Prostaglandin D2 and its metabolites induce caspase-dependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism

Many inflammatory mediators retard granulocyte apoptosis. Most natural PGs studied herein (e.g., PGE(2), PGA(2), PGA(1), PGF(2 alpha)) either delayed apoptosis or had no effect, whereas PGD(2) and its metabolite PGJ(2) selectively induced eosinophil, but not neutrophil apoptosis. This novel proapoptotic effect does not appear to be mediated via classical PG receptor ligation or by elevation of intracellular cAMP or Ca(2+). Intriguingly, the sequential metabolites Delta(12)PGJ(2) and 15-deoxy-Delta(12,) Delta(14)-PGJ(2) (15dPGJ(2)) induced caspase-dependent apoptosis in both granulocytes, an effect that did not involve de novo protein synthesis. Despite the fact that Delta(12)PGJ(2) and 15dPGJ(2) are peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators, apoptosis was not mimicked by synthetic PPAR-gamma and PPAR-alpha ligands or blocked by an irreversible PPAR-gamma antagonist. Furthermore, Delta(12)PGJ(2) and 15dPGJ(2) inhibited LPS-induced I kappa B alpha degradation and subsequent inhibition of neutrophil apoptosis, suggesting that apoptosis is mediated via PPAR-gamma-independent inhibition of NF-kappa B activation. In addition, we show that TNF-alpha-mediated loss of cytoplasmic I kappa B alpha in eosinophils is inhibited by 15dPGJ(2) in a concentration-dependent manner. The selective induction of eosinophil apoptosis by PGD(2) and PGJ(2) may help define novel therapeutic pathways in diseases in which it would be desirable to specifically remove eosinophils but retain neutrophils for antibacterial host defense. The powerful proapoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2) in both granulocyte types suggest that these natural products control the longevity of key inflammatory cells and may be relevant to understanding the control and resolution of inflammation.     

Effects of PPAR-gamma ligands on vascular smooth muscle marker expression in hypertensive and normal arteries

Having previously demonstrated that glucose transporter-4 (GLUT4) expression was reduced in aortas and carotid arteries of deoxycorticosterone acetate (DOCA) salt-hypertensive rats, we hypothesized that troglitazone (TG), through activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), would stabilize GLUT4 expression and possibly preserve the differentiated phenotype in vascular smooth muscle cells. In DOCA salt-hypertensive rats treated with TG (100 mg/day), there was a significant (P < 0.001) decrease in systolic blood pressure (BP; 149.9 +/- 4.4 mmHg) compared with the untreated DOCA salt-hypertensive rats (202.2 +/- 10.34 mmHg). Separate trials with rosiglitazone (RS; 3 mg/day) demonstrated a significant (P < 0.001) decrease in BP (DOCA salt, 164.2 +/- 9.8 vs. DOCA-RS, 124.9 +/- 3.7 mmHg) comparable to that with TG. Expression of GLUT4, h-caldesmon, and smooth muscle myosin heavy chain SM2 was significantly decreased in aortas of DOCA salt-hypertensive rats and was reversed by TG to levels similar to those in aortas of sham-treated rats. TG (50 microM) induced GLUT4 and h-caldesmon expression in 24-h culture of explanted carotid arteries of DOCA salt-hypertensive rats, and the endogenous PPAR-gamma ligand 15-deoxy-Delta(12-14)-prostaglandin J(2) (PGJ(2); 20 microM) and TG (50 microM) similarly increased GLUT4, h-caldesmon, and SM2 protein expression in explanted aortas. The expression of activated, phosphorylated Akt was increased by PGJ(2) and TG with no significant effect on total Akt levels. Inhibition of phosphorylated Akt expression using the phosphatidylinositol 3-kinase inhibitor LY-294002 (16 microM) abrogated the increased expression of h-caldesmon and SM2. These data demonstrate that PPAR-gamma agonists maintain or induce expression of markers of the contractile phenotype independently of their effects on hypertension, and that this effect may be mediated through activation of phosphatidylinositol 3-kinase/Akt.     

15-deoxy-delta12,14-prostaglandin J2-induced apoptosis in amnion-like WISH cells.

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-delta12,14PGJ2 (15d-PGJ2), delta12PGJ2, ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ2 (10 microM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50) of approximately 7 microM 15d-PGJ2. Delta12-PGJ2 also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ2 and delta12PGJ2, which may be independent of PPAR-gamma activation, requires further elucidation.     

Prostaglandin D(2) and J(2) induce apoptosis in human leukemia cells via activation of the caspase 3 cascade and production of reactive oxygen species

The presence of prostaglandins (PGs) has been demonstrated in the processes of carcinogenesis and inflammation. In the present study, we found that 12-o-tetradecanoylphorbol 13-acetate (TPA) induced cyclooxygenase 2 (COX-2), but not COX-1, protein expression in HL-60 cells, and the addition of arachidonic acid (AA) in the presence or absence of TPA significantly reduced the viability of HL-60 cells, an effect that was blocked by adding the COX inhibitors, NS398 and aspirin. The AA metabolites, PGD(2) and PGJ(2), but not PGE(2) or PGF(2alpha), reduced the viability of the human HL60 and Jurkat leukemia cells according to the MTT assay and LDH release assay. Apoptotic characteristics including DNA fragmentation, apoptotic bodies, and hypodiploid cells were observed in PGD(2)- and PGJ(2)-treated leukemia cells. A dose- and time-dependent induction of caspase 3 protein procession, and PARP and D4-GDI protein cleavage with activation of caspase 3, but not caspase 1, enzyme activity was detected in HL-60 cells treated with PGD(2) or PGJ(2). Additionally, DNA ladders induced by PGD(2) and PGJ(2) were significantly inhibited by the caspase 3 peptidyl inhibitor, Ac-DEVD-FMK, but not by the caspase 1 peptidyl inhibitor, Ac-YVAD-FMK, in accordance with the blocking of caspase 3, PARP, and D4-GDI protein procession. An increase in intracellular peroxide levels by PGD(2) and PGJ(2) was identified by the DCHF-DA assay, and anti-oxidant N-acetyl cysteine (NAC), mannitol (MAN), and tiron significantly inhibited cell death induced by PGD(2) and PGJ(2) by reducing reactive oxygen species (ROS) production. The PGJ(2) metabolites, 15-deoxy-Delta(12,14)-PGJ(2) and Delta(12)-PGJ(2), exhibited effective apoptosis-inducing activity in HL-60 cells through ROS production via activation of the caspase 3 cascade. The proliferator-activated receptor-gamma (PPAR-gamma) agonists, rosiglitazone (RO), troglitazone (TR), and ciglitazone (CI), induced apoptosis in cells which was blocked by the addition of the PPAR-gamma antagonists, GW9662 and BADGE, via blocking of caspase 3 and PARP cleavage. However, neither GW9662 nor BADGE showed any protective effect on PGD(2)- and PGJ(2)-induced apoptosis. A differential apoptotic effect of PGs through ROS production, followed by activation of the caspase 3 cascade, was demonstrated.     

15-Deoxy-delta(12,14)-prostaglandin J(2) down-regulates CXCR4 on carcinoma cells through PPARgamma- and NFkappaB-mediated pathways

The chemokine receptor CXCR4 plays a key role in the metastasis of colorectal cancer and its growth at metastatic sites. Here, we have investigated the mechanisms by which CXCR4 on cancer cells might be regulated by eicosanoids present within the colorectal tumor microenvironment. We show that prostaglandins PGE(2), PGA(2), PGD(2), PGJ(2) and 15dPGJ(2) each down-regulates CXCR4 receptor expression on human colorectal carcinoma cells to differing degrees. The most potent of these were PGD(2) and its metabolites PGJ(2) and 15dPGJ(2). Down-regulation was most rapid with the end-product 15dPGJ(2) and was accompanied by a marked reduction in CXCR4 mRNA. 15dPGJ(2) is known to be a ligand for the nuclear receptor PPARgamma. Down-regulation of CXCR4 was also observed with the PPARgamma agonist rosiglitazone, while 15dPGJ(2)-induced CXCR4 down-regulation was substantially diminished by the PPARgamma antagonists GW9662 and T0070907. These data support the involvement of PPARgamma. However, the 15dPGJ(2) analogue CAY10410, which can act on PPARgamma but which lacks the intrinsic cyclopentenone structure found in 15dPGJ(2), down-regulated CXCR4 substantially less potently than 15dPGJ(2). The cyclopentenone grouping is known to inhibit the activity of NFkappaB. Consistent with an additional role for NFkappaB, we found that the cyclopentenone prostaglandin PGA(2) and cyclopentenone itself could also down-regulate CXCR4. Immunolocalization studies showed that the cellular context was sufficient to trigger a focal nuclear pattern of NFkappaB p50 and that 15dPGJ(2) interfered with this p50 nuclear localization. These data suggest that 15dPGJ(2) can down-regulate CXCR4 on cancer cells through both PPARgamma and NFkappaB. 15dPGJ(2), present within the tumor microenvironment, may act to down-regulate CXCR4 and impact upon the overall process of tumor expansion.     

Mechanism of constrictive vascular remodeling by homocysteine: role of PPAR

To test the hypothesis that homocysteine induces constrictive vascular remodeling by inactivating peroxisome proliferator-activated receptor (PPAR), aortic endothelial cells (ECs) and smooth muscle cells (SMCs) were isolated. Collagen gels were prepared, and ECs or SMCs (10(5)) or SMCs + ECs (10(4)) were incorporated into the gels. To characterize PPAR, agonists of PPAR-alpha [ciprofibrate (CF)] and PPAR-gamma [15-deoxy-12,14-prostaglandin J(2) (PGJ(2))] were used. To determine the role of disintegrin metalloproteinase (DMP), cardiac inhibitor of metalloproteinase (CIMP) was used in collagen gels. Gel diameter at 0 h was 14.1 +/- 0.2 mm and was unchanged up to 24 h as measured by a digital micrometer. SMCs reduce gel diameter to 10.5 +/- 0.4 mm at 24 h. Addition of homocysteine to SMCs reduces further the gel diameter to 8.0 +/- 0.2 mm, suggesting that SMCs induce contraction and that the contraction is further enhanced by homocysteine. Addition of ECs and SMCs reduces gel diameter to 12.0 +/- 0.3 mm, suggesting that ECs play a role in collagen contraction. Only PGJ(2), not CF, inhibits SMC contraction. However, both PGJ(2) and CF inhibit contraction of ECs and SMCs + ECs. Addition of anti-DMP blocks SMC- as well as homocysteine-mediated contraction. However, CIMP inhibits only homocysteine-mediated contraction. The results suggest that homocysteine may enhance vascular constrictive remodeling by inactivating PPAR-alpha and -gamma in ECs and PPAR-gamma in SMCs.     

15-deoxy-delta12,14 prostaglandin J2 synergizes with phorbol ester to induce proliferation in Swiss 3T3 cells independently of peroxisome proliferator-activated receptor gamma and PGD2 receptors

15-deoxy-Delta(12,14) prostaglandin J(2) (15dPGJ(2)), a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, induced synergistic stimulation of DNA synthesis in the presence of phorbol dibutyrate (PDB) in Swiss 3T3 cells. This effect was dose-dependent and the maximum response was obtained at 2 microM 15dPGJ(2), although higher concentrations of 15dPGJ(2) were cytotoxic. Furthermore, 15dPGJ(2) synergizes with PDB to induce cell-cycle progression and cyclin D(1) expression. Rosiglitazone and ciglitazone, two other agonists of PPARgamma, did not synergize with PDB to induce DNA synthesis, suggesting that activation of PPARgamma is not involved in 15dPGJ(2)-induced DNA synthesis. 15dPGJ(2) neither increased the levels of cAMP, nor changed the phosphorylation state of CREB, nor induced calcium mobilization, indicating that 15dPGJ(2) effects are independent of prostaglandin D(2) receptor (DP1 and DP2). Moreover, 15dPGJ(2) did not induce activation of PKB/AKT or activation of extracellular signal-regulated kinase (ERK). These results establish a proliferative role for 15dPGJ(2) in Swiss 3T3 cells independent of the activation of PPARgamma or the PGD(2) receptors.     

15-Deoxy-Delta(12,14)-prostaglandin J2 inhibits the expression of proinflammatory genes in human blood monocytes via a PPAR-gamma-independent mechanism

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in inhibition of the expression of proinflammatory cytokines and inducible enzymes such as cyclooxygenase-2 (COX-2). Using real-time RT-PCR the present study investigates the impact of two PPAR-gamma agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and ciglitazone, on the expression of several proinflammatory genes in lipopolysaccharide (LPS)-stimulated human blood monocytes. Stimulation of cells with LPS resulted in a profound induction of the expression of COX-2, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Treatment of cells with 15d-PGJ(2) (10 microM) was associated with a nearly complete inhibition of the expression of all genes that remained unaltered in the presence of the PPAR-gamma antagonist bisphenol A diglycidyl ether (BADGE; 100 microM). By contrast, treatment of cells with another potent PPAR-gamma agonist, ciglitazone (50 microM), and the PPAR-alpha agonist WY-14,643 (100 microM) did not suppress LPS-induced expression of the investigated genes. Stimulation of monocytes with LPS resulted in an 88% inhibition of PPAR-gamma mRNA expression that was fully restored by 15d-PGJ(2) but only to a partial extent by ciglitazone and WY-14,643. Again, BADGE did not alter the effect of 15d-PGJ(2). Collectively, our results show that alterations of gene expression by 15d-PGJ(2) in LPS-stimulated human blood monocytes are mediated by PPAR-gamma-independent mechanisms. Moreover, it is concluded that both inhibition of proinflammatory gene expression and restoration of LPS-induced decrease of PPAR-gamma expression may contribute to the biological action of 15d-PGJ(2).     

Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.     

Novel side chain modified Delta8(14)-15-ketosterols

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).     

Cytotoxicities, cell cycle and caspase evaluations of 1,6-diaryl-3(Z)-hexen-1,5-diynes, 2-(6-aryl-3(Z)-hexen-1,5-diynyl)anilines and their derivatives

Compounds 3, 6-7, 9-10, 15-17, and 20-21 showed growth inhibition effects on a full panel of 60 human cancer cell lines, and most of the average IC50 values of the indicated analogues were from < 0.01 to 96.6 microM, in which analogues 16 and 17 revealed the highest cytotoxic activity with the cancer cell lines at 10(-7) M concentration range. During the cell cycle analysis, a moderate to high apoptotic progress induction was shown by 3, 9, 16-17, and 20 compared with the control, which 2-(6-(2-thienyl)-3(Z)-hexen-1,5-diynyl)aniline 16 showed the highest apoptotic effect. Structures 16-17 displayed a significant G2/M phase arrest in the cell growth cycle compared with other derivatives, which the proportions of the G2/M phase cells were accumulated to 71.5% and 82.6%, respectively. Moreover, the colorimetric assay of 16-17 also provided advanced evidence to the relationship between the compounds and the caspase-3 enzyme, which was one of the major promoters of apoptotic effect.     

RNA interference-mediated silencing of laminin receptor 1 (LR1) suppresses migration and invasion and down-regulates matrix metalloproteinase (MMP)-2 and MMP-9 in trophoblast cells: implication in the pathogenesis of preeclampsia

Shallow trophoblast invasion is a common pathological feature of preeclampsia. The 67 kDa laminin receptor 1 (LR1) is a laminin-binding protein that has been reported to be down-regulated in preeclamptic placentas. The aim of the present study was to determine the functional role of LR1 in the migration and invasion of the trophoblast cell line, JEG3 cells. RNA interference mediated by plasmid expressing LR1 short hairpin RNA (shRNA) was utilized to knockdown LR1 expression in JEG3 cells. We found that the mRNA and protein expression levels of LR1 were significantly reduced in LR1-specific shRNA transfected cells compared with the untransfected and control shRNA transfected cells. The wound healing and Transwell invasion assays demonstrated that LR1 knockdown remarkably suppressed the migration and invasion potential of JEG3 cells. The gelatin zymography assay showed that LR1 knockdown greatly reduced matrix metalloproteinase (MMP)-2 and MMP-9 activities in the culture supernatants. Western blot analysis showed that LR1 shRNA significantly decreased expression levels of MMP-2, MMP-9 and phospho-extracellular signal-regulated kinase, but increased expression levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in comparison to the control vector-transfected cells. In conclusion, our data support an important role for LR1 in regulating trophoblast invasion and migration, and suggest a possible pathological mechanism of preeclampsia.     

PGJ2 and delta 12PGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ2 and delta 12PGJ2 (1 microM to 30 microM) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [3H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC50 values for PGJ2 and delta 12PGJ2 were approximately 8 microM and 6 microM, respectively. [3H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ2 and delta 12PGJ2, but not PGE1, reduced isoproterenol (10 microM)-induced accumulation of cyclic AMP, suggesting that PGJ2 and delta 12PGJ2 may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [3H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ2 and delta 12PGJ2 inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ2 and delta 12PGJ2 failed to inhibit GTP gamma S (10 microM)- nor Ca2+ (1 mM)-induced accumulation of inositol phosphates. The site of PGJ2 or delta 12PGJ2 in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ2 and delta 12PGJ2 inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.