Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells

The anti-inflammatory activity of the predominant procyanidin dimer in cocoa, dimer B2, was investigated in this study. Pretreatment of the procyanidin dimer B2 reduced COX-2 expression induced by the endotoxin lipopolysaccharide (LPS) in differentiated human monocytic cells (THP-1) in culture. To further elucidate the underlying mechanism of COX-2 inhibition by procyanidin, we examined their effects on the activation of extracellular signal-regulated protein kinase (ERK), Jun-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), which are upstream enzymes known to regulate COX-2 expression in many cell types. Pretreatment with procyanidin dimer B2 decreased the activation of ERK, JNK, and p38 MAPK. In addition, procyanidin dimer B2 suppressed the NF-kappaB activation through stabilization of IkappaB proteins, suggesting that these signal-transducing enzymes could be potential targets for procyanidin dimer B2. By affecting the expression rather than the activity of COX-2, these in vitro data reported herein give further evidence on the anti-inflammatory protection by procyanidins.     

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

Adenosine A 2B receptors modulate cAMP levels and induce CREB but not ERK1/2 and p38 phosphorylation in rat skeletal muscle cells

The present study examined the existence of the adenosine A(1),A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4)M) increased the cAMP concentration in cultured muscle cells with an EC(50) of (95% confidence interval)=15 (5.9-25.1) micro M, whereas CGS 21680 (10(-9)-10(-4)M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6)M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC(50) of (95% confidence interval)=1.7 (0.40-7.02) micro M, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1),A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A(2B) receptor.     

R(+)-methanandamide-induced cyclooxygenase-2 expression in H4 human neuroglioma cells: possible involvement of membrane lipid rafts

Cannabinoids induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells via a pathway independent of cannabinoid- or vanilloid receptor activation. The underlying mechanism was recently shown to involve increased synthesis of ceramide, which in turn leads to activation of p38 and p42/44 mitogen-activated protein kinases (MAPKs). The present study investigates a possible contribution of membrane lipid rafts to cannabinoid-induced COX-2 expression. To address this issue, we tested the influence of methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, on COX-2 expression by the endocannabinoid analogue R(+)-methanandamide (R(+)-MA). Incubation of H4 cells with MCD was associated with a loss of lipid raft integrity and a substantial inhibition of R(+)-MA-induced COX-2 expression and subsequent formation of prostaglandin E2. Moreover, MCD was shown to suppress signal transduction steps upstream to COX-2 induction by R(+)-MA. Accordingly, the cholesterol depletor suppressed R(+)-MA-induced formation of ceramide as well as phosphorylation of p38 and p42/44 MAPKs. Together, our results suggest that R(+)-MA induces COX-2 expression in human neuroglioma cells via a pathway linked to lipid raft microdomains.     

Prostaglandin E2 induces cyclooxygenase-2 expression in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 mitogen-activated protein kinases

Prostaglandins (PGs) have been implicated in lowering intraocular pressure (IOP). A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing impaired COX-2 expression in the non-pigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. The present study investigates the effect of the major COX-2 product, PGE(2), on the expression of its synthesizing enzyme in human NPE cells (ODM-2). PGE(2) led to an increase of COX-2 mRNA and protein expression, whereas the expression of COX-1 remained unchanged. Upregulation of COX-2 expression by PGE(2) was accompanied by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, and was abrogated by inhibitors of both pathways. Moreover, PGE(2)-induced COX-2 expression was suppressed by the intracellular calcium chelator, BAPTA/AM, and the protein kinase C inhibitor bisindolylmaleimide II, whereas the protein kinase A inhibitor H-89 was inactive in this respect. Induction of COX-2 expression was also elicited by butaprost (EP(2) receptor agonist) and 11-deoxy PGE(1) (EP(2)/EP(4) receptor agonist), but not by EP(1)/EP(3) receptor agonists (17-phenyl-omega-trinor PGE(2), sulprostone). Consistent with these findings, the EP(1)/EP(2) receptor antagonist, AH-6809, and the selective EP(4) receptor antagonist, ONO-AE3-208, significantly reduced PGE(2)-induced COX-2 expression. Collectively, our results demonstrate that PGE(2) at physiologically relevant concentrations induces COX-2 expression in human NPE cells via activation of EP(2)- and EP(4) receptors and phosphorylation of p38 and p42/44 MAPKs. Positive feedback regulation of COX-2 may contribute to the production of outflow-facilitating PGs and consequently to regulation of IOP.     

The expression of cyclooxygenase-2 (COX-2) in amnion and decidua following spontaneous labor

Objective: Prostaglandins production rises dramatically during term and preterm labor. The source of this production is thought to be the fetal membranes and maternal decidua. The enzyme responsible for the conversion of arachidonic acid to the prostaglandins and related endoperoxides is variously known as prostaglandin synthase or cyclooxygenase (COX). An inducible form of this enzyme, COX-2, has been described in several tissues. The purpose of this study was to investigate a possible role for COX-2 in labor by comparing the COX-2 content in amnion and decidua from laboring and non-laboring patients. Study Design: Fetal membranes from seven normal labor and ten elective cesarean sections at term were collected immediately following delivery. The maternal age and gravity were similar between the groups. The amnion and decidua were identified, washed in sterile saline, frozen in liquid nitrogen and stored in −70°C. COX-2 expression was determined using Western Blot analysis with a purified COX-2 antibody. A scanning densitometer was used to quantify the bands. Results were expressed as mean ±S.D. ng/l50μg protein. Results: The concentration of COX-2 in amnion of laboring women showed a twofold increase ( 240.0 ± 17.6 vs. 120.7 ± 5.1) compared to the non-labored group (p<0.05). The concentration in the decidua showed no significant increase during labor (38.1 ± 7.5 vs. 26.4 ± 2.1, p > 0.05).Conclusion: We evaluated the role of COX-2 in normal labor. Our study demonstrate that COX-2 is significantly induced in the amnion following spontaneous labor. These findings suggest that the induction of amnion COX-2 may be involved in the process of human labor.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.     

Protease-activated receptor-2 regulates vascular endothelial growth factor expression in MDA-MB-231 cells via MAPK pathways

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor that is cleaved and activated by serine proteases including the coagulation protease factor VIIa (FVIIa). There is evidence that PAR2 function contributes to angiogenesis, but the mechanisms involved are poorly defined. Here we show that PAR2 activation in human breast cancer cells leads to the upregulation of vascular endothelial growth factor (VEGF). Activation of PAR2 with agonist peptide (AP), trypsin or FVIIa results in a robust increase of VEGF message and protein. Incubation of cells with PAR1-AP, PAR3-AP, PAR4-AP, or thrombin has only a modest effect on VEGF production. Cleavage blocking antibodies show that FVIIa-mediated VEGF production is PAR2 mediated. Mitogen-activated protein kinase (MAPK) pathway inhibitors U0126 and SB203580 inhibit PAR2-mediated VEGF production. Incubation of cells with PAR2-AP leads to significant extracellular regulated kinase1/2 (ERK1/2) and p38 MAPK phosphorylation and activation. Collectively, these data suggest that PAR2 signaling through MAPK pathways leads to the production of proangiogenic VEGF in breast cancer cells.     

6-Shogaol and 6-gingerol, the pungent of ginger, inhibit TNF-alpha mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes

In this study, we demonstrated that the two ginger-derived components have a potent and unique pharmacological function in 3T3-L1 adipocytes via different mechanisms. Both pretreatment of 6-shogaol (6S) and 6-gingerol (6G) significantly inhibited the tumor necrosis factor-α (TNF-α) mediated downregulation of the adiponectin expression in 3T3-L1 adipocytes. Our study demonstrate that (1) 6S functions as a PPARγ agonist with its inhibitory mechanism due to the PPARγ transactivation, and (2) 6G is not a PPARγ agonist, but it is an effective inhibitor of TNF-α induced c-Jun-NH₂-terminal kinase signaling activation and thus, its inhibitory mechanism is due to this inhibitory effect.     

Protein kinase Cdelta-mediated CREB activation regulates ghrelin-induced cyclooxygenase-2 expression and prostaglandin E2 production in human colonic epithelial cells

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone release and appetite. Our recent study showed that ghrelin could stimulate protein kinase C-mediated activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 secretion in human colonic epithelial cells transfected with a functional ghrelin receptor. In the present study, the effect of ghrelin stimulation on cyclooxygenese-2 expression and prostaglandin E2 production was examined. The data indicate that ghrelin significantly increased the levels of cyclooxygenase-2 (COX-2) protein as well as its promoter activity, which leaded to profound increase in prostaglandin E2 secretion. In order to examine the involvement of NF-kappaB and cAMP responsive element-binding protein (CREB) in this response, the NF-kappaB inhibitory protein IkappaBalpha or a dominant negative mutant of CREB was co-transfected into cells and the data show that transfection of either IkappaBalpha or DN-CREB significantly attenuated ghrelin-induced COX-2 expression. Moreover ghrelin stimulated phosphorylation of CREB, which was mediated primarily via protein kinase Cdelta activation. Furthermore, inhibition of PKCdelta function significantly attenuated ghrelin-induced COX-2 expression. In addition, ghrelin stimulates phosphorylation of PKCdelta. Together, these results indicate that in addition to NF-kappaB, protein kinase Cdelta-mediated CREB activation plays an important role in the cellular responses of ghrelin.     

Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications

Can drinking several cups of green tea a day keep the doctor away? This certainly seems so, given the popularity of this practice in East Asian culture and the increased interest in green tea in the Western world. Several epidemiological studies have shown beneficial effects of green tea in cancer, cardiovascular, and neurological diseases. The health benefits associated with green tea consumption have also been corroborated in animal studies of cancer chemoprevention, hypercholesterolemia, artherosclerosis, Parkinson's disease, Alzheimer's disease, and other aging-related disorders. However, the use of green tea as a cancer chemopreventive or for other health benefits has been confounded by the low oral bioavailability of its active polyphenolic catechins, particularly epigallocatechin-3-gallate (EGCG), the most active catechin. This review summarizes the purported beneficial effects of green tea and EGCG in various animal models of human diseases. Dose-related differences in the effects of EGCG in cancer versus neurodegenerative and cardiovascular diseases, as well as discrepancies between doses used in in vitro studies and achievable plasma understanding of the in vivo effects of green tea catechins in humans, before the use of green tea is widely adopted as health-promoting measure.