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The molecular pathways by which the cyclopentenone prostaglandins (PGA and PGJ series) inhibit cell growth and tumorigenicity are poorly understood. These cellular responses may be caused by specific regulation of growth-related and stress-induced genes. A variety of prostaglandins were tested for their ability to regulate insulin-like growth factor-I (IGF-I) and Waf1 gene expression in C6 rat glioma cells. The prostaglandins (in order of potency) PGJ 2 >PGA 1 >PGA 2 ≈PGD 2 ≫PGE 2 all significantly repressed IGF-I gene expression. With the exception of PGE 2 , the same prostaglandins that repressed IGF-I also induced Waf1 gene expression. However, the order of potency for Waf1 induction was different than for IGF-I repression: PGA 2 >PGA 1 ≈PGJ 2 >PGD 2 . The different order of potency of the prostaglandins in regulating IGF-I and Waf1 gene expression suggests that different intracellular signals may be involved in regulating the two genes. Augmentation of glutathione levels by pretreatment of cells with N -acetyl- l -cysteine attenuated the effect of PGA 2 on IGF-I and Waf1 gene expression. Conversely, depletion of the intracellular glutathione pool by pretreatment with buthionine sulfoximine potentiated the effect of PGA 2 on the expression of both genes. These results suggest that conjugation with glutathione prevents the regulation of gene expression by PGA 2 . We also tested the effect of several simpler compounds that contain a five-membered ring system on IGF-I and Waf1 gene expression. 2-Cyclopenten-1-one, but not cyclopentanone or cyclopentene, repressed IGF-I and induced Waf1 gene expression, demonstrating the requirement for an α , β -unsaturated carbonyl for regulation of the two genes. The dione compound 4-cyclopentene-1,3-dione, which has two potentially reactive carbons rather than one, was considerably more potent than 2-cyclopenten-1-one in repressing IGF-I gene expression (IC 50 =30 μ M for 4-cyclopentene-1,3-dione as compared with 167 μ M for 2-cyclopenten-1-one). Additional results indicated that diethyl maleate, which has two α , β -unsaturated carbonyls in a non-cyclic configuration, also repressed IGF-I gene expression (IC 50 =214 μ M) and induced Waf1 gene expression, indicating that the cyclic structure is not required for either effect.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor family of transcription factors, a large and diverse group of proteins that mediate ligand-dependent transcriptional activation and repression. Expression of PPAR-gamma is an early and pivotal event in the differentiation of adipocytes. Several agents that promote differentiation of fibroblast lines into adipocytes have been shown to be PPAR-gamma agonists, including several prostanoids, of which 15-deoxy-delta-prostaglandin J2 is the most potent, as well as members of a new class of oral antidiabetic agents, the thiazolidinediones, and a variety of non-steroidal anti-inflammatory drugs (NSAIDs). Here we show that PPAR-gamma agonists suppress monocyte elaboration of inflammatory cytokines at agonist concentrations similar to those found to be effective for the promotion of adipogenesis. Inhibition of cytokine production may help to explain the incremental therapeutic benefit of NSAIDs observed in the treatment of rheumatoid arthritis at plasma drug concentrations substantially higher than are required to inhibit prostaglandin G/H synthase (cyclooxygenase).

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen. PPAR-gamma has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-delta prostaglandin J2 (15d-PGJ2) bind to PPAR-gamma and stimulate transcription of target genes. Prostaglandin D2 metabolites have not yet been identified in adipose tissue, but are major products of arachidonic-acid metabolism in macrophages, raising the possibility that they might serve as endogenous PPAR-gamma ligands in this cell type. Here we show that PPAR-gamma is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-gamma ligands. PPAR-gamma inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-kappaB. These observations suggest that PPAR-gamma and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-gamma ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.

Cyclopentenone prostanoids inhibit virus replication by turning on an intracellular defence response that involves the induction of cytoprotective heat-shock proteins, the modification of viral glycoprotein maturation and the control of NF-kappa B activation. These molecules represent an interesting model for the development of novel antiviral drugs that can affect different targets during the virus life cycle.

Four cyclopentenone prostaglandins (CPPGs) and PGE 2 caused significant dose-dependent inhibition in growth of human oral squamous carcinoma cells (SCC-15). The rank order of their potency was PGJ 2 >PGA 1 >16, 16-dimethyl PGA 1 >PGA 2 >PGE 2 . In a follow-up experiment it was found that the mean per cent inhibition in cell growth by PGJ 2 and Δ 12 -PGJ 2 at 10 −5 M was 61.22 and 63.81, while that of 5-fluorouracil and methotrexate was 36.67 and 38.86, respectively. Δ 12 -PGJ 2 and PGJ 2 induced significant dose-dependent inhibition in nuclear DNA synthesis (i.e. cell proliferation). Combining vitamin E succinate with lower concentrations of CPPGs enhanced significantly their inhibitory effect on nuclear DNA synthesis of cancer cells.

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.

Prostaglandins (PGs) function as intracellular signal mediators in the regulation of a variety of physiological and pathological processes, including inflammation and immune responses. Cyclopentenone PGs are characterized by antiviral activity against several viruses, including human immunodeficiency virus type 1 (HIV-1), and by the ability to induce heat shock protein expression through activation of the heat shock transcription factor. Here we report that PGA1is a potent inhibitor of nuclear factor-κB (NF-κB) activation in human cells and of NF-κB-dependent HIV-1 transcription in long terminal repeat-chloramphenicol acetyltransferase transient transfection experiments. PGA1acts by inhibiting phosphorylation and preventing degradation of the NF-κB inhibitor IκB-α. Inhibition does not require protein synthesis, is dependent on the presence of a reactive cyclopentenonic moiety, and is associated with heat shock transcription factor activation. Because NF-κB is critically involved in the activation of immunoregulatory and viral genes, inhibition of its activity could be a major component of the immunosuppressive and antiviral activity of PGs.

Inflammation is a primordial response to infection and injury that seeks to neutralise and eliminate foreign organisms and/or material. Thus, inflammation is no trivial event. Life depends upon it. In general, the innate inflammatory response initiates within minutes and resolves within hours. Chronic inflammation, on the other hand, persists for weeks, months or even years and, unlike the acute response, is the side of host immunity we need to avoid. Notwithstanding, dispersed among this black and white view of inflammation are shades of grey. We are constantly reminded that defining inflammation is not so easy and that while acute inflammation can resolve, it can also be recurrent and that, over time, chronic inflammation can also resolve or persist with devastating consequences to the host. In this presentation, one of these many aspects of the inflammatory response – how acute inflammation resolves, will be discussed. In doing so it will be argued that resolution is as active process, whose failure may predispose the host to chronic inflammatory diseases and autoimmunity. At the very least it is hoped we can highlight resolution as a critical facet of the inflammatory response and serve to underline the importance of not altering its normal course of action when developing novel anti-inflammatory drugs. Ultimately, it will be proposed here that resolution is controlled by endogenous pro-resolution factors, which, for the future, may represent a treasure trove for drug discovery in terms of designing drugs that mimic their mode of action or enhance their synthesis. Specifically, I will recount the earlier work done on cyclooxygenase and lipoxygenase-derived lipid mediators (cyclopentenone prostaglandins, prostaglandin D 2 , lipoxins and aspirin-triggered epi-lipoxins) in limiting the continuity of the inflammatory response; bring the audience up to date with a more recent understanding of some of the cellular players in this setting and introduce some new soluble mediators (resolvins) and their receptors as potentially novel pro-resolving agents. In this setting, macrophage phenotype as a critical determinant of inflammation longevity will also be discussed. Disclosure of Interest None Declared

Cyclopentenone prostaglandins (PGs) inhibit the replication of a wide variety of enveloped DNA and RNA viruses. The antiviral activity is associated with alterations in the synthesis, maturation, and intracellular translocation of viral proteins. In the present report, we describe the effects of cyclopentenone PGs PGA1 and delta 12-PGJ2 on poliovirus (PV) replication in HeLa cells. Both PGs were found to inhibit PV replication dose dependently. Virus yield was significantly reduced at nontoxic concentrations, which did not suppress RNA or protein synthesis in uninfected or PV-infected cells. Both the pattern of PV proteins synthesized and the kinetics of viral protein synthesis and degradation appeared to be similar in PGA1-treated cells and control cells. Antiviral PGs have been shown to selectively inhibit virus protein synthesis during the replication of several viruses, including vesicular stomatitis virus (VSV), and this effect has been recently associated with the induction of a 70-kDa heat shock protein (HSP70). PGA1 and delta 12-PGJ2 were found to induce HSP70 synthesis in uninfected or VSV-infected HeLa cells. PV infection was found to inhibit PG-induced HSP70 synthesis in these cells, suggesting that the lack of ability of cyclopentenone PGs to block PV protein synthesis could be related to an impaired heat shock response in PV-infected cells. The finding that PV protein synthesis was not inhibited by PGs suggests that cyclopentenone PGs could interfere with a late event in the virus replication cycle, such as protein assembly and maturation of PV virions.

Cyclopentenone prostaglandins (PGs) inhibit virus replication in several DNA and RNA virus models, in vitro and in vivo. In the present report we demonstrate that the cyclopentenone prostaglandins PGA(1) and PGJ(2) at nontoxic concentrations can dramatically suppress HIV-1 replication during acute infection in CEM-SS cells. PGs did not affect HIV-1 adsorption, penetration, reverse transcriptase activity nor viral DNA accumulation in HIV-1 infected cells. A dramatic reduction in HIV-1 mRNA levels was detected up to 48-72 h after infection (p.i.) in PG-treated cells, and HIV-1 protein synthesis was greatly reduced by a single PG-treatment up to 96 h p.i. Repeated PGA(1)-treatments were effective in protecting CEM-SS cells by the cytopathic effect of the virus, and in dramatically reducing HIV-1 RNA levels up to 7 d after infection. The antiviral effect was not mediated by alterations in the expression of alpha-, beta-, or gamma-interferon,TNFalpha, TNFbeta, IL6, and IL10 in HIV-infected CEM-SS cells. The fact that prostaglandins are used clinically in the treatment of several diseases, suggests a potential use of cyclopentenone PGs in the treatment of HIV-infection.

The cyclopentenone prostaglandins (PGs), such as delta 12-PGJ2 and PGA1, are potent inhibitors of growth in a variety of cultured cells, including human epidermal cells. To clarify the mechanism of the cytotoxicity of these PGs, we examined the effects of delta 12-PGJ2 on the function and expression of E-cadherin, which plays a major role in the maintenance of intercellular adhesion, in transformed human epidermal cells in culture (HSC-1). A 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 did not affect the cell-binding activity of E-cadherin expressed in HSC-1 cells. Immunoblot analysis using a monoclonal antibody specific to human E-cadherin revealed that a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 induced E-cadherin expression in HSC-1 cells. Immunofluorescence using a monoclonal antibody against human E-cadherin demonstrated that E-cadherin was localized to the cell-cell contact regions in HSC-1 cells. Following a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2, E-cadherin was also detected in a uniform pattern along cell junctions, although cell morphology was changed by the presence of cytotoxic PGs. These results suggest that the cytotoxicity of cyclopentenone PGs is related, at least in part, to E-cadherin expression in transformed human epidermal cells.

A new horizon in chemoprevention research is the recent discovery of molecular links between inflammation and cancer. The implication of inflammatory tissue damage in pathophysiology of human maligancy is under intense investigation both at the research level and in clinical practice. Numerous studies have been reported with the global ‘omics’ profiling technologies, such as DNA microarray, proteomics, metabolomics, lipidomics, etc., to identify and characterize a series of critical molecules/changes in the inflammatory signaling. It is by gaining this type of mechanistic understanding of a disease that researchers will unlock the keys to discovering new diagnostics and therapeutic strategies for the management of inflammation-associated cancer. Aberrant upregulation of cyclooxygenase-2 (COX-2), a key enzyme in the arachidonic acid cascade, has been frequently observed in various precancerous and malignant tissues. Therefore, the normalization of inappropriately overamplified signaling cascades implicated in chronic inflammation-associated carcinogenesis by use of COX-2 specific inhibitors has been recognized as a rational and pragmatic strategy in molecular target-based chemoprevention. Proinflammatory stimuli trigger the activation of intracellular signal transduction network comprising proline-directed serine/threonine kinases, and their downstream transcription factors, such as NF-κB, resulting in an overexpression of COX-2. Cyclopentenone prostaglandins of J series as products of cyclooxygenase-2 (COX-2)-mediated arachidonic acid cascades have been reported to possess multifaceted cellular functions, including antiinflammatory and cytoprotective effects. A typical example is 15-deoxy-Δ 12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor-γ (PPARγ), that modulates the activities of various intracellular signaling molecules. Because of the electrophilic α,β-unsaturated carbonyl moiety present in its cyclopentenone ring structure, 15d-PGJ2 acts as a Michael addition acceptor and can readily interact with critical cellular nucleophiles, such as cysteine thiol groups of proteins. Many of the biological effects induced by 15d-PGJ2 are mediated by targeting redox-sensitive transcription factors and their regulators, including IκB kinase-NF-κB, AP-1, Nrf2-Keap1, HIF-1α, STAT3and p53 tumor suppressor. The regulation of the aforementioned signaling molecules by 15d-PGJ2 is not likely to be dependent on PPARγ activation, but rather involves direct modification or oxidation of their critical cysteine residues acting as a redox-sensor. Though 15d-PGJ2 exerts cytoprotective effects in normal cells, elevated production of this cyclopentenone prostaglandin has been implicated in tumorigenesis through induction of angiogenesis and metastasis. As one of the terminal products of COX-2-mediated arachidonic acid metabolism, 15d-PGJ2 upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line by activation of Akt and subsequently AP-1. Some malignant cells might exploit this visious cycle of COX-2 up-expression for maintaining invasiveness and taking survival advantage. Cysteine thiols present in various transcription factors and their regulators function as redox sensors in fine-tuning of transcriptional regulation. Thus, oxidation or covalent modification of thiol groups present in the redox-sensitive transcription factors and their regulating molecules can provide a unique strategy for molecular target-based chemoprevention. Citation Information: Cancer Prev Res 2011;4(10 Suppl):CN07-01.

Cyclopentenone prostaglandins (PGs) induce the synthesis of heat shock proteins (HSPs) in mammalian cells. Since arachidonic acid metabolites are implicated in the control of fever, we investigated the effect of PG treatment on thermal injury in human K562 erythro-leukemia cells. Prostaglandin A1 (PGA1) was found to protect cells after severe heat shock and to induce a thermotolerant state, which persisted for 24-48 h. Prostaglandins of the B, E, and F type were not effective. Kinetics of thermotolerance induction was comparable to heat-induced heat resistance. Establishment of a thermotolerant state was not a direct effect of PGA1, since it was dependent on de novo protein synthesis and was associated with HSP70 induction. This activity of PGA1 could be part of a protective control mechanism during fever.

Cyclopentenone prostaglandins (PGs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report on the effect of prostaglandin A1 (PGA1) on the multiplication of a positive strand RNA virus, Sindbis virus, in Vero cells under one-step multiplication conditions. PGA1 was found to inhibit Sindbis virus production dose-dependently, and virus yield was reduced by more than 90% at the concentration of 8 micrograms/ml, which was non-toxic to the cells and did not inhibit DNA, RNA or protein synthesis in Vero cells. The cyclopentenone prostaglandin delta 12-PGJ2 was also shown to be a potent inhibitor of Sindbis virus replication. Virus-induced reduction of [3H]uridine uptake by cells was partially prevented by PGA1 treatment, which also caused a 1 h delay in the peak of virus RNA synthesis. SDS-PAGE analysis of [35S]methionine-labeled proteins showed that PGA1 moderately inhibited the synthesis of the viral structural proteins E1, E2 and C, and induced the synthesis of a 72 kDa M(r) protein, identified as a heat-shock protein related to the HSP70 group, in both virus-infected and uninfected cells. Actinomycin D treatment completely prevented PGA1-antiviral activity, indicating that a cellular product is responsible for this action. PGA1-induced HSP70 is a good candidate for this role.

The histone deacetylase inhibitors (HDIs) valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat) are used clinically for the treatment of epilepsy and cutaneous T-cell lymphoma, respectively. HDIs have been shown to be more effectively utilized in combination with other agents despite the promising anti-tumor effect of HDIs alone. Therefore, additional mechanism-based therapeutic strategies for use of HDIs in combination with other agents should be investigated to overcome resistance to HDIs or to increase the efficacy of HDIs. The natural endogenous ligand of peroxisome proliferator-activated receptor γ (PPARγ) 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a member of cyclopentenone prostaglandins and known to be a potent anti-neoplastic agent. Thus, we examined whether VPA or SAHA in combination with 15d-PGJ2 could show synergistic anti-tumor activitiy in colon cancer DLD-1 cells. Cell viability was determined using a Cell Counting Kit-8 assay. Apoptosis and reactive oxygen species (ROS) generation were determined using flow cytometry analysis. Western blotting and real-time RT-PCR analysis were carried out to investigate the expressions of apoptosis-related molecules. Small interfering RNA or transient transfection with plasmids was used for gene silencing, gene overexpression or luciferase assays. Mice bearing DLD-1 xenograft were divided into 4 groups (n=5) and injected everyday (i.p.) with diluent, VPA (100 mg/kg), 15d-PGJ2 (5 mg/kg) or a combination for 25 days. Cotreatment with VPA or SAHA at clinically achievable concentrations and 15d-PGJ2 synergistically induced cell death and drastically caused caspase-dependent apoptosis in DLD-1 cells. The apoptosis induced by VPA/15d-PGJ2 cotreatment was due to alterations of apoptosis-related genes such as an antiapoptotic Bcl-2 family member Bcl-XL, X-chromosome-linked inhibitor of apoptosis (XIAP), C/EBP homologous protein (CHOP) and death receptor 5 (DR5) via ROS generation, subsequent endoplasmic reticulum stress and/or histone deacetylase inhibition. Moreover, VPA/15d-PGJ2 cotreatment induced ROS-dependent apoptosis in other malignant tumor cells and was more effective than a VPA or 15d-PGJ2 monotherapy in vivo. These results suggest that cotreatment with the clinically relevant HDIs and 15d-PGJ2 may warrant clinical investigation as a rational combination therapy against a broad spectrum of malignant tumors. 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 5415.

Treatment of human K562 erythroleukemia cells with the antiproliferative prostaglandin A1 results in the elevated transcription of two heat shock genes, HSP70 and HSP90. Parallel with increased heat shock gene transcription is the activation of heat shock transcription factor. Heat shock transcription factor levels are induced within 60 min after prostaglandin A1 addition to levels similar to that achieved during heat shock. The requirement for protein synthesis for prostaglandin A1 activation of heat shock transcription factor suggests that effects on nascent protein synthesis may be involved in the signaling mechanism. Although it is unclear whether the activation of a heat shock response by prostaglandins is relevant to the biochemical properties of these natural substances, cells pretreated with prostaglandin A1 are protected against a subsequent heat shock, indicative of a thermotolerant state.

Although the effects of anti-inflammatory drugs have been well studied, little is known about the signaling pathways involved in the inflammatory response. The recent finding that cyclopentenone prostaglandins inhibit IκB kinase provides a new explanation for the anti-inflammatory effects of these molecules.

Abstract 1645 Poster Board I-671 Human Dectin-1 (hDectin-1) is a member of the C-type lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these pathogens. Activation of Dectin-1 via extracts such as zymosan or more specifically with curdlan can lead to DC activation characterized by upregulation of surface molecules or secretion of cytokines. Futhermore, Dectin-1 is important for recruitment of leukocytes and production of inflammatory mediators at the site of infection.. Peroxisome proliferator-activated receptor (PPAR) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have been shown to have profound modulatory effects on B and T lymphocytes as well as DCs. Cyclopentenone prostaglandins are produced during the late phase of inflammation due to up-regulation of cyclooxygenase 2 (COX2), a key enzyme for the synthesis of cyclopentenone prostaglandins, that mediate their effects by activation of PPAR-gamma dependent and independent pathways. In our study we analyzed the effects of troglitazone (TGZ), a high affinity synthetic ligand of PPAR-gamma, on the Dectin-1 mediated activation of antigen presenting cells (APC). TGZ was added to the cell culture medium during the differention of monocyte derived DC. On day 5-6 of culture the cells were additionally treated with curdlan or zymosan and phenotypical and functional analyses were performed. Activation of PPAR-gamma resulted in a reduced stimulation of APC via Dectin-1 characterized by down-regulation of CD1a, CD83 and costimulatory molecules on the cell surface and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation and recruitment including IL-1beta, TNF-a, IL-6, IL-12, MIP-1a and Rantes. Western blot analyses revealed that these inhibitory effects were mediated by the inhibition of the mitogen-activated protein (MAP) kinase pathways characterized by a reduced phosphorylation of ERK1/2 and p38. Furthermore, we found that Dectn-1 induced c-Jun phosphorylation was reduced by pretreatment with TGZ. In addition, Dectin-1 induced nuclear translocation of NF-kappaB family members RelB and cRel was dramatically reduced in APC incubated with TGZ. The observed inhibition of signaling pathways was not due to a reduced expression of Dectin-1, phosphorylation of Syk or increased secretion of IL-10 and TGF-beta. TGZ had no effect on the Dectin-1 induced phosphorylation and nuclear expression of NFAT. Our data demonstrate that PPAR-gamma ligands inhibit Dectin-1 mediated activation by interfering with the MAP kinase, c-Jun and NF-kappaB signaling pathways, thus confirming their important role as a negative feedback mechanisms in the regulation of potentially harmful inflammation signaling pathways. Disclosures No relevant conflicts of interest to declare.

PGs of A and J series are active compounds exerting anti-proliferative and anti-viral activities of PGs. They are a group of PGs having a reactive α, β-unsaturated ketone group in their cyclopentane ring and thus named cyclopentenone PGs. Some cyclopentenone PGs occur in nature. Clavulones and punaglandins, acetoxy and halogenated derivatives of PGA, are isolated from octocoral, and Δ12-PGJ2 was identified as a PGD2 metabolite in human urine. These PGs dose-dependently inhibit growth of cultured cells. The structure activity relationship of these PGs is quite different from those of other PGs, and unlike other PGs, they do not express their actions by acting on a cell surface receptor. They are actively transported into cells, and accumulate in nuclei. These cellular uptake and nuclear accumulation correlate well with their anti-proliferative actions. Flow cytometric analysis revealed that they block cell cycle progression at a specifc point in the G1 phase. Concurrent with this block, the PGs induce and suppress expression of several proteins. In addition to the anti-proliferative actions, these PGs show antiviral activity at concentrations not toxic to host cells. The structure activity relationship for this activity is similar to that for the anti-proliferative activity, and studies have suggested that the PGs suppress expression of some viral genes to prevent virus replication. Thus, there are accumulating evidences that the cyclopentenone PGs exert their actions by regulating specific genes in nuclei. Based on these in vitro studies, pre-clinical trials are in progress using synthetic analogues of these PGs on their anti-tumor and anti-viral activities.

The cytotoxic action of various prostanoids was examined on a transformed human epidermal cell line (HSC-1), and methyl(5S,6S,7Z)-5,6-diacetoxy-7-((2S)-4-chloro-2-hydroxy-2-((2Z)-2-octenyl)-5-oxo-3-cyclopentenylidene)heptanoate (YM-11), which is a punaglandin compound, was found to be most active. YM-11 exerted a dose-dependent inhibition of HSC-1 cell growth over 0.03 µM (0.01 µg/ml), and at 0.3 µM(0.1 µg/ml) its growth was completely inhibited. The IC50 value of YM-11 on HSC-1 cell growth was calculated as 0.15 µM(0.05 µg/ml). Methyl(E)-7-(5-chloro-2-hydroxy-2-octyl-5-oxo-3-cyclopentenylidene)heptanoate (YM-3), which is also a punaglandin derivative, showed remarkable cytotoxicity on HSC-1 cells with an IC50 of 0.24 µM(0.08 µg/ml). Concerning other cytotoxic prostaglandins (PGs), the IC50 values of Δ7-PGA1, Δ12-PGJ2 and PGD2 were 1.5 µM (0.5 µg/ml), 2.1 µM(0.75 µg/ml) and 5.7 µM (2 µg/ml), respectively. On the basis of the present data and previous in vitro and in vivo evidence, punaglandin derivatives may be useful antineoplastic agents for skin cancer.

Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ2, and delta 12-PGJ2, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA2 and delta 12-PGJ2 in primary cultures of purified rat hepatocytes. As a model ligand, [3H]PGA1 bound to L-FABP specifically, reversibly, rapidly, and with high affinity. Its dissociation constants were 134 nM (high affinity) and 3.6 microM (low affinity). The high-affinity binding of [3H]PGA1 was 9- and approximately 13-fold more avid than the binding of the conventional fatty acid ligands, oleic acid and arachidonic acid, respectively. The abilities of different prostaglandins to compete with the high-affinity binding of [3H]PGA1 correlated with their growth inhibitory activities reported previously and here. The growth inhibitory cyclopentenone prostaglandins (PGA1, PGA2, delta 12-PGJ2, and PGJ2) were the best competitive ligands, intermediate competitors were the weak growth inhibitors PGE1 and PGD2, and the poorest competitors were PGE2 and PGF2 alpha, which stimulate rather than inhibit DNA synthesis in rat hepatocytes in primary culture. The in vitro actions of L-FABP are compatible with those of a specific and dissociable carrier of growth inhibitory prostaglandins in rat hepatocytes and suggest that the carcinogen may usurp the cellular machinery of the growth inhibitory prostaglandins.

Graft-versus-host disease (GVHD) mediated by alloreactive donor T cells is the most dreaded complication after allogeneic bone marrow transplantation (BMT). Interaction of donor T cells with host antigen presenting cells is considered to be central to the development of GVHD. Furthermore, GVHD development depends on the inflammatory response induced by the conditioning regimen. Interfering with inflammatory signaling pathways could be an approach to prevent GVHD without compromising the graft-versus-malignancy reaction. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear receptor transcription factor that regulates cell growth, differentiation and homeostasis. PPARγ agonists, such as 15-deoxy -Δ12,14-Prostaglandin J2 (15d-PGJ2) have been shown to have potent immunomodulatory effects on B and T lymphocytes as well as DCs, and recent studies have demonstrated protective effects of PPARγ agonists on animal models of autoimmunity including experimental allergic encephalomyelitis (EAE), asthma, arthritis, colitis and diabetes. There is very few data addressing the role of PPARγ agonists on GVHD. The aim of this study was to investigate the protective effect and mechanism of 15d-PGJ2 on experimental GVHD. GVHD was induced following lethal irradiation (1075 rad) using the fully MHC-mismatched strain combination BALB/c to B6. Recipients were treated with 1 mg/kg of 15d-PGJ2 i.p on days −1, +1, +3, +5 and +7 post-BMT. Treatment with 15d-PGJ2 resulted in a significantly reduced GVHD mortality (p=0.01 log rank test) with a Median Survival Time (MST) of 23 days in the control group. Median survival was not reached in the 15d-PGJ2 treatment group at the end of the observation period (day +50 post BMT). Furthermore, we found a significant reduction in donor T cell expansion of 15d-PGJ2-treated animals on day +6 post-BMT. To further delineate the mechanisms underlying its protective effect, we analyzed the effects of 15d-PGJ2 on alloantigen-driven proliferation in MLR cultures in vitro as well as development and maturation of mouse bone marrow derived- dendritic cells (DC) in vitro. Treatment with 15d-PGJ2 resulted in a dose-dependent inhibition of allo-antigen driven proliferation. Furthermore, using a 12-day culture system 15d-PGJ2 led to a significant reduction of mature DC as determined by the number of DC co-expressing CD40, CD80, CD86. Treatment for 24 hours with 5uM of 15d-PGJ2 significantly inhibited activation of the NF-κB pathway as demonstrated by abrogation of p65 and IkB-α phosphorylation in response to alloactivation and also in 15d-PGJ2 treated DC using western blot analysis. In conclusion our data support the notion that the inhibitory effect of 15d-PGJ2 on the development of GVHD involves NF-κB-dependent inhibition of DC maturation and expansion of donor T cells. Given its potent antitumor effects cyclopentenone prostaglandins could be ideal candidates for prevention of GVHD while maintaining Graft versus malignancy effects.

Human albumin catalyzed the in vitro transformation of prostaglandin D2 into three novel dehydration products identified as 9-deoxy-11-keto-15 alpha-hydroxy-delta-5,9,12-prostenoic acid; 15-deoxy-11-keto-9 alpha-hydroxy-delta 5,9,12,14-prostenoic acid. Results suggest that albumin can influence, qualitatively and quantitatively, the metabolism of eicosanoids.

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 act specifically on cells in the G1 phase and induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima, M., Narumiya, S., and Fujiwara, M. (1988) J. Pharmacol. Exp. Ther. 245, 294-298). In this study, we characterized proteins induced by these PGs in HeLa S3 cells of synchronized growth and examined its association with the cell cycle block. HeLa S3 cells transiently expressed two 68-kDa proteins of isoelectric points of 5.5 and 5.6 in the G1 phase of cell cycle. When G1-enriched cells were incubated with either PGA2 or delta 12-PGJ2, synthesis of these proteins was markedly enhanced. Enhancement by delta 12-PGJ2 was persistent and irreversible, whereas that by PGA2 was reversible. delta 12-PGJ2 also enhanced the synthesis of two additional 68-kDa proteins with isoelectric points of 5.8 and 5.9. On two-dimensional gel electrophoresis, these proteins overlapped exactly with the 68-kDa heat shock proteins induced in cells treated at 43 degrees C for 90 min. They were also indistinguishable from the heat shock proteins in limited proteolysis. When delta 12-PGJ2 was incubated with G2/M phase cells, it induced only a small and transient increase in the 68-kDa proteins. These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the G1 phase HeLa S3 cells and this induction is closely associated with the G1 block of cell cycle progression caused by these PGs.

15-Deoxy-Δ 12,14 -prostaglandin J 2 regulates mesangial cell proliferation and death. Background Proliferation of intrinsic glomerular cells is a common response to renal injury. Acutely, proliferation may be beneficial, but sustained glomerular hypercellularity after injury is associated with progressive renal failure. To identify endogenous factors that may be responsible for regulating glomerular cell number, the effects of J-series cyclopentenone prostaglandins (PGs) on human glomerular mesangial cell proliferation and death were examined. Methods Human mesangial cells were grown in the presence or absence of PGJ 2 or its metabolite 15-Deoxy-Δ 12,14 -PGJ 2 (15dPGJ 2 ). The number of viable cells was measured by the reduction of the tetrazolium MTS to a colored formazan product. Apoptosis was assessed by caspase-3 activation and DNA fragmentation. Results PGJ 2 at concentrations up to 10 μmol/L caused mesangial proliferation. 15dPGJ 2 also caused mesangial proliferation at low concentrations (≤2.5 μmol/L), but induced mesangial cell death at higher concentrations (>5 μmol/L). Cell death occurred in part through apoptosis, measured as an increase in caspase-3 activity and DNA fragmentation in 15dPGJ 2 -treated cells. Cell death was associated with a decline in baseline phosphorylation of the survival factor Akt and increased Akt degradation, whereas 15dPGJ 2 -induced mesangial proliferation was blocked by inhibition of the PI 3-kinase/Akt pathway. 15dPGJ 2 is a potent PPARγ agonist. Like 15dPGJ 2 , treatment of mesangial cells with thiazolidinedione-type PPARγ ligands (10 to 20 μmol/L) caused significant cell death, but at lower concentrations also caused a small degree of proliferation. Conclusions J-series prostaglandins thus may be involved in the initiation of glomerular hypercellularity through Akt-dependent proliferation, and restoration of normal glomerular architecture through PPARγ-mediated apoptosis. Manipulation of these prostaglandins may be relevant to the treatment of progressive glomerular disease.

Regulation of adipose cell mass is a critical homeostatic process in higher vertebrates. The conversion of fibroblasts into cells of the adipose lineage is induced by expression of the orphan nuclear receptor PPAR gamma. This suggests that an endogenous PPAR gamma ligand may be an important regulator of adipogenesis. By assaying arachidonate metabolites for their capacity to activate PPAR response elements, we have identified 15-deoxy-delta 12, 14-prostaglandin J2 as both a PPAR gamma ligand and an inducer of adipogenesis. Similarly, the thiazolidinedione class of antidiabetic drugs also bind to PPAR gamma and act as potent regulators of adipocyte development. Thus, adipogenic prostanoids and antidiabetic thiazolidinediones initiate key transcriptional events through a common nuclear receptor signaling pathway. These findings suggest a pivotal role for PPAR gamma and its endogenous ligand in adipocyte development and glucose homeostasis and as a target for intervention in metabolic disorders.

Effects of cyclopentenone prostaglandins, delta 12-prostaglandin (PG) J2 and PGA2 on the expression of N-myc in relation to the effects on cell cycle progression were investigated using human neuroblastoma cell line GOTO. Both PGs suppressed N-myc expression within several hours prior to inducing G1 arrest. The N-myc suppression with delta 12-PGJ2 was continued but with PGA2 it was gradually released, followed by the release of G1 arrest. These results suggest that delta 12-PGJ2 and PGA2 inhibit cell cycle progression in strong association with N-myc suppression and delta 12-PGJ2 is more potent and has a longer effect than PGA2.

Prostaglandins (PGs) A1 and J2 were found to potently suppress the proliferation of human K562 erythroleukemia cells and to induce the synthesis of a 74-kDa protein (p74) that was identified as a heat shock protein related to the major 70-kDa heat shock protein group. p74 synthesis was stimulated at doses of PGA1 and PGJ2 that inhibited cell replication, and its accumulation ceased upon removal of the PG-induced proliferation block. PGs that did not affect K562 cell replication did not induce p74 synthesis. p74 was found to be localized mainly in the cytoplasm of PG-treated cells, but moderate amounts were found also in dense areas of the nucleus after PGJ2 treatment. p74 synthesis was not necessarily associated with cytotoxicity or with inhibition of cell protein synthesis. The results described support the hypothesis that synthesis of the 70-kDa heat shock proteins is associated with changes in cell proliferation. The observation that PGs can induce the synthesis of heat shock proteins expands our understanding of the mechanism of action of these compounds whose regulatory role is well known in many physiological phenomena, including the control of fever production.

Prostaglandins of the A series strongly inhibit the production of Sendai virus in African green monkey kidney cells and are able to prevent the establishment of persistent infection ("carrier" state). This action is specific for prostaglandin A and is not due to alteration in the host cell metabolism or in the virus infectivity. The possibility that this effect is mediated by interferon is discussed.

Human rhinoviruses (HRV) are recognized as the major etiologic agents of the common cold. Starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (HT) on HRV replication in HeLa cells. We report that a 20-min HT at 45°C is effective in suppressing HRV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins is not affected by HT, indicating that the target for treatment is a posttranslational event. The antiviral effect is a transient cell-mediated event and is associated with the synthesis of the 70-kDa heat shock protein hsp70. Unlike poliovirus, rhinovirus infection does not inhibit the expression of hsp70 induced by heat. The possibility that hsp70 could play a role in the control of rhinovirus replication is suggested by the fact that a different class of HSP inducers, the cyclopentenone prostaglandins PGA 1 and Δ 12 -PGJ 2 , were also effective in inhibiting HRV replication in HeLa cells. Inhibition of hsp70 expression by actinomycin D prevented the antiviral activity of prostaglandins in HRV-infected cells. These results indicate that the beneficial effect of respiratory hyperthermia may be mediated by the induction of a cytoprotective heat shock response in rhinovirus-infected cells.

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmax app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s−1·mg protein−1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

40 p.-5 fig.-1 tab., Cyclopentenone prostaglandins (cyPG) are endogenous lipid mediators involved in the resolution of inflammation and the regulation of cell proliferation and cellular redox status. Upon exogenous administration they have shown beneficial effects in models of inflammation and tissue injury, as well as potential antitumoral actions, which have raised a considerable interest in their study for the development of therapeutic tools. Due to their electrophilic nature, the best-known mechanism of action of these mediators is the covalent modification of proteins at cysteine residues through Michael addition. Identification of cyPG targets through proteomic approaches, including MS/MS analysis to pinpoint the modified residues, is proving critical to characterize their mechanisms of action. Among the targets of cyPG are proinflammatory transcription factors, proteins involved in cell defense, such as the regulator of the antioxidant response Keap1 and detoxifying enzymes like GST, and key signaling proteins like Ras proteins. Moreover, cyPG may interact with redox-active small molecules, such as glutathione and hydrogen sulfide. Much has been learned about cyPG in the past few years and this knowledge has also contributed to clarify both pharmacological actions and signaling mechanisms of these and other electrophilic lipids. Given the fact that many cyPG targets are involved in or are targets for redox regulation, there is a complex interplay with redox-induced modifications. Here we address the modification of protein cysteine residues by cyPG elucidated by proteomic studies, paying special attention to the interplay with redox signaling., Work in the authors’ laboratory is supported by grants from MINECO SAF2009-11642 and SAF2012-36519, RETIC RIRAAF RD07/0064/0007 and RD12/0013/0008 from ISCIII and COST action CM1001. CLO is the recipient of a FPI fellowship from MINECO.