Cyclooxygenase-2 Promoter Activity Research Articles

A positive feedback loop between STAT3 and cyclooxygenase‐2 gene may contribute to Helicobacter pylori‐associated human gastric tumorigenesis

Persistent infection with Helicobacter pylori (H. pylori) contributes to gastric diseases including chronic gastritis and gastric cancer. However, the pathogenesis of this carcinogenic bacterium has not been completely elucidated. Here, we report that H. pylori rapidly triggers STAT3 signaling and induces STAT3-dependent COX-2 expression both in vitro and in vivo. STAT3 upregulates COX-2 by binding to and increasing the activity of COX-2 promoter. COX-2 in turn regulates IL-6/STAT3 signaling under basal conditions and during H. pylori infection. These findings suggest that a positive feedback loop between STAT3 and COX-2 exists in the basal condition and H. pylori infectious condition. Immunohistochemical staining revealed that H. pylori-positive gastritis tissues exhibited markedly higher levels of pSTAT3(Tyr705) than H. pylori-negative ones. High pSTAT3(Tyr705) levels are correlated with intestinal metaplasia and dysplasia, suggesting pSTAT3(Tyr705) may be useful in the early detection of gastric tumorigenesis. Additionally, a strong positive correlation between STAT3/pSTAT3(Tyr705) levels and COX-2 expression was identified in gastritis and gastric cancer tissues. Together, these findings provide new evidence for a positive feedback loop between STAT3 signaling and COX-2 in H. pylori pathogenesis and may lead to new approaches for early detection and effective therapy of gastric cancer

Rutin inhibits B[a]PDE-induced cyclooxygenase-2 expression by targeting EGFR kinase activity

Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR–/–mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin.

Telomerase reverse transcriptase inhibition stimulates cyclooxygenase 2 expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects

Background:Telomerase and telomerase reverse transcriptase (hTERT) confer cancer cells sustained proliferation and survival potentials. Targeting telomerase or hTERT is a novel anti-cancer strategy. However, telomerase/hTERT inhibition alone has minimal clinical efficacy. We explored the relationship between hTERT and cyclooxygenase 2 (COX2) and evaluated synergistic anti-cancer effects of targeting both hTERT and COX2.Methods:hTERT was depleted in gastric and cervical cancer cells using small interfering RNA (siRNA) and analysed for COX2 expression using quantitative PCR and immunoblotting. Viable cells and apoptotic cells in gastric cancer cells treated with hTERT siRNA or/and the COX2 inhibitor celecoxib were measured using Trypen blue exclusion and flow cytometry. The in vivo anti-cancer effect of hTERT depletion or/and celecoxib was evaluated using mouse xenograft models.Results:Knocking down hTERT expression in cancer cells led to robust increases in mRNA and protein levels of COX2. The COX2 promoter activity increased substantially in hTERT-depleted cells. hTERT depletion led to the activation of p38 mitogen-activated protein kinase responsible for the stimulation of COX2 gene transcription. hTERT depletion or celecoxib alone did not affect cancer cell survival, whereas their combination synergistically killed them both in vitro and in vivo.Conclusion:hTERT induces COX2 expression and simultaneously targeting hTERT and COX2 synergistically kills cancer cells.

Abstract 582: hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects.

Abstract Telomerase and telomerase reverse transcriptase (hTERT) confer cancer cells sustained proliferation and survival potentials through telomere lengthening dependent and independent activities. Targeting telomerase or hTERT is thus a novel anti-cancer strategy. However, telomerase/hTERT inhibition alone has minimal clinical efficacy. We explored the relationship between hTERT and oncogenic cyclooxygenase 2 (COX2) and evaluated the synergistic anti-cancer effects of targeting both hTERT and COX2. hTERT depletion in gastric and cervical cancer cells led to robust increases in mRNA and protein levels of COX2. The COX2 promoter activity increased substantially in hTERT-depleted cancer cells. Mechanistically, hTERT depletion led to the activation of p38 MAPK that was responsible for the stimulation of COX2 gene transcription. hTERT depletion or the COX2 inhibitor celecoxib alone did not affect gastric cancer cell survival, while their combination synergistically killed them both in vitro and in vivo. Taken together, hTERT induces COX2 expression in cancer cells and simultaneously targeting hTERT and COX2 synergistically kills cancer cells, which may help path a new avenue in telomerase-based cancer therapeutics. Citation Format: Tiantian Liu, Magnus Björkholm, Dawei Xu. hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 582. doi:10.1158/1538-7445.AM2013-582 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5101: Tanshinone II-A inhibits angiogenesis through down regulation of COX-2 in human colorectal cancer.

Abstract Tanshinone II-A Inhibits Angiogenesis through Down Regulation of COX-2 in Human Colorectal Cancer Qi Li*, Li-Hong Zhou, Hua Sui, Yan Wang, Xuan Liu, Ning-Ning Liu, Pei-Hao Yin Clinic Oncology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai China *correspondence to Qi Li, Lzwf@hotmail.com Angiogenesis plays a significant role in colorectal cancer (CRC). cyclooxygenase-2 (COX-2) is involved with multiple aspects of CRC angiogenesis. The aim was to investigate the inhibitory effects of Tan II-A(Tanshinone II-A, Tan II-A) on tumor growth in mice, as well as alteration of expression of COX-2 and VEGF in CRC. We established the mice xenograft model of C26 CRC cell line, and injected 0.5, 1, 2mg/kg of Tan II-A and 1mg/kg of 5-FU in different individuals respectively in vivo. Then, we assayed tumor weight and volume, evaluated microvascular density and expression of VEGF. COX-2 promoter and COX-2 plasmids were transfected into HCT-116 cells, followed by detection of COX-2 promoter activity by chemiluminescence, detection of COX-2 mRNA expression by fluorescence quantitative PCR. Taken together, the results showed Tan II-A could inhibit tumor growth and VEGF level in vivo. Cell experiment showed marked inhibitory effect of Tan II-A on COX-2 and VEGF of HCT-116 cell lines in a dose-dependent manner. That means Tan II-A can effectively inhibit tumor growth and angiogenesis of human colorectal cancer via inhibiting the expression level of COX-2 and VEGF. Keywords: Tanshinone II-A - colorectal carcinoma - angiogenesis - cyclooxygenase 2 - vascular endothelial growth factor Citation Format: Qi Li. Tanshinone II-A inhibits angiogenesis through down regulation of COX-2 in human colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5101. doi:10.1158/1538-7445.AM2013-5101

The transcription factor Snail expressed in cutaneous squamous cell carcinoma induces epithelial–mesenchymal transition and down-regulates COX-2

Cutaneous spindle cell squamous cell carcinoma (SCC) is a rare, but highly malignant variant of SCC. The presence of spindle-shaped cells with a sarcomatous appearance, which are derived from squamous cells, suggests that these cells are produced as a result of epithelial–mesenchymal transition (EMT). EMT is a complex process in which epithelial cells lose their polarity and cell–cell contacts, while also acquiring increased motility and invasiveness. Snail regulates EMT by binding to proximal E-boxes in the promoter region of E-cadherin and repressing its transcription. When examining the expression of EMT markers and Snail in spindle cell SCCs, we found that cyclooxygenase-2 (COX-2) expression was down-regulated. Since it has been shown that COX-2 is constitutively overexpressed in a variety of malignancies, including colon, gastric, and lung carcinomas, the down-regulation of COX-2 expression was unexpected. The presence of E-box-like sequences in the promoter region of COX-2 prompted us to perform a more detailed analysis. We introduced a Snail expression vector into keratinocyte-derived cell lines (HaKaT, HSC5, and A431 cells), and isolated stable transfectants. We determined that COX-2 expression was down-regulated in cells expressing Snail. Consistent with these observations, reporter assays revealed that COX-2 promoter activity was repressed upon Snail overexpression. Thus Snail down-regulates COX-2 in these cells.

Tanshinone II-A Inhibits Angiogenesis through Down Regulation of COX-2 in Human Colorectal Cancer

Angiogenesis plays a significant role in colorectal cancer (CRC) and cyclooxygenase-2 (COX-2) appears to be involved with multiple aspects of CRC angiogenesis. Our aim was to investigate the inhibitory effects of Tan II-A (Tanshinone II-A, Tan II-A) on tumor growth in mice, as well as alteration of expression of COX-2 and VEGF in CRC. We established the mice xenograft model of C26 CRC cell line, and injected 0.5, 1, 2mg/kg of Tan II-A and 1mg/kg of 5-FU in respectively in vivo. Then, we assayed tumor weight and volume, and evaluated microvascular density and expression of VEGF. COX-2 promoter and COX-2 plasmids were transfected into HCT-116 cells, followed by detection of COX-2 promoter activity by chemiluminescence, and detection of COX-2 mRNA expression by fluorescence quantitative PCR. Taken together, the results showed Tan II-A could inhibit tumor growth and suppress the VEGF level in vivo. HCT-116 cell experiments showed marked inhibitory effects of Tan II-A on COX-2 and VEGF in a dose-dependent manner. The results indicate that Tan II-A can effectively inhibit tumor growth and angiogenesis of human colorectal cancer via inhibiting the expression level of COX-2 and VEGF.

Inhibitive effect of proanthocyanidins on cyclooxygenase-2 expression in A549 cells induced by cytokine interleukin-1 beta

Cyclooxygenase-2 (COX-2), an important enzyme, plays a pathological role in diseases, which can be inhibited by proanthocyanidins (PCs) effectively. In this paper, we investigated the inhibitive mechanism of COX-2 performed by PCs. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify the mRNA expression level of COX-2 in A549 cell, which was induced by interleukin-1 beta (IL-1β). The pGL3 luciferase reporter vector containing the COX-2 gene promoter fragment (pGL3/COX-2p) was transfected into A549 cell induced by IL-1β, the interference on the COX-2 promoter activity from PCs was analyzed using a dualluciferase reporter assay, and the expressions of the nuclear factor κB composed of subunit p65 (NF-κB/p65) and the inhibitor-κB (I-κB) were measured by the Western blotting and immunocytochemistry. The results exhibited that PCs not only inhibited the transcript of COX-2 mRNA and the COX-2 promoter activity, but also suppressed the nuclear translocation of NF-κB/p65 protein and the degradation of I-κB protein.

Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells

Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca2+ concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca2+ signals is the activity of the Ca2+- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression -both mRNA and protein- was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression -both mRNA and protein- was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal cells of non-pregnant but not of early-pregnant rats. These results might be related to differential roles that COX-2 plays in the endometrium.

Effect of NF-κB on inhibition of non-small cell lung cancer cell cyclooxygenase-2 by brucine

To study the molecular mechanism of cyclooxygenase-2 (COX-2), one of effective ingredient of brucine, in inducing non-small cell lung cancer cell apoptosis. COX-2 promoter, transcription factor deletion mutants and COX-2 mRNA 3'-UTR-containing report plasmids were transfected with Renillia to non-small cell lung cancer A549 cell, in order to detect the activity of report gene luciferase and minimum cis-acting element of COX-2 promoter inhibited by brucine. The influence of brucine on IkappaB phosphorylation and the nuclear translocation of p65 were detected by immunoblotting assay. Brucine significantly suppressed LPS-induced COX-2 promoter activation, but revealed minor impact on COX-2 mRNA stability. NF-kappaB in the vicinity of COX-2 promoter-262 was an important cis-acting element of brucine for inhibiting the activity of COX-2 promoter. Brucine was found to inhibit the phosphorylation of IkappaBalpha as well as the nuclear translocation of p65. Brucine can improve A549 cells apoptosis by inhibiting the activity of NF-kappaB and the subsequent COX-2 gene expression.

Delivery of interferon alpha using a novel Cox2-controlled adenovirus for pancreatic cancer therapy

Combination therapy with interferon alpha (IFN) is correlated with improved survival in patients with pancreatic ductal adenocarcinoma (PDAc) but frequently presents side effects. We designed a novel targeted adenovirus with replication restricted to cyclooxygenase 2 (Cox2)-overexpressing PDAcs and hypothesize that the locally delivered therapeutic gene IFN can augment oncolytic effects while minimizing systemic toxicity. IFN-expressing vectors were tested invitro with the use of 4 PDAc cell lines with cytocidal effect measured by crystal violet and colorimetrically and IFN production assayed by ELISA. Cox2 promoter activity was checked by a luciferase reporter assay. Invivo, subcutaneous tumor xenografts with 2 PDAc cell lines in nude mice were treated with a single intratumoral viral dose. All PDAc cell lines were Cox2-positive. Oncolysis from the novel Cox2-controlled virus was comparable or superior to Adwt, the wild-type virus without safety features. The absence of cytocidal effect in Cox2-negative cells with the novel virus indicated cancer specificity. Invivo, stronger tumor suppression from the novel virus was seen when compared with nonreplicating IFN-expressing vectors. We demonstrated the potent therapeutic effects of a novel tumor-specific conditionally replicative IFN-expressing adenovirus. With potential to locally deliver IFN and avoid systemic toxicity, this strategy may therefore expand the application of this robust and promising therapy.

Arachidonic acid regulation of the cytosolic phospholipase A2α/cyclooxygenase-2 pathway in mouse endometrial stromal cells

ObjectiveTo investigate the role of arachidonic acid (AA) in mouse endometrial stromal cells.DesignExperimental animal study.SettingUniversity research laboratory.Animal(s)Sexually mature female CD1-strain mice.Intervention(s)Primary culture of endometrial stromal cells.Main Outcome Measure(s)Western blot and real-time polymerase chain reaction for gene expression and/or phophorylation analysis. Luciferase assay for Cox-2 promoter analysis.Result(s)AA-derived prostaglandins play important roles during embryo implantation and decidualization. However, the function of AA itself in reproduction is largely unknown. In this study, exogenous AA stimulated cPLA2α phosphorylation and COX-2 expression, mainly through ERK1/2 in mouse endometrial stromal cells, and p38 inhibitor modestly inhibited cPLA2α phosphorylation induced by AA. The induction of COX-2 by AA was diminished by short interfering RNA against C/EBPβ and inhibitory C/EBPβ (LIP). C/EBPβ binding site at −872–−864 of Cox-2 promoter contributes to Cox-2 promoter activation induced by C/EBPβ transfection. The expression of C/EBPβ protein induced by AA was inhibited by p38 inhibitor, and the phosphorylation of C/EBPβ induced by AA was inhibited by p38 inhibitor and ERK1/2 inhibitor. A nonmetabolized analogue of AA (ETYA) also enhanced cPLA2α phosphorylation and COX-2 expression. The activation of cPLA2α/COX-2 by AA was not inhibited by COX inhibitor indomethacin.Conclusion(s)AA can induce cPLA2α/COX-2 pathway activation in mouse endometrial stromal cells. To investigate the role of arachidonic acid (AA) in mouse endometrial stromal cells. Experimental animal study. University research laboratory. Sexually mature female CD1-strain mice. Primary culture of endometrial stromal cells. Western blot and real-time polymerase chain reaction for gene expression and/or phophorylation analysis. Luciferase assay for Cox-2 promoter analysis. AA-derived prostaglandins play important roles during embryo implantation and decidualization. However, the function of AA itself in reproduction is largely unknown. In this study, exogenous AA stimulated cPLA2α phosphorylation and COX-2 expression, mainly through ERK1/2 in mouse endometrial stromal cells, and p38 inhibitor modestly inhibited cPLA2α phosphorylation induced by AA. The induction of COX-2 by AA was diminished by short interfering RNA against C/EBPβ and inhibitory C/EBPβ (LIP). C/EBPβ binding site at −872–−864 of Cox-2 promoter contributes to Cox-2 promoter activation induced by C/EBPβ transfection. The expression of C/EBPβ protein induced by AA was inhibited by p38 inhibitor, and the phosphorylation of C/EBPβ induced by AA was inhibited by p38 inhibitor and ERK1/2 inhibitor. A nonmetabolized analogue of AA (ETYA) also enhanced cPLA2α phosphorylation and COX-2 expression. The activation of cPLA2α/COX-2 by AA was not inhibited by COX inhibitor indomethacin. AA can induce cPLA2α/COX-2 pathway activation in mouse endometrial stromal cells.

Identification of Cis-acting Elements and Signaling Components of High Affinity IgE Receptor that Regulate the Expression of Cyclooxygenase-2

Allergic and inflammatory responses are functionally linked through a cascade of signaling events that connect the aggregation of the high affinity IgE receptor (FcHRI) on mast cells and the initiation of cyclooxygenase-2 (COX-2) expression. In this study, we identified the cis-acting elements in the cox-2 promoter that control the expression of COX-2 in RBL-2H3 mast cells. We also investigated how the inflammatory reaction is controlled by the allergic reaction by determining the signaling components employed by FcHRI in the transcriptional regulation of cox-2. Among cis-acting components present in the cox-2 promoter, the CREB binding site, as well as the AP-1 and proximal NF-IL6 binding sites to a lesser extent, were required for the transcriptional regulation of the cox-2 promoter. However, NF-NB and Ets-1 binding sites exerted negative effects on the cox-2 promoter activity. Among the signaling components of FcHRI, Fyn, PI 3-kinase, Akt, and p38 MAPK positively mediated the COX-2 expression. Conventional PKCs and atypical PKCs exerted opposite regulatory effects on the cox-2 promoter activity. Blockade of MEK/ERK pathway inhibited the cox-2 promoter activity and the COX-2 expression. These results reveal intricate functional interactions among different cis-acting elements in the transcriptional regulation of cox-2. FynoPI 3-kinaseoAkt pathway directly stimulate. On the other hand, LynoSyk pathway exerts auxiliary or compensatory influences on COX-2 expression via PKC and MEK/ERK.

BAI, a 3-aminoindazole derivative, inhibits interleukin-1β-induced expression of cyclooxygenase-2 in A549 human airway cells

Cyclooxygenase (COX)-2 and its products, including PGE2, are key inflammatory mediators. In this study, we have assessed the pharmacological characteristics of BAI, a 3-aminoindazole derivative and a novel cyclin-dependent kinase (CDK) inhibitor, for regulation of COX-2 expression induced by interleukin (IL)-1β in A549 human airway cells. Treatment with BAI strongly inhibited IL-1β-induced expression of COX-2 at both the protein and mRNA levels. Results of luciferase experiments also revealed that BAI treatment reduced IL-1β-induced COX-2 promoter activity. Distinctly, treatment with BAI did not affect IL-1β-induced phospho-rylation of extracellular signal-regulated protein kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal protein kinase-1/2 (JNK-1/2) and proteolysis of IκB-α, an inhibitor of nuclear factor (NF)-κB, but inhibited IL-1β-induced phosphorylation of histone H1, a target for phosphorylation by CDKs. siRNA transfection experiments demonstrated that knockdown of CDK2 and CDK4 led to a slight reduction of IL-1β-induced histone H1 phosphorylation but had no effect on IL-1β-induced COX-2 expression. Interestingly, additional cell culture experiments showed the ability of BAI to repress the PMA-induced COX-2 expression in A549 cells and serum-dependent COX-2 expression in NCI-H292 cells, a human laryngeal cell line. Collectively, these results demonstrate firstly that BAI downregulates IL-1β-induced COX-2 expression through transcriptional repression, which appears to be independent of CDK2, CDK4, MAPKs and NF-κB, in A549 cells. It is suggested that BAI may be a potential candidate for treatment of the airway inflammatory diseases where COX-2 overexpression is problematic.

Black soybean (Glycine max cv. Heugmi) seed coat extract suppresses TPA or UVB-induced COX-2 expression by blocking mitogen activated protein kinases pathway in mouse skin epithelial cells

Although previous studies have reported that black soybean has chemopreventive potential, the underlying mechanisms remain unclear. Upregulation of cyclooxygenase-2 (COX-2) has been known to be a key mediator in the development of skin cancer. The present study investigated the effect of black soybean (Glycine max cv. Heugmi) seed coat extract (BSE) on 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet-B-(UVB)-induced COX-2 expression, and its underlying mechanisms. The TPA- or UVB-induced COX-2 expression in mouse skin epithelial cells were dose-dependently inhibited by BSE treatment. BSE suppressed the COX-2 promoter activity. BSE also attenuated the transactivation of activator protein-1 (AP-1) and nuclear factor (NF)-κB, transcription factors of COX-2 expression in mouse skin epithelial cells transfected stably with AP-1 and NF-κB luciferase promoter, respectively. Furthermore, BSE inhibited the activation of MAPKKs/MAPKs pathways that otherwise induced by TPA or UVB. Collectively, BSE suppresses TPA or UVB-induced COX-2 expression by blocking the expressions of MAPKKs/MAPKs pathways, which may contribute to its chemopreventive potential.

Enantioselective endocrine-disrupting effects of bifenthrin on hormone synthesis in rat ovarian cells

Bifenthrin (BF), a broad-spectrum and widely used synthetic pyrethroid, is a typical chiral pesticide. More attention is being paid to the health risk assessment of the enantioselective toxicity of BF isomers. In this study, we used rat ovarian granulosa cells as in vitro model to investigate effects of BF enantiomers on the biosynthesis of two hormones, progesterone and prostaglandin E2 (PGE2), which are critical for mammalian reproduction. We showed that 1 S- cis-BF, but not 1 R- cis-BF significantly decreased the secretion of progesterone and PGE2 in granulosa cells. 1 S-isomer of BF reduced the expression of genes P450scc, StAR, PBR and DBI, as well as COX-2, which are involved in regulating the rate-limiting steps of progesterone or PGE2 biosynthesis. The transcriptional activation of StAR and COX-2 promoter were disrupted by 1 S- cis-BF. Furthermore, activity of protein kinase C (PKC), an important signaling mediator of progesterone and PGE2 synthesis, was differentially inhibited by 1 S- cis-BF. The data of molecular docking revealed that one hydrogen bond was formed between 1 S- cis-BF and PKC protein. In conclusion, we firstly reported in this study the enantioselective disrupting effects of BF isomers on progesterone and PGE2 synthesis via PKC pathway in rat ovarian cells. Our findings suggest that the enantioselective toxicity of chiral pesticides should be considered for evaluating mammalian reproductive health, a toxicologic endpoint of great concern in health risk assessment.

Regulation of cyclooxygenase-2 expression in human bladder epithelial cells infected with type I fimbriated uropathogenic E. coli

The type 1 fimbriae of uropathogenic Escherichia coli (UPEC) have been described as important for the establishment of bladder infections and urinary tract infections (UTI). Urinary prostaglandin (PG) levels and cyclooxygenase (COX)-2 expression in urine particulates may increase with infectious and inflammatory processes, including UTIs. We investigated the mechanisms underlying the modulation of COX-2 expression through the invasion of type 1 fimbriated UPEC strain J96 (J96-1) in human bladder 5637 cells. Bladder 5637 cells infected with J96-1 induced increases in the expression of COX-2 and secretion of PGE(2) . By using specific inhibitors and short hairpin RNA (shRNA), we have demonstrated that the activation of extracellular signal-related kinase (ERK), c-Jun-NH(2) -terminal kinase (JNK) and p38 MAPK pathways is critical for J96-1-induced COX-2 expression. Luciferase reporters and chromatin immunoprecipitation assays suggest that J96-1 invasion increases NF-κB- and AP-1-DNA-binding activities in 5637 cells. Inhibition of NF-κB and AP-1 activations blocked the J96-1-induced COX-2 promoter activity and expression. The effect of J96-1 on 5637 cell signalling and COX-2 expression is mediated by Toll-like receptor (TLR)-4. In summary, our findings provide the molecular pathways underlying type 1 fimbriated J96-dependent COX-2 expression in 5637 cells, providing insight into the function of UPEC invasion in bladder epithelial cells.

Quercetin Suppresses Cyclooxygenase-2 Expression and Angiogenesis through Inactivation of P300 Signaling

Quercetin, a polyphenolic bioflavonoid, possesses multiple pharmacological actions including anti-inflammatory and antitumor properties. However, the precise action mechanisms of quercetin remain unclear. Here, we reported the regulatory actions of quercetin on cyclooxygenase-2 (COX-2), an important mediator in inflammation and tumor promotion, and revealed the underlying mechanisms. Quercetin significantly suppressed COX-2 mRNA and protein expression and prostaglandin (PG) E(2) production, as well as COX-2 promoter activation in breast cancer cells. Quercetin also significantly inhibited COX-2-mediated angiogenesis in human endothelial cells in a dose-dependent manner. The in vitro streptavidin-agarose pulldown assay and in vivo chromatin immunoprecipitation assay showed that quercetin considerably inhibited the binding of the transactivators CREB2, C-Jun, C/EBPβ and NF-κB and blocked the recruitment of the coactivator p300 to COX-2 promoter. Moreover, quercetin effectively inhibited p300 histone acetyltransferase (HAT) activity, thereby attenuating the p300-mediated acetylation of NF-κB. Treatment of cells with p300 HAT inhibitor roscovitine was as effective as quercetin at inhibiting p300 HAT activity. Addition of quercetin to roscovitine-treated cells did not change the roscovitine-induced inhibition of p300 HAT activity. Conversely, gene delivery of constitutively active p300 significantly reversed the quercetin-mediated inhibition of endogenous HAT activity. These results indicate that quercetin suppresses COX-2 expression by inhibiting the p300 signaling and blocking the binding of multiple transactivators to COX-2 promoter. Our findings therefore reveal a novel mechanism of action of quercetin and suggest a potential use for quercetin in the treatment of COX-2-mediated diseases such as breast cancers.

Regulation of bombesin-stimulated cyclooxygenase-2 expression in prostate cancer cells

BackgroundCyclooxygenase-2 (COX-2) and the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), have been implicated in the progression of hormone-refractory prostate cancer; however, a mechanistic link between the bioactive peptide and COX-2 expression in prostate cells has not been made.ResultsWe report that BBS stimulates COX-2 mRNA and protein expression, and the release of prostaglandin E2 from the GRP receptor (GRPR)-positive, androgen-insensitive prostate cancer cell line, PC-3. BBS-stimulated COX-2 expression is mediated, in part, by p38MAPK and PI3 kinase (PI3K)/Akt pathways, and blocked by a GRPR antagonist. The PI3K/Akt pathway couples GRPR to the transcription factor, activator protein-1 (AP-1), and enhanced COX-2 promoter activity. Although BBS stimulates nuclear factor-kappaB (NF-κB) in PC-3, NF-κB does not regulate GRPR-mediated COX-2 expression. The p38MAPK pathway increases BBS-stimulated COX-2 expression by slowing the degradation of COX-2 mRNA. Expression of recombinant GRPR in the androgen-sensitive cell line LNCaP is sufficient to confer BBS-stimulated COX-2 expression via the p38MAPK and PI3K/Akt pathways.ConclusionsOur study establishes a mechanistic link between GRPR activation and enhanced COX-2 expression in prostate cancer cell lines, and suggests that inhibiting GRPR may, in the future, provide an effective therapeutic alternative to non-steroidal anti-inflammatory drugs for inhibiting COX-2 in patients with recurrent prostate cancer.

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH2-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.