15d-PGJ2 Research Articles

Part 4: Conflict of interest management before, during, and after the 2010 International Consensus Conference on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations

Part 4: Conflict of interest management before, during, and after the 2010 International Consensus Conference on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science with Treatment Recommendations

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA(2)-IVA, the calcium-independent iPLA(2)-VIA, and the secreted sPLA(2)-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of phospholipase A(2) (PLA(2))and PLD(2). A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-prostaglandinJ(2) (15-d PGJ(2)), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.

Cyclopentenone Prostaglandins with Dienone Structure Promote Cross-Linking of the Chemoresistance-Inducing Enzyme Glutathione Transferase P1-1

Glutathione transferase P1-1 (GSTP1-1) plays crucial roles in cancer chemoprevention and chemoresistance and is a key target for anticancer drug development. Oxidative stress or inhibitor-induced GSTP1-1 oligomerization leads to the activation of stress cascades and apoptosis in various tumor cells. Therefore, bivalent glutathione transferase (GST) inhibitors with the potential to interact with GST dimers are been sought as pharmacological and/or therapeutic agents. Here we have characterized GSTP1-1 oligomerization in response to various endogenous and exogenous agents. Ethacrynic acid, a classic GSTP1-1 inhibitor, 4-hydroxy-nonenal, hydrogen peroxide, and diamide all induced reversible GSTP1-1 oligomerization in Jurkat leukemia cells through the formation of disulphide bonds involving Cys47 and/or Cys101, as suggested by reducing and nonreducing SDS-polyacrylamide gel electrophoresis analysis of cysteine to serine mutants. Remarkably, the electrophilic prostanoid 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) induced irreversible GSTP1-1 oligomerization, specifically involving Cys101, a residue present in the human but not in the murine enzyme. 15d-PGJ(2)-induced GSTP1-1 cross-linking required the prostaglandin (PG) dienone structure and was associated with sustained c-Jun NH(2)-terminal kinase activation and induction of apoptosis. It is noteworthy that 15d-PGJ(2) elicited GSTP1-1 cross-linking in vitro, a process that could be mimicked by other dienone cyclopentenone PG, such as Δ(12)-PGJ(2), and by the bifunctional thiol reagent dibromobimane, suggesting that cyclopentenone PG may be directly involved in oligomer formation. Remarkably, Δ(12)-PGJ(2)-induced oligomeric species were clearly observed by electron microscopy showing dimensions compatible with GSTP1-1 tetramers. These results provide the first direct visualization of GSTP1-1 oligomeric species. Moreover, they offer novel strategies for the modulation of GSTP1-1 cellular functions, which could be exploited to overcome its role in cancer chemoresistance.

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells

In human cells the length of telomeres depends on telomerase activity. This activity and the expression of the catalytic subunit of human telomerase reverse transcriptase (hTERT) is strongly up-regulated in most human cancers. hTERT expression is regulated by different transcription factors, such as c-Myc, Mad1 and Sp1. In this study, we demonstrated that 15d-PG J2 and rosiglitazone (an endogenous and synthetic peroxisome proliferators activated receptor γ (PPARγ) ligand, respectively) inhibited hTERT expression and telomerase activity in CaCo-2 colon cancer cells. Moreover, both ligands inhibited c-Myc protein expression and its E-box DNA binding activity. Additionally, Mad1 protein expression and its E-box DNA binding activity were strongly increased by 15d-PG J2 and, to a lesser extent, by rosiglitazone. Sp1 transcription factor expression and its GC-box DNA binding activity were not affected by both PPARγ ligands. Results obtained by transient transfection of CaCo-2 cells with pmaxFP-Green-PRL plasmid constructs containing the functional hTERT core promoter (including one E-box and five GC-boxes) and its E-box deleted sequences, cloned upstream of the green fluorescent protein reporter gene, demonstrated that 15d-PG J2, and with minor effectiveness, rosiglitazone, strongly reduced hTERT core promoter activity. E-boxes for Myc/Mad/Max binding showed a higher activity than GC-boxes for Sp1. By using GW9662, an antagonist of PPARγ, we demonstrated that the effects of 15d-PG J2 are completely PPARγ independent, whereas the effects of rosiglitazone on hTERT expression seem to be partially PPARγ independent. The regulation of hTERT expression by 15d-PG J2 and rosiglitazone, through the modulation of the Myc/Max/Mad1 network, may represent a new mechanism of action of these substances in inhibiting cell proliferation.

Redox Signaling, Alkylation (Carbonylation) of Conserved Cysteines Inactivates Class I Histone Deacetylases 1, 2, and 3 and Antagonizes Their Transcriptional Repressor Function

Cells use redox signaling to adapt to oxidative stress. For instance, certain transcription factors exist in a latent state that may be disrupted by oxidative modifications that activate their transcription potential. We hypothesized that DNA-binding sites (response elements) for redox-sensitive transcription factors may also exist in a latent state, maintained by co-repressor complexes containing class I histone deacetylase (HDAC) enzymes, and that HDAC inactivation by oxidative stress may antagonize deacetylase activity and unmask electrophile-response elements, thus activating transcription. Electrophiles suitable to test this hypothesis include reactive carbonyl species, often derived from peroxidation of arachidonic acid. We report that alpha,beta-unsaturated carbonyl compounds, e.g. the cyclopentenone prostaglandin, 15-deoxy-Delta12,14-PGJ(2) (15d-PGJ(2)), and 4-hydroxy-2-nonenal (4HNE), alkylate (carbonylate), a subset of class I HDACs including HDAC1, -2, and -3, but not HDAC8. Covalent modification at two conserved cysteine residues, corresponding to Cys(261) and Cys(273) in HDAC1, coincided with attenuation of histone deacetylase activity, changes in histone H3 and H4 acetylation patterns, derepression of a LEF1.beta-catenin model system, and transcription of HDAC-repressed genes, e.g. heme oxygenase-1 (HO-1), Gadd45, and HSP70. Identification of particular class I HDACs as components of the redox/electrophile-responsive proteome offers a basis for understanding how cells stratify their responses to varying degrees of pathophysiological oxidative stress associated with inflammation, cancer, and metabolic syndrome.

Effects of 15-deoxy-△12,14-prostagliandxin J2 on the expression of type I collagen in hypertrophic scar of rabbit ears

Objective To investigate the effect of 15d-PGJ2 on the expression of type Ⅰ collagen in hypertrophic scar of rabbit ears, and the possibility of hypertrophic scar treated by 15d-PGJ2. Methods Eighteen New Zealand white rabbits were used to establish the hypertrophic scar models on the rabbit ears. The wounds were created as follows: 2 cm × 3 cm wounds with total skin loses on the ventral side, 2wounds for each ear, total 72 wounds. The wounds were randomly divided into the 15d-PGJ2 treatment group and NS control group. A total of 20 μl 15d-PGJ2 or NS was injected into the ear scar once a day for 7 days. At the 14th and 21st day after the injection, 18 scars in each group were harvested. The expression levels of type Ⅰ collagen was detected by immunohistochemistry, fluorescence quantitative polymerase chain reaction (PCR) and Western blotting. Results Compared with the NS control group, the 15d-PGJ2-treated scars appeared to be smaller, softer, flatter and lighter in color. The expression of type Ⅰcollagen was mainly distributed in matrix, fibroblast cytoplasma and the vascular wall. The mRNA and protein levels of type Ⅰ collagen were significantly decreased in the 15d-PGJ2-treated group as compared with those in the NS control group at different time points (P < 0. 05). Conclusion 15d-PGJ2, the ligand of PPAR-γ, can reduce the expression of type Ⅰ collagen in hypertrophic scar of rabbit ears and plays an important role in the prevention and treatment of hypertrophic scar. It offers a new way for treating the hypertrophic scar clinically. Key words: Hypertrophic scar; PPAR-γ; Collagen

Peroxisome Proliferator-Activated Receptor (PPAR)γ Can Inhibit Chronic Renal Allograft Damage

Chronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor known to have antidiabetogenic and immune effects, and PPARgamma forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARgamma-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 +/- 5.8 (controls) vs. 8.1 +/- 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 +/- 1.8 (controls) vs. 2.6 +/- 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-beta1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARgamma activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARgamma-dependent manner. The combination of PPARgamma- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARgamma activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist.

Abstract 679: Targeting LC3-SQSTM1 (p62) signaling axis to treat apoptosis-resistant and metastatic breast cancers

Abstract Development of therapy resistance to conventional treatments of breast cancer is a major health concern. Of these, triple negative breast cancers are among the most aggressive and therapy resistant. Early breast tumors, which are sensitive to the apoptosis inducing effects of radiation and chemotherapy, become resistant to apoptosis inducing therapies in their advanced stages. Therefore, there is an urgent need to develop alternative approaches to induce non-apoptotic death in apoptosis resistant breast tumors. In this connection, we showed that certain sulfhydryl (-SH) reactive compounds containing α,β-ketones cause misfolding of nascent proteins, endoplasmic reticulum (ER) stress and non-apoptotic cytoplasmic vacuolation death of breast cancer cells. Importantly, we found that 15-deoxy-Δ 12,14-prostaglandin J2 (15d-PGJ2), a cyclooxygenase-2 derived prostaglandin, induced non-apoptotic cell death in apoptosis resistant and rapidly growing MDA-MB-231 breast cancer cells. In contrast, identical doses of 15d-PGJ2 failed to induce similar cytoplasmic vacuolation and cell death in slow growing benign human mammary epithelial cells (HME). Other cyclopentenone prostaglandins such as PGA1 and PGA2 or PGD2, the precursor of 15d-PGJ2, were less potent in inducing cytoplasmic vacuolation death suggesting the uniqueness of 15d-PGJ2 in mediating non-apoptotic cell death in MDA-MB-231 cells. In addition to induction of ER stress marker (Bip), 15d-PGJ2 treatment also induced microtubule-associated protein 1 light chain 3 (LC3) and sequestosome1 (p62) proteins in MDA-MB-231 but not in normal HME cells. Interestingly, our results show that 15d-PGJ2 treatment further increased p62 protein to levels far higher than the concentrations found in untreated breast cancer cells, that are already elevated, compared to HME cells. This suggests that autophagy may be inhibited by 15d-PGJ2. We also found that Manumycin A (Man A), a structurally different molecule with -SH reactive unsaturated α,β-ketone group also induced cytoplasmic vacuolation leading to cell death similarly to 15d-PGJ2 in MDA-MB-231 cells by inducing LC3, p62 and ER stress marker Bip. To study whether Man A might be effective in reducing breast tumor burden, we used a nude xenograft model. Tumors receiving Man A showed remarkable reduction in tumor growth and contained extensive cytoplasmic vacuoles and pyknotic nuclei. Our results showed for the first time that in treatment resistant rapidly dividing breast cancer cells with a high requirement for new protein synthesis, the apoptotic and autophagy fate is avoided, but the cells can be killed by a facile non-autophagic and non-apoptotic mechanism, triggered by specific -SH reactive inducers of protein misfolding and aggregation through a possible LC3-SQSTM1(p62) signaling axis. 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 679.

Abstract 5415: Histone deacetylase inhibitors and 15-deoxy-Δ12,14-prostaglandin J2 synergistically induce apoptosis

Abstract 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.

Abstract 1483: 15-Deoxy-D12,14-prostaglandin J2 upregulates 15-hydroxyprostaglandin dehydrogenase expression through activation of AP-1 signaling in human colon cancer cells

Abstract Abnormal up-regulation of cyclooxygenase-2 (COX-2) expression has been considered as a key oncogenic event in inflammation-associated human colon carcinogenesis. It was reported that 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a physiological antagonist of COX-2 in its catabolism, is ubiquitously lost in human colon cancer, and hence considered as a tumor suppressor. However, the molecular mechanisms underlying regulation of 15-PGDH expression in colon carcinogenesis remain largely unresolved. In the present study, we found that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the terminal products of COX-mediated arachidonic acid metabolism, upregulates the expression of 15-PGDH in the human colon cancer HCT116 cells in a time and concentration-dependent manners. 15d-PGJ2 treatment (10 μM) significantly upregulated the expression of 15-PGDH and its mRNA transcript. 15-PGDH activity was also elevated by 15d-PGJ2 treatment. In a subsequent experiment, we performed cDNA microarray analysis with HCT116 cells exposed to 15d-PGJ2 (10 µM) for 3 h. We observed that C/EBP delta, FOS-like antigen 1, and c-Jun (AP-1) were the most highly induced genes upon treatment with 15d-PGJ2. Based on these findings, we determined whether 15d-PGJ2 could activate the DNA binding of AP-1, a representative transcription factor that preferentially binds to the promoter region of 15-PGDH. 15d-PGJ2 caused increased DNA binding activity of AP-1 in HCT116 cells. Moreover, siRNA knock down of c-Jun, a major subunit of AP-1, significantly reduced 15-PGDH expression as well as AP-1 DNA binding induced by 15d-PGJ2. To elucidate the upstream signaling pathways responsible for AP-1 activation and 15-PGDH expression induced by 15d-PGJ2, we examined the effect of this cyclopentenone prostaglandin on the activation of mitogen-activated protein kinases (MAPKs), such as ERK, p38 and JNK, and also Akt. We observed that a pharmacological inhibition of Akt/PKB with LY294002 abrogated the 15d-PGJ2-induced 15-PGDH expression and AP-1 DNA binding activity, while inhibitors of MAPKs failed to affect these events. Taken together, these finding suggest that 15d-PGJ2 upregulates the expression of 15-PGDH via Akt-mediated AP-1 activation. 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 1483.

Cyclopentenone prostaglandin-induced unfolding and aggregation of the Parkinson disease-associated UCH-L1.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) has been implicated in Parkinson's disease (PD) and is present in neurofibrillary tangles or Lewy bodies. However, the molecular basis for UCH-L1s involvement in proteinacious fibril formation is still elusive, especially in regard to the pathogenicity of the I93M mutation. Here we show that modification of UCH-L1 by cyclopentenone prostaglandins causes unfolding and aggregation. A single thiol group on Cys152 reacts with the alpha,beta-unsaturated carbonyl center in the cyclopentenone ring of prostaglandins, resulting in a covalent adduct. We also show that the PD-associated I93M mutant of UCH-L1 is well-folded, structurally similar to the wild-type protein, and aggregates upon conjugation by cyclopentenone prostaglandins. Our findings suggest a possible mechanistic link between UCH-L1 modification by cyclopentenone prostaglandins and the etiology of neurodegeneration.

15-Deoxy-Δ12,14-prostaglandin J2 stabilizes, but functionally inactivates p53 by binding to the cysteine 277 residue

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative cyclopentenone prostaglandin, has many interesting biological effects. In this study, treatment of human breast cancer cells (MCF-7) with 15d-PGJ(2) led to accumulation of p53 protein. However, the p53 DNA binding and its transcriptional activity were significantly reduced. 15d-PGJ(2) directly modified p53 as verified by reacting recombinant p53 with biotinylated 15d-PGJ(2). 9,10-Dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) lacking the electrophilic alpha,beta-unsaturated functionality failed to inhibit p53 DNA binding as well as to modify p53. Moreover, by conducting an in vitro [(35)S]-labeled p53 translation assay, we identified cysteine 277 as a putative site of p53 modification by 15d-PGJ(2). The DNA-binding ability of a mutant p53 in which cysteine 277 was substituted by alanine was virtually unaffected by 15d-PGJ(2). Likewise, p53 binding activity of biotinylated 15d-PGJ(2) was abolished in mutant cells. In addition, cells expressing wild-type p53 exhibited p53 protein stability to a greater extent than mutant C277A cells. In conclusion, 15d-PGJ(2) can undergo nucleophilic addition to p53, presumably at the cysteine 277 residue, rendering this tumor suppressor less susceptible to proteasomal degradation.

Effect of 15-deoxy-△~(12,14)-prostagliandxin J2 on hypertrophic scar in rabbit ear

Objective To investigate the effect of 15d-PGJ2 on the expression of collagen type,CTGF and a-SMA in the hypertrophic scar in the rabbit ear,and the possibility of hypertrophic scar treated by 15d-PGJ2.Methods 18 New Zealand white rabbits were used to establish a hypertrophic scar model on the rabbit ear.The wounds were established as follows:2 cm × 3 cm wounds with total skin loses on the ventral side,2 wounds for each ear,totally 72 wounds.The wounds were randomly divided into the 15d-PGJ2 treatment group and NS control group.20μl 15d-PGJ2 or NS was injected into the ear scar once a day for 7 days.At 7,14 and 21 days after the injection,12 scars of each group were harvested.The expression of collagen type Ⅰ,CTGF and a-SMA was detected by immunohistochemical method.Results Excessive dermal scars on rabbit ears that were similar to human hypertrophic scar appeared in the two groups.Compared with the NS-treated scars group,the 15d-PGJ2-treated scars appeared to be smaller,softer,flatter and lighter in color.The expression of collagen type Ⅰ,CTGF and a-SMA in the 15d-PGJ2 group was significantly decreased as compared with that in the control group at different time points(P＜0.05).Conclusion 15d-PGJ2,the ligand of PPAR-r,can reduce the expression of collagen type Ⅰ,CTGF and a-SMA of hypertrophic scar in the rabbit ears and plays an important role in the prevention and treatment of hypertrophic scar.It may provide a new approach for the treatment of hypertrophic scar in clinical setting. Key words: Hypertrophic scar; Peroxisome proliferator activated receptor-r; PPAR-r; Collagen type Ⅰ; Connective tissue growth factor; CTGF; a-Smooth muscle actin; a-SMA

Peroxisome Proliferator-Activated Receptor (PPAR)-Gamma Agonists Inhibit Dectin-1 mediated Activation of Human Dendritic Cells (DC).

Abstract 1645Poster Board I-671Human 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. DisclosuresNo relevant conflicts of interest to declare.

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification

The cyclopentenone prostaglandin (cyPG) PGA(1) displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA(1) derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA(1) (PGA(1)-biotinamide, PGA(1)-B), to establish its validity to identify cyPG-protein interactions of potential therapeutic interest. PGA(1) and PGA(1)-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA(1)-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA(1)-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA(1)-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA(1) that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.

Anti-Atherosclerotic Molecules Targeting Oxidative Stress and Inflammation

The accumulation of lipids within arteries remains to be the initial impulse for the pathogenesis of atherosclerosis; however, both inflammation and oxidative stress are considered to play a critical role in this process. Several lipid lowering drugs are used as the first line therapy in atherosclerosis; however, different agents have been found to exhibit beneficial effects which are independent of their lipid lowering activity. Both statins and fibrates have been reported to exert anti-inflammatory and anti-oxidative effects in addition to their anti-atherosclerotic actions. Furthermore, anti-hypertensive, anti-diabetic and anti-platelet drugs, which reduce oxidative stress and inflammation, have been shown to attenuate atherosclerosis. In addition, novel substances such as HDL-related agents, cyclopentenone prostaglandins, lipoprotein-associated phospholipase A(2) inhibitors, 5-lipoxygenase pathway inhibitors, acyl CoA: cholesterol acyltransferase inhibitors, analogues of probucol and lysophosphatidic acid antagonists have been developed for the treatment of atherosclerosis as a consequence of their actions on oxidative stress and inflammation. The present article reviews the involvement of inflammation and oxidative stress in the pathogenesis of atherosclerosis and focuses on the mechanisms of some clinically used as well as potential anti-atherosclerotic substances with anti-inflammatory and anti-oxidative properties.

(Arylthio)cyclopentenones derivatives prevent glutamate-induced HT22 cell death through a PPARγ-dependent pathway

We show that cyclopentenone derivatives, which are synthetic analogs of cyclopentenone prostaglandins, are neuroprotective against glutamate-induced oxidative stress in HT22 cells. This effect was antagonized by a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, T0070907. Pull-down assays revealed that biotinylated (arylthio)cyclopentenone (GIF-0643-biotin) and other biotin-bound cyclopentenones bind to PPARγ. The results also indicate that the GIF-0643-biotin–PPARγ complex is localized to the nucleus. Moreover, retinoid X receptor-α (RXR) co-precipitated with the GIF-0643-biotin–PPARγ complex, indicating that (arylthio)cyclopentenone can activate PPARγ-RXR heterodimers. These findings suggest that (arylthio)cyclopentenone derivatives prevent excitotoxicity by modulating gene expression via activation of the PPARγ-RXR hetrodimer.

Effects of 15‐Deoxy‐Δ12,14‐prostaglandin J2 on the Expression of p53 in MCF‐7 Cells

Cyclopentenone prostaglandins (cyPGs) exert diverse cellular functions, such as anti-inflammatory and cytoprotective effects, via multiple mechanisms. CyPGs, especially those of the A and J series, are characterized by the presence of a chemically reactive alpha,beta-unsaturated carbonyl group. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative cyPG of the J series, has been reported to directly inhibit the activity of redox-sensitive transcription factors, such as activator protein-1 and nuclear factor-kappaB. In the present study, we examined the effects of 15d-PGJ(2) on activation of p53 tumor suppressor in human breast cancer (MCF-7) cells. MCF-7 cells treated with 15d-PGJ(2) exhibited elevated p53 protein expression in time- and concentration-related manners, whereas prostaglandin A(2) (PGA(2)) and the nonprostaglandin derivative 2-cyclopenten-1-one exerted an effect to a lesser extent than did 15d-PGJ(2). In addition, MCF-7 cells exposed to 15d-PGJ(2) significantly accumulated p53 in both cytosolic and nuclear fractions. Despite the elevated levels of p53, its DNA-binding activity was reduced in 15d-PGJ(2)-treated MCF-7 cells. Moreover, isolated MCF-7 nuclear extracts directly treated with 15d-PGJ(2) exhibite diminished DNA-binding ability of p53, while the same concentration of PGA(2) or 2-cyclopenten-1-one was much less inhibitory. Thus, the electrophilic carbon center located in the alpha,beta-unsaturated carbonyl moiety of the cyclopentenone ring might be critical for the control of DNA-binding activity as well as cellular levels of p53 by 15d-PGJ(2).

Neuroprotective effects of (arylthio)cyclopentenone derivatives on manganese-induced apoptosis in PC12 cells

Parkinson's disease is characterized by degeneration of dopaminergic neurones in the substantia nigra. Chronic manganese poisoning shares many features of Parkinson's disease, and also induces extrapyramidal syndromes that resemble those of Parkinson's disease due to dopamine depletion in the central nervous system. This study was undertaken to develop novel neuroprotective drugs via the identification of compounds that inhibit manganese-induced apoptosis. Here, we report that (arylthio)cyclopentenone derivatives, which are synthetic analogs of cyclopentenone prostaglandins, prevent manganese-induced apoptosis in PC12 cells. A highly sensitive assay of caspase-3/7 activity was used for screening newly synthesized prostaglandin analogs. The results showed that some cyclopentenone derivatives (GIF-0642, GIF-0643, GIF-0644, GIF-0745, and GIF-0747) inhibit manganese-induced caspase-3/7 activation in a concentration-dependent manner. Effective compounds all have an arylthio group, indicating that this structure plays an important role in the anti-apoptotic effects of (arylthio)cyclopentenone derivatives. The anti-apoptotic effects of these compounds were confirmed by verifying their ability to inhibit the DNA fragmentation and caspase-9 activation induced by manganese. Furthermore, GIF-0747 prevented manganese-induced cytochrome c release from mitochondria. These results suggest that (arylthio)cyclopentenone derivatives may be good candidates for treating neurodegenerative diseases.