Bisphenol A-induced epithelial to mesenchymal transition is mediated by cyclooxygenase-2 up-regulation in human endometrial carcinoma cells

Many studies have highlighted the correlation between the increase of bisphenol A (BPA) level in the environment and the incidence of tumor in humans. In human carcinogenesis, the overexpression of cyclooxygenase-2 (COX-2) and epithelial-mesenchymal transition (EMT) are closely related with tumor development. In this study, human endometrial carcinoma cells line (RL95-2) was used to investigate whether BPA can induce EMT and COX-2 expression. The results show that BPA increased growth rate and colony-forming efficiency in a dose-dependent manner, induced EMT and COX-2 gene expression and promoted the migration and invasion ability of RL95-2 cells. Furthermore, our study showed that the expression of COX-2 was essential for BPA-induced cell migration and invasion. The results of this study provide new insights into the mechanism of endometrial cancer cell growth and invasion and potential therapeutic strategy.     

Involvement of protein kinase C-gamma in IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

The signaling pathway of protein kinase C (PKC) is known to play a role in mediating the action of various cytokines. Here we examined the signal transduction pathway of PKC activation and the role of PKC isoforms in interleukin-1beta (IL-1beta)-mediated cyclooxygenase-2 (COX-2) expression in human pulmonary epithelial cell line (A549). The tyrosine kinase inhibitors (genistein and tyrphostin AG126) and phosphatidylcholine-phospholipase C inhibitor (D-609) prevented IL-1beta-induced prostaglandin E(2) (PGE(2)) release and COX-2 expression, whereas U-73122 (a phosphatidylinositol-phospholipase C inhibitor) and propranolol (a phosphatidate phosphohydrolase inhibitor) had no effect. The PKC inhibitors (Go 6976 and Ro 31-8220) and NF-kappaB inhibitor, pyrrolidine dithiocarbamate, also attenuated IL-1beta-induced PGE(2) release and COX-2 expression. Western blot analysis using PKC isoenzyme-specific antibodies indicated that A549 cells expressed PKC-alpha, -gamma, -iota, -lambda, -zeta, and -micro. IL-1beta caused the translocation of PKC-gamma but not other isoforms from cytosol to the membrane fraction. Moreover, the translocation of PKC-gamma was inhibited by genistein or D-609, but not by U-73122. IL-1beta caused the translocation of p65 NF-kappaB from cytosol to the nucleus as well as the degradation of IkappaB-alpha in cytosol. Furthermore, the translocation of p65 NF-kappaB was inhibited by genistein, Go 6976, Ro 31-8220, or pyrrolidine dithiocarbamate. These results indicate that in human pulmonary epithelial cells, IL-1beta might activate phosphatidylcholine-phospholipase C through an upstream tyrosine phosphorylation to elicit PKC activation, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE(2) release. Of the PKC isoforms present in A549 cells, only activation of PKC-gamma is involved in regulating IL-1beta-induced responses.     

Induction of cyclooxygenase-2 overexpression in human gastric epithelial cells by Helicobacter pylori involves TLR2/TLR9 and c-Src-dependent nuclear factor-kappaB activation

Gastric epithelial cells were incubated with a panel of clinical isolates of Helicobacter pylori, including nonulcer dyspepsia with gastritis (HS, n = 20), gastric ulcer (HU, n = 20), duodenal ulcer (HD, n = 21), and gastric cancer (HC, n = 20). HC strains induced a higher cyclooxygenase-2 (COX-2) expression than those from HS, HD, and HU. The bacterial virulence factors and the host cellular pathways were investigated. Virulence genes of iceA, vacA, babA2, cagA 3' repeat region, and hrgA failed to show any association with the disease status and COX-2 expression. Methylation-specific polymerase chain reaction revealed HC strains not affecting the methylation status of COX-2 promoter. Nuclear factor (NF)-kappaB, NF-interleukin 6, and cAMP response element were found to be involved in COX-2 induction. We explored a novel NF-kappaB activation pathway. The mutants of TLR2 and TLR9, but not TLR4, inhibited H. pylori-induced COX-2 promoter activity, and neutralizing antibodies for TLR2 and TLR9 abolished H. pylori-induced COX-2 expression. Phosphatidylinositol-specific phospholipase C (PI-PLC), protein kinase C (PKC), and Src inhibitors inhibited COX-2 induction. The dominant-negative mutants of NIK and various IkappaB kinase complexes, including IKKbeta (Y188F), IKKbeta (Y199F), and IKKbeta (FF), inhibited the COX-2 promoter activity. Phosphorylation of GST-IKKbeta (132-206) at Tyr188 and Tyr199 by c-Src was found after H. pylori infection. In summary, H. pylori induces COX-2 expression via activations of NF-kappaB, NF-interleukin 6, the cAMP response element. In NF-kappaB activation, H. pylori acts through TLR2/TLR9 to activate both the cascade of PI-PLCgamma/PKCalpha/c-Src/IKKalpha/beta and the cascade of NIK/IKKalpha/beta, resulting in the IkappaBalpha degradation and the expression of COX-2 gene. The COX-2 overexpression may contribute to the carcinogenesis in patients colonized with these strains.     

Extract from Nandina domestica inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in human pulmonary epithelial A549 cells

Extract from fruits of Nandina domestica THUNBERG (NDE) has been used to improve cough and breathing difficulty in Japan for many years. To explore whether NDE may alleviate respiratory inflammation, we investigated its effect on expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E? (PGE?) in human pulmonary epithelial A549 cells in culture. Treatment with lipopolysaccharide (LPS; 6?μg/mL) resulted in an increase of COX-2 expression and PGE? production in A549 cells. Both the LPS-induced COX-2 expression and PGE? production were significantly inhibited by NDE (1-10?μg/mL) in a concentration-dependent manner. NDE did not affect COX-1 expression nor COX activity. These results suggest that NDE downregulates LPS-induced COX-2 expression and inhibits PGE? production in pulmonary epithelial cells. Furthermore, higenamine and nantenine, two major constituents responsible for tracheal relaxing effect of NDE, did not mimic the inhibitory effect of NDE on LPS-induced COX-2 expression in A549 cells. To identify active constituent(s) of NDE responsible for the anti-inflammatory effect, NDE was introduced in a polyaromatic absorbent resin column and stepwise eluted to yield water fraction, 20% methanol fraction, 40% methanol fraction, 99.8% methanol fraction, and 99.5% acetone fraction. However, none of these five fractions alone inhibited LPS-induced COX-2 expression. On the other hand, exclusion of water fraction from NDE abolished the inhibitory effect of NDE on LPS-induced COX-2 expression. These results suggest that constituent(s) present in water fraction is required but not sufficient for the anti-inflammatory activity of NDE, which may result from interactions among multiple constituents.     

YC-1 attenuates LPS-induced proinflammatory responses and activation of nuclear factor-kappaB in microglia

BACKGROUND AND PURPOSE: An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC-1, a known activator of soluble guanylyl cyclase, against LPS-induced inflammatory responses in microglia. EXPERIMENTAL APPROACH: To understand the inhibitory effects of YC-1 on LPS-induced neuroinflammation, primary cultures of rat microglia and the microglia cell line BV-2 were used. To examine the mechanism of action of YC-1, LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, iNOS, COX-2 and cytokine expression were analyzed by Griess reaction, ELISA, Western blotting and RT-PCR, respectively. The effect of YC-1 on LPS-induced activation of nuclear factor kappa B (NF-kappaB) was studied by NF-kappaB reporter assay and immunofluorocytochemistry. KEY RESULTS: YC-1 inhibited LPS-induced production of NO and PGE2 in a concentration-dependent manner. The protein and mRNA expression of iNOS and COX-2 in response to LPS application were also decreased by YC-1. In addition, YC-1 effectively reduced LPS-induced expression of the mRNA for the proinflammatory cytokines, TNF-alpha and IL-1beta. Furthermore, YC-1 inhibited LPS-induced NF-kappaB activation in microglia. CONCLUSIONS AND IMPLICATIONS: YC-1 was able to inhibit LPS-induced iNOS and COX-2 expression and NF-kappaB activation, indicating that YC-1 may be developed as an anti-inflammatory neuroprotective agent.     

Involvement of nuclear factor-kappaB in lipoteichoic acid-induced cyclooxygenase-2 expression in RAW 264.7 macrophages

We have investigated the role of protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) in cyclooxygenase-2 (COX-2) expression caused by Staphylococcus aureus lipoteichoic acid in RAW 264.7 macrophages. A phosphatidylcholine-phospholipase C (PC-PLC) inhibitor (D-609) and a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor (U-73122) attenuated lipoteichoic acid-induced COX-2 expression, while a phosphatidate phosphohydrolase inhibitor (propranolol) had no effect. Two PKC inhibitors (Go 6976 and Ro 31-8220) and the NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), also attenuated lipoteichoic acid-induced COX-2 expression. Lipoteichoic acid resulted in a decrease in PKC activity in the cytosol and an increase in PKC activity in membranes. The lipoteichoic acid-induced translocation of p65 NF-kappaB from the cytosol to the nucleus was inhibited by D-609, U-73122, Go 6976, Ro 31-8220, and PDTC, but not by propranolol. The results suggested that lipoteichoic acid might have activated PC-PLC and PI-PLC to induce PKC activation, which in turn initiated NF-kappaB activation, and finally induced COX-2 expression in RAW 264.7 macrophages.     

TCDD-induced cyclooxygenase-2 expression is mediated by the nongenomic pathway in mouse MMDD1 macula densa cells and kidneys

Cyclooxygenase-2 (Cox-2) plays a critical role in TCDD-induced hydronephrosis in mouse neonates. In this study we found that induction of Cox-2 by TCDD in MMDD1, a mouse macula densa cell line, is accompanied with a rapid increase in the enzymatic activity of cytosolic phospholipase A2 (cPLA2) as well as activation of protein kinases. Calcium serves as a trigger for such an action of TCDD in this cell line. These observations indicate that the basic mode of action of TCDD to induce the rapid inflammatory response in MMDD1 is remarkably similar to those mediated by the nongenomic pathway of aryl hydrocarbon receptor (AhR) found in other types of cells. Such an action of TCDD to induce Cox-2 in MMDD1 was not affected by “DRE decoy oligonucleotides” treatment or by introduction of a mutation on the DRE site of Cox-2 promoter, suggesting that this route of action of TCDD is clearly different from that mediated by the classical genomic pathway. An in vivo study with Ahr^nls mouse model has shown that TCDD-induces Cox-2 and renin expression in the kidneys of the Ahr^nls mice as well as Ahr^+/− mice, but not in the Ahr^−/− mice, indicating that this initial action of TCDD in mouse kidney does not require the translocation of AhR into the nucleus, supporting our conclusion that induction of Cox-2 by TCDD in mouse kidney is largely mediated by the nongenomic pathway of TCDD-activated AhR.     

Glucose regulation of dipeptidyl peptidase IV gene expression is mediated by hepatocyte nuclear factor-1alpha in epithelial intestinal cells

1. Dipeptidyl peptidase IV (DPP-IV) is a new drug target in the treatment of Type 2 diabetes. Dipeptidyl peptidase IV enzyme activity is significantly altered in Type 2 diabetic patients with hyperglycaemia, but the underlying molecular mechanisms remain unclear. 2. The first aim of the present study was to clarify whether glucose regulates DPP-IV enzyme activity. To address this, DPP-IV gene expression and enzyme activity were measured in Caco2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) concentrations of glucose. We observed that high glucose inhibited DPP-IV gene expression and enzyme activity. 3. The second aim of the present study was to investigate whether hepatocyte nuclear factor (HNF)-1alpha contributes to glucose regulation of DPP-IV gene expression. To explore this question, associations between the gene expression of DPP-IV and HNF-1alpha were examined in Caco-2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) glucose. We found that the pattern of glucose-regulated DPP-IV gene expression is similar to that of HNF-1alpha. Moreover, to elucidate whether glucose regulation of DPP-IV gene expression is affected when HNF-1alpha is inhibited, we produced two stable cell lines in which a dominant-negative mutant HNF-1alphaR271G or basic vectors were stably expressed. We found that glucose regulation of DPP-IV gene expression was compromised in HNF-1alphaR271G cells, but was well maintained in basic vector cells. 4. These results suggest that glucose regulation of DPP-IV gene expression is mediated by HNF-1alpha.     

Antitumorigenic effects of peroxisome proliferator-activated receptor-gamma in non-small-cell lung cancer cells are mediated by suppression of cyclooxygenase-2 via inhibition of nuclear factor-kappaB

Pharmacological activators of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibit growth of non-small-cell lung cancer (NSCLC) cell lines in vitro and in xenograft models. Because these agents engage off-target pathways, we have assessed the effects of PPARgamma by overexpressing the protein in NSCLC cells. We reported previously that increased PPARgamma inhibits transformed growth and invasiveness and promotes epithelial differentiation in a panel of NSCLC expressing oncogenic K-Ras. These cells express high levels of cyclooxygenase-2 (COX-2) and produce high levels of prostaglandin E(2) (PGE(2)). The goal of these studies was to identify the molecular mechanisms whereby PPARgamma inhibits tumorigenesis. Increased PPARgamma inhibited expression of COX-2 protein and promoter activity, resulting in decreased PGE(2) production. Suppression of COX-2 was mediated through increased activity of the tumor suppressor phosphatase and tensin homolog, leading to decreased levels of phospho-Akt and inhibition of nuclear factor-kappaB activity. Pharmacological inhibition of PGE(2) production mimicked the effects of PPARgamma on epithelial differentiation in three-dimensional culture, and exogenous PGE(2) reversed the effects of increased PPARgamma activity. Transgenic mice overexpressing PPARgamma under the control of the surfactant protein C promoter had reduced expression of COX-2 in type II cells and were protected against developing lung tumors in a chemical carcinogenesis model. These data indicate that high levels of PGE(2) as a result of elevated COX-2 expression are critical for promoting lung tumorigenesis and that the antitumorigenic effects of PPARgamma are mediated in part through blocking this pathway.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide and cyclooxygenase-2 by chiisanoside via suppression of nuclear factor-kappaB activation in RAW 264.7 macrophage cells

In the present study, the effects of several triterpenes isolated from the leaves of Acanthopanax chiisanensis (Araliaceae), namely, chiisanoside, isochiisanoside, 22-hydroxychiisanoside and chiisanogenin (the aglycone of chiisanoside) were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by the RAW 264.7 macrophage cell line. Of the triterpenes tested, chiisanoside was found to most potently inhibit NO and PGE2 production. In addition, chiisanoside significantly reduced the release of inflammatory cytokines like TNF-alpha and IL-1beta. Consistent with these observations, the protein and mRNA expression levels of iNOS and COX-2 enzyme were found to be inhibited by chiisanoside in a concentration-dependent manner. Furthermore, chiisanoside inhibited the nuclear factor-kappaB (NF-kappaB) activation induced by LPS and this was associated with a reduction in p65 protein in the nucleus and with the phosphorylations of ERK1/2 and JNK MAP kinases. Taken together, our data indicate that the anti-inflammatory properties of chiisanoside might be the result from the inhibition of iNOS, COX-2, TNF-alpha and IL-1beta expression through the down-regulation of NF-kappaB binding activity.     

Byakangelicol,isolated from Angelica dahurica,inhibits both the activity and induction of cyclooxygenase-2 in human pulmonary epithelial cells

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1beta (IL-1beta)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01-1 microM), and byakangelicol (10-50 microM) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 microM, did not affect the activity and expression of COX-1 enzyme. IL-1beta-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 microM), while byakangelicol (50 microM) had no effect. Treatment of cells with byakangelicol (50 microM) or pyrrolidine dithiocarbamate (PDTC; 50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol, translocation of p65 NF-kappaB from the cytosol to the nucleus and the NF-kappaB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1beta-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Ceramide is involved in r(+)-methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells

Cannabinoids have recently been shown to induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells. Using this cell line, the present study investigates the contribution of the second messenger ceramide to this signaling pathway. Incubation of cells with the endocannabinoid analog R(+)-methanandamide (R(+)-MA) was associated with an increase of intracellular ceramide levels. Enhancement of ceramide formation by R(+)-MA was abolished by fumonisin B1, a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (l-cycloserine, ISP-1) were inactive in this respect. R(+)-MA caused a biphasic activation of the p38 and p42/44 mitogen-activated protein kinases (MAPKs), with phosphorylation peaks occurring after 15-min and 4- to 8-h treatments, respectively. Inhibition of ceramide synthesis with fumonisin B1 was associated with a suppression of R(+)-MA-induced delayed phosphorylations of p38 and p42/44 MAPKs and subsequent COX-2 expression. The involvement of ceramide in COX-2 expression was corroborated by findings showing that C2-ceramide and neutral sphingomyelinase from Bacillus cereus caused concentration-dependent increases of COX-2 expression that were suppressed in the presence of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580, a p38 MAPK inhibitor) or 2'-amino-3'-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor). In contrast, dihydro-C2-ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression. Collectively, our results demonstrate that R(+)-MA induces COX-2 expression in human neuroglioma cells via synthesis of ceramide and subsequent activation of p38 and p42/44 MAPK pathways. Induction of COX-2 expression via ceramide represents a hitherto unknown mechanism by which cannabinoids mediate biological effects within the central nervous system.     

Activation of phosphoinositide 3-kinase in response to high glucose leads to regulation of reactive oxygen species-related nuclear factor-kappaB activation and cyclooxygenase-2 expression in mesangial cells

Hyperglycemia causes glomerular mesangial cell proliferation and increases matrix synthesis, contributing to early diabetic glomerulopathy. Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes have been identified. However, the role of cyclooxygenase-2 in early diabetes-induced mesangial cell proliferation remains unknown. The authors tested the hypothesis that hyperglycemia modulates an intrarenal cyclooxygenase-2 expression, which might mediate the mesangial cell proliferation via a possible phosphoinositide 3-kinase/Akt pathway. Expression of cyclooxygenase-2, but not cyclooxygenase-1, could be induced in mesangial cells cultured under high glucose. Antioxidants (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) and phosphoinositide 3-kinase inhibitors [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin] effectively inhibited this high glucose-induced response. Moreover, high glucose markedly triggered the activation of phosphoinositide 3-kinase and Akt in mesangial cells, suggesting that a phosphoinositide 3-kinase/Akt pathway is involved in the high glucose-induced responses. Phosphoinositide 3-kinase inhibitors could also effectively attenuate the high glucose-triggered intracellular reactive oxygen species generation and nuclear factor-kappaB activation. Likewise, blocking the phosphoinositide 3-kinase or Akt activity with the dominant-negative vectors DN-p85 or DN-Akt, respectively, also greatly diminished the high glucose-triggered reactive oxygen species generation and nuclear factor-kappaB activation. Treatment of mesangial cells with LY294002 and cyclooxygenase-2 inhibitors [N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS398) and aspirin] effectively inhibited the high glucose-induced mesangial cell proliferation. These results suggest that high glucose may trigger the reactive oxygen species-regulated nuclear factor-kappaB activation and cyclooxygenase-2 expression and cell proliferation in mesangial cells through a phosphoinositide 3-kinase-dependent pathway.     

Pentoxifylline inhibits ICAM-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells

Airway epithelium participates in inflammatory reactions by producing chemokines and expressing cell-surface adhesion molecules which aid in the selective recruitment of effector cells. Previous studies showed that proinflammatory cytokines, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha), induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and the production of the chemokines interleukin 8 (IL-8) and monocyte chemoattractant protein (MCP-1) on pulmonary epithelial cell lines in vitro. In this study, the dose response of four cytokines, IL-1alpha, IL-1beta, TNF alpha and TNF beta, in inducing ICAM-1 expression and production of IL-8 and MCP-1 on pulmonary A549 epithelial cells was examined. Both IL-1alpha and IL-1beta induced ICAM-1 expression and IL-8 and MCP-1 production at lower doses than TNF alpha or TNF beta. Pentoxifylline, an anti-inflammatory agent used to treat vascular diseases, was tested for its ability to inhibit the activation of airway epithelial cells by these cytokines. Pentoxifylline completely inhibited the surface expression of ICAM-1 and the production of IL-8 and MCP-1 by cytokine-activated epithelial cells. As elevated levels of chemokines are often present in bronchial lavage fluids of patients suffering from various acute respiratory diseases, pentoxifylline may be useful for preventing the rapid development of immune reactions leading to lung injury.