sulprostone and Inflammation

A wide range of studies has demonstrated the potent anticancer activity of Chinese herbs. Here, we evaluated the anticancer activity and molecular mechanisms of Actinidia chinensis root extract (acRoots) on hepatocellular carcinoma (HCC). HepG2 HCC cells were treated with various concentrations of acRoots for 72 h and examined by mRNA expression profiling, revealing alterations in cellular immunity, inflammation, proliferation, cell cycle, and metabolic signaling responses. Further analysis of the altered genes in cellular immunity and inflammation gene clusters identified prostaglandin E receptor 3 (EP3) as a key regulator of gene expression in response to acRoots. Further analysis revealed inhibition of cell growth, migration, and invasion in HCC in response to acRoots, along with increased apoptosis due to downregulation of EP3 expression. Treatment with acRoots and EP3 antagonist L-798106 led to decreases in VEGF, EGFR, MMP2, and MMP9 expression in HCC cells, along with significant effects on growth, migration, invasion, and apoptosis; the effects were reversed/blocked by the EP3 agonist sulprostone. Taken together, these data clearly demonstrated that acRoots inhibit HCC cell invasion and metastasis via inhibition of EP3 expression, resulting in decreased activation of VEGF, EGFR, MMP2, and MMP9.

Topics: Actinidia; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dinoprostone; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Liver Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Phytotherapy; Plant Extracts; Plant Roots; Receptors, Prostaglandin E, EP3 Subtype; Signal Transduction; Sulfonamides

Activation of EP2 receptors by prostaglandin E2 (PGE2) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gαs and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE2 had no effect on expression of CXCL10, TGF-β1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1β but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE2 or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.

Topics: Animals; Animals, Newborn; Brain; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Inflammation; Microglia; Models, Biological; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin E, EP2 Subtype; Signal Transduction

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Blood Platelets; Cells, Cultured; Citrates; Collagen; Dinoprostone; Drug Synergism; Hirudins; Humans; Inflammation; Platelet Aggregation; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Vasoconstrictor Agents

Although inflammatory breast cancer (IBC) is recognized as the most lethal variant of locally advanced breast cancer, few molecular signatures of IBC have been identified that can be used as targets to develop therapeutics that effectively inhibit the aggressive phenotype displayed by IBC tumors.. Real-time polymerase chain reaction analysis, Western blot analysis, modified Boyden chamber invasion assays, vasculogenic mimicry (VM) assays, and gelatin zymography were used in the current studies. Agonists and antagonists of the prostanoid receptors EP3 and EP4 and of EP4 short-hairpin RNA (shRNA) knockdown approaches were used as tools to assess the role of prostanoid receptors EP3 and EP4 in the regulation of specific biologic activities of IBC cells.. The current studies revealed that the IBC breast cancer cell lines SUM149 and SUM190 express high levels of cyclooxygenase-2 messenger RNA and protein, produce abundant levels of prostaglandin E(2), and produce both EP3 and EP4 receptor proteins. Studies using the EP4 antagonist GW627368X and shRNA molecular knockdown approaches revealed a role for EP4 in regulating invasion of IBC cells. EP3, but not EP4, regulated the ability of SUM149 cells to undergo VM, which is the ability to form capillary-like structures, a characteristic exhibited by very aggressive tumor types. Inhibition of VM by sulprostone was associated with an inhibition of matrix metalloprotease-2 (MMP-2) enzyme activity.. The prostanoid receptors EP3 and EP4 differentially regulate activities exhibited by IBC cells that have been associated with the aggressive phenotype of this lethal variant of breast cancer. Whereas EP4 regulates invasion, EP3 regulates VM and the associated increased MMP-2 enzyme activity.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclooxygenase 2; Dinoprostone; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Isoindoles; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neovascularization, Pathologic; Prostaglandins E; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Sulfonamides

Interleukin-23, a recently described cytokine produced by activated antigen-presenting cells, including dendritic cells, is a p19/p40 heterodimer. The p40 subunit is shared with IL-12, the major Th1-driving cytokine, while p19 is distantly related to IL-12 p35. IL-23 has pro-inflammatory actions, inducing IL-17 secretion from activated CD4+ T cells, and stimulating the proliferation of memory CD4+ T cells. Here, we examined the effects of PGE2, a well-known immunomodulator, on the production of IL-23 by bone marrow- derived dendritic cells (BM-DCs). Our results indicate that PGE2 increases the production of functional IL-23 from immature BM-DCs in a time- and dose-dependent manner. PGE2 induces both the expression of p19 and p40, without affecting p35 expression. The effect of PGE2 is mediated through the specific receptors EP2/4 and is mimicked by cAMP-inducing agents, such as forskolin and dbcAMP. Although PGE2 also induces IL-1beta and IL-6 expression in non-stimulated DCs, the stimulatory effect of PGE2 on IL-23 production is not mediated through IL-1beta or IL-6. GM-CSF, the pro-inflammatory cytokine required for the generation of BM-DCs, amplifies the IL-23 inducing activity of PGE2 in a synergistic manner. Recent studies described both pro- and anti-inflammatory effects of PGE2, and our results suggest an additional mechanism for its pro-inflammatory role, particularly significant for autoimmune diseases, such as rheumatoid arthritis.

Topics: Alprostadil; Animals; Arthritis; Bone Marrow Cells; Bucladesine; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Colforsin; Culture Media, Conditioned; Cyclic AMP; Dendritic Cells; Dinoprostone; Gene Expression Regulation; Inflammation; Interleukin-12; Interleukin-12 Subunit p35; Interleukin-12 Subunit p40; Interleukin-17; Interleukin-23; Interleukin-23 Subunit p19; Interleukins; Lipopolysaccharides; Male; Mice; Misoprostol; Plasmacytoma; Prostaglandin Antagonists; Protein Subunits; Receptors, Cell Surface; Second Messenger Systems; Specific Pathogen-Free Organisms; Toll-Like Receptor 2; Toll-Like Receptor 4

In this study, we investigated the role of PGE(2) in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE(2) accelerated ProNectin F(TM) (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin F(TM). We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE(2) effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin F(TM) and the pharmacological inhibitor of PKA, H-89, blocked PGE(2)-mediated adhesion. Consistent with mRNA expression of the G(s)-coupled EP4- and G(i)-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated G(s)-activity, which was inhibited by a G(i) inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE(2) accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclases; Animals; Cell Adhesion; Cyclic AMP; Dinoprostone; Dose-Response Relationship, Drug; Fibronectins; Inflammation; Isoquinolines; Mastocytoma; Mice; Protein Binding; Protein Kinases; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Tumor Cells, Cultured

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.

Topics: Adenylyl Cyclases; Animals; Calcium; Cells, Cultured; Cyclic AMP; Dinoprostone; Fluorescent Dyes; Fura-2; Gene Expression Regulation, Enzymologic; Gliosis; Inflammation; Interleukin-1; Lipopolysaccharides; Menstruation-Inducing Agents; Microglia; Rats; Rats, Wistar; Receptors, Prostaglandin E; Reverse Transcriptase Polymerase Chain Reaction; Second Messenger Systems

Topics: Alprostadil; Animals; Bradykinin; Capillary Permeability; Dinoprostone; Drug Synergism; Inflammation; Rabbits; Receptors, Prostaglandin; Receptors, Prostaglandin E; Skin; Vasodilation