacologic blockade of p44/42 or p38 MAPK significantly inhibited PAR-2-activated mRNA and protein expression of COX-2 in both cells. COX-2 protein expression was also inhibited by the blocker of NF-κB pathway in both cells. PAR-2 may regulate COX-2 expression in human bile duct cancer via the MAPKs and NF-κB pathways.

Citral, a component of lemongrass oil, activates PPARα and γ and suppresses COX-2 expression

Lemongrass is a widely used herb as a food flavoring, as a perfume, and for its analgesic and anti-inflammatory purposes; however, the molecular mechanisms of these effects have not been elucidated. Previously, we identified carvacrol from the essential oil of thyme as a suppressor of cyclooxygenase (COX)-2, a key enzyme for prostaglandin synthesis, and also an activator of peroxisome proliferator-activated receptor (PPAR), a molecular target for “lifestyle-related” diseases. In this study, we evaluated the essential oil of lemongrass using our established assays for COX-2 and PPARs. We found that COX-2 promoter activity was suppressed by lemongrass oil in cell-based transfection assays, and we identified citral as a major component in the suppression of COX-2 expression and as an activator of PPARα and γ. PPARγ-dependent suppression of COX-2 promoter activity was observed in response to citral treatment. In human macrophage-like U937 cells, citral suppressed both LPS-induced COX-2 mRNA and protein expression, dose-dependently. Moreover, citral induced the mRNA expression of the PPARα-responsive carnitine palmitoyltransferase 1 gene and the PPARγ-responsive fatty acid binding protein 4 gene, suggesting that citral activates PPARα and γ, and regulates COX-2 expression. These results are important for understanding the anti-inflammatory and anti-lifestyle-related disease properties of lemongrass.

Persistent Cyclooxygenase-2 Inhibition Downregulates NF-κB, Resulting in Chronic Intestinal Inflammation in the Min/+ Mouse Model of Colon Tumorigenesis

Cyclooxygenase-2 (COX-2) inhibition prevents adenoma formation in humans and mouse models of colon cancer. The selective COX-2 inhibitor celecoxib reduces COX-2 and prostaglandin E(2) (PGE(2)) expression and adenomas in the intestine of Min/+ mice after treatment for several weeks, but prolonged treatment increases PGE(2) production, resulting in drug-resistant tumor formation and transforming growth factor beta (TGFbeta)-dependent intestinal fibrosis. In this study, we examined pathways that regulate COX-2 expression and suppress chronic intestinal inflammation. We show that NF-kappaB signaling was inhibited in the ileum of Min/+ mice receiving long-term treatment with celecoxib. This effect was associated with inhibition of TGFbeta-associated kinase-1 and IkappaB kinase alpha/beta activities and reduced expression of the Toll-like receptor (TLR) 2 and TLR4 that enhance colonic barrier function. Additionally, we observed reduced activities of protein kinases c-Jun NH(2)-terminal kinase 1 and protein kinase A and transcription factor cyclic AMP-responsive element binding protein, regulators of COX-2 expression, which cross-talk with NF-kappaB. In ileum subjected to long-term celecoxib treatment, we noted relatively higher expression of COX-2, vascular endothelial growth factor, and interleukin-1beta in Paneth cells, whereas NF-kappaB and COX-2 were more strongly expressed by an expanded population of stromal myofibroblasts. Our findings argue that celecoxib resistance is an acquired adaptation to changes in the crypt microenvironment that is associated with chronic intestinal inflammation and impaired acute wound-healing responsiveness.

Effect of endothelin-1 on cyclooxygenase-2 expression in human hormone refractory prostate cancer cells

The present study aimed to explore the effects and possible mechanisms of recombinant human endothelin (ET)-1 on cyclooxygenase (COX)-2 expression in human hormone refractory prostate cancer PC3 cells. PC3 cells were treated with 100 nmol/l ET-1 for the indicated times (3, 6, 9, 12 and 24 h) and concentrations (0.1, 1, 10 and 100 nmol/l) for 24 h. Moreover, 100 nmol/l ET-1 was used to treat PC3 cells alone or in combination with endothelin A receptor (ET(A)R) antagonist BQ123 (1 μmol/l), endothelin B receptor (ET(B)R) antagonist BQ788 (1 μmol/l), MAPK/extracellular signal-regulated kinase kinase (MEK)-selective inhibitor, PD98059 (10 μmol/l), p38 mitogen-activated protein kinase (MAPK) antagonist SB203580 (5 μmol/l) or epidermal growth factor receptor (EGFR) antagonist AG1478 (0.1 μmol/l) for 24 h. COX-2 mRNA and protein expression was detected in the PC3 cells by reverse transcription-polymerase chain reaction and Western blot analysis. ET-1 induced a time- and dose-dependent increase in the mRNA and protein expression of COX-2 in the PC3 cells. BQ123, LY294002, SC203580 and AG1478 prevented the expression of COX-2 in the PC3 cells (P<0.05), while BQ788 did not. ET-1 induced the up-regulation of COX-2 in the PC3 cells. ET(A)R may be involved in the process. Several signaling pathways, including p42/44 MAPK, p38 MAPK and EGFR, are therefore implicated in the regulation of COX-2 expression.

Embryonic Expression of Cyclooxygenase-2 Causes Malformations in Axial Skeleton

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.

T2016 Regulation of COX-2 Expression by MicroRNA-542-3p

T2016 Regulation of COX-2 Expression by MicroRNA-542-3p

Abstract 1350: Macrophages induce COX-2 expression in breast cancer cells: Role of IL-1β dependent signal transduction pathways

Abstract Macrophages are a major component of the inflammatory infiltrate observed in many tumors including carcinoma of the breast. Evidence suggests that tumor associated macrophages (TAMs) are a part of an inflammatory “loop” that promotes tumor progression. In the breast, the presence of high numbers of TAMs is associated with a poor prognosis. However, the mechanisms underlying the interactions between TAMs and breast cancer cells are not clearly understood. In this regard, cyclooxygenase-2 (COX-2) is over expressed in approximately 40% of breast cancers, and is associated with a poor prognosis. COX-2 derived prostaglandin E2 (PGE2) promotes the growth and metastasis of breast cancers. To determine whether TAMs regulate COX-2 expression in breast cancer cells, PMA-treated THP-1 cells grown on porous inserts were co-cultured with HCC1954 breast cancer cells. The presence of THP-1 cells led to increased COX-2 expression in the breast cells and elevated levels of PGE2 in conditioned media. Similar results were observed when THP-1 cells were incubated with HCC1937 cells and when peripheral blood-derived mononuclear cells were incubated with HCC1954 cells. To identify potential paracrine factors that mediated COX-2 induction in breast cancer cells, levels of pro-inflammatory cytokines in THP-1/HCC1954 co-culture conditioned media were measured. The levels of IL-1β rose rapidly in co-culture conditioned media. Moreover, the expression of IL-1β in both breast cancer cells and macrophages was increased as a result of co-culture. This increased expression was blocked in both cells by IL-1 receptor antagonist (IL-1ra) suggesting an autocrine loop. Importantly, the addition of IL-1ra or IL-1β neutralizing antibody blocked the induction of COX-2 in HCC1954 cells. These data suggest that IL-1β is a major mediator of COX-2 induction. Co-culture conditioned media triggered production of reactive oxygen species (ROS) in naïve HCC1954 cells. Blocking ROS production by DPI (a NADPH oxidase inhibitor) or knocking down the expression of p67 subunit of the NADPH oxidase complex led to inhibition of COX-2 induction in HCC1954 cells. ROS production in HCC1954 cells triggered activation of Src kinase, and subsequently the downstream MAP kinases (ERK, p38, and JNK). Blocking the activity of Src or MAP kinases, utilizing specific inhibitors or siRNA, attenuated COX-2 induction. In summary, we identified that IL-1β was a major mediator of COX-2 induction in HCC1954 cells co-cultured with THP-1 macrophages. We also showed that co-culture induced COX-2 expression was a consequence of IL-1β dependent ROS→Src→MAP kinases signaling. These findings provide new insights into the mechanisms of interactions between TAMs and breast cancer cells and potential intervention targets in TAM-positive breast cancers. 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 1350.

Re: Cyclooxygenase-2, prostaglandin E2 and acute myeloid leukemia

We thank Dr Yves Denizot et al. for their letter as well as expressingcommendable feedbacks on our recently published work (1). In ourstudies, we demonstrate that vascular endothelial growth factor(VEGF)-C may promote leukemic angiogenesis by inducing cyclo-oxygenase (COX)-2 expression in subsets of leukemias. Our resultsalso present a close correlation of VEGF-C and COX-2 expression inthe bone marrow biopsies from acute leukemia patients. Several stud-ies have worked on the regulation of COX-2 expression in leukemicblast cells (2). We do appreciate the delineation of COX-2 and itslipidicmetabolitesontheproliferationanddifferentiationofleukemiacells (3,4). Since there are no specific questions or concerns raised intheletter,wewilltakethisopportunitytocommentonthefactthattherole of VEGF family in hematological malignancies may be broaderin nature than usually thought.VEGF-A is the well-documented blast-derived proangiogenicfactor in acute myeloid leukemia (AML). In certain VEGFR2-expressing leukemias, VEGF-A acts as an autocrine and paracrinegrowthfactor(5).AlthoughVEGF-A/VEGFR2signalinginleukemo-genesis and leukemic progression has been unveiled, the contributionof VEGF-C to neoplastic progression in hematological malignancieshas remained obscure until recently (1,6,7). Upregulation of COX-2by VEGF-C/VEGFR3 pathway in subsets of leukemias highlightedthe pathogenic role of VEGF-C in leukemias by altering COX-2-derived eicosanoids biosynthesis. These eicosanoids, including pros-taglandin E2, have proleukemic autocrine or paracrine actions, as func-tional receptors for E series of prostaglandins (EP receptors) areexpressed on both leukemic blast and endothelial cells (8,9). The con-tribution of VEGF-C/VEGFR3 signaling to COX-2 upregulation inAML thus supports its role in leukemic progression by influencing notonly the tumor itself but also the surrounding microenvironment as well.The stromal microenvironment of bone marrow may foster leuke-mogenesis and progression through complex network of soluble me-diators (10). Although leukemic blast-associated VEGF-C expressionlevels display no prognostic significance in recent clinical studiesshouldnotbeunderestimated, asthe intrinsiclevels ofVEGF-Cintheneoplasticbonemarrowmayoriginatefromleukemicblastsaswellasbone marrow stromal cells such as endothelial cells (6). Our findingson VEGF-C/COX-2 axis in AML has enhanced the understanding ofthedynamicnetworkofgrowthfactorsandcytokinesintheneoplasticbone marrow, which would potentially lead to the development ofnovel therapeutic initiatives.Acknowledgements

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.

MiRNA-26b regulates the expression of cyclooxygenase-2 in desferrioxamine-treated CNE cells

Here we report that miR-26b is involved in COX-2 overexpression in desferrioxamine (DFOM)-treated carcinoma of nasopharyngeal epithelial (CNE) cells. The level of miR-26b in DFOM-treated CNE cells is inversely proportional to the expression level of the COX-2 protein. Overexpression of miR-26b in DFOM-treated CNE cells inhibits cell proliferation. A luciferase reporter gene experiment suggests that the 3′ untranslated region of COX-2 carries a binding site for miR-26b. Overexpression of miR-26b marginally reduces the levels of COX-2 protein in DFOM-treated CNE cells. Moreover, knockdown of COX-2 expression had a similar effect to overexpression of miR-26b. Taken together, these results suggest that miR-26b regulates COX-2 expression in DFOM-treated cells.

LKB1/PEA3/ΔNp63 Pathway Regulates PTGS‐2 (COX‐2) Transcription in Lung Cancer Cells Upon Cigarette Smoke Exposure

This is the first study to show that cigarette smoking induced the LKB1/PEA 3/ΔNp63-dependent transcriptional regulation of inflammatory molecules, such as COX-2/PTGS-2. Using mainstream smoke extract (MSE) and sidestream smoke extract (SSE) as modeling tools for primary and secondhand smoking, we found that both MSE and SSE downregulated protein levels for LKB1, while upregulated protein levels for PEA 3 and COX-2 in a dose-dependent manner. Using the endogenous ChIP analysis, we further found that the C/EBPβ, NFκB, NF-Y (CHOP), PEA 3 (ETS) and ΔNp63 proteins bound to the specific area (-550 to -130) of the COX-2 promoter, while forming multiple protein complexes in lung cancer cells exposed to MSE and SSE. Our results define a novel link between various transcription factors occupying the COX-2 promoter and cellular response to cigarette smoke exposure bringing a new component, ΔNp63α, showing a critical role for cooperation between various chromatin components in regulation of COX-2 expression and, therefore strengthening the central role of inflammatory process in tumorigenesis of epithelial cells, especially after cigarette smoke exposure (both primary and secondhand).