Does the inhibition of c-myc expression mediate the anti-tumor activity of PPAR's ligands in prostate cancer cell lines?

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands seem to induce anticancer effects on prostate cancer cells, but the mechanism is not clear. The effect of PPARgamma ligands omega-6 fatty acids and ciglitazone (2-15 microM)--on proliferation, and apoptosis of LNCaP, PC-3, DU145, CA-K and BPH-K cells was studied. PPARgamma ligands led to: (1) reduction of proliferation (20-50%) of all the studied cell lines, (2) stimulation of differentiation of prostate cancer cells through an increased expression (1.5-3-fold: LNCaP, DU145, BPH-K) or reexpression (PC-3, CA-K) of E-cadherin with parallel inhibition of N-cadherin expression (PC-3, CA-K) and (3) down-regulation (1-2-fold) of beta-catenin and c-myc expression. The selective PPARgamma antagonist GW9662 abolished the effect of those ligands on prostate cancer cells. These results suggest that inhibition of beta-catenin and in effect c-myc expression through activation of PPARgamma may help prostate cancer cells to restore several characteristics of normal prostate cells phenotype.     

Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.     

Peroxisome proliferator-activated receptor gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2, and ciglitazone, induce growth inhibition and cell cycle arrest in hepatic oval cells

There is growing evidence to show that hepatic oval cells contribute to liver regeneration, dysplastic nodule formation, and hepato-carcinogenesis. Peroxisome proliferator-activated receptors (PPARs) and their ligands play an important role in cell growth, inflammatory responses, and liver pathogenesis including fibrosis and cancer. However, little is known about the role of PPARgamma/its ligands in the growth and differentiation of hepatic oval cells. In this study, we found that OC15-5, a rat hepatic oval cell line, expressed PPARgamma at mRNA and protein levels, and a natural ligand for PPARgamma, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), and a synthetic ligand, ciglitazone, inhibited growth of OC15-5 cells by arresting at G1-S in a dose-dependent manner. Apoptosis was also induced in OC15-5 cells by 15d-PGJ2 treatment. In OC15-5 cells treated with 15d-PGJ2, the expression of CDK inhibitor, p27(Kip1), was up-regulated, while that of p21(WAF1/Cip1), p18(INK4C) CDK2, CDK4, and cyclin E was unchanged. In addition, delayed up-regulation of AFP expression was observed in OC15-5 cells after 15d-PGJ2 or ciglitazone treatment. This is the first report to show that the PPARgamma ligand was involved in the growth, cell cycle, and differentiation of hepatic oval cells, raising the possibility that the PPARgamma ligands may regulate liver regeneration and hepato-carcinogenesis.     

Peroxisome proliferator-activated receptor gamma ligands inhibit retinoblastoma phosphorylation and G1--> S transition in vascular smooth muscle cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that is activated by binding certain fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZD). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell proliferation and migration both in vitro and in vivo. The precise mechanism of its antiproliferative activity, however, has not been elucidated. We report here that PPARgamma ligands inhibit rat aortic vascular smooth muscle cell proliferation by blocking the events critical for G(1) --> S progression. Flow cytometry demonstrated that both TRO and another TZD, rosiglitazone, prevented G(1) --> S progression induced by platelet-derived growth factor and insulin. Movement of cells from G(1) --> S was also inhibited by the non-TZD, natural PPARgamma ligand 15-deoxy-(12,14)Delta prostaglandin J(2) (15d-PGJ(2)), and the mitogen-activated protein kinase pathway inhibitor PD98059. Inhibition of G(1) --> S exit by these compounds was accompanied by a substantial blockade of retinoblastoma protein phosphorylation. TRO and rosiglitazone attenuated both the mitogen-induced degradation of p27(kip1) and the mitogenic induction of p21(cip1). 15d-PGJ(2) and PD98059 inhibited both the degradation of p27(kip1) and the induction of cyclin D1 in response to mitogens. These effects resulted in the inhibition of mitogenic stimulation of cyclin-dependent kinases activated by cyclins D1 and E. These data demonstrate that PPARgamma ligands are antiproliferative drugs that act by modulating cyclin-dependent kinase inhibitors; they may provide a new therapeutic approach for proliferative vascular diseases.     

Design, synthesis, and biological activity of novel PPARgamma ligands based on rosiglitazone and 15d-PGJ2

To develop novel PPARgamma ligands, we synthesized thirteen 3-{4-(2-aminoethoxy)phenyl}propanoic acid derivatives, which are designed based on the structures of rosiglitazone and 15d-PGJ2. Among these compounds, compound 9 was found to be as potent as rosiglitazone in a binding assay and a preadipocyte differentiation test. Molecular modeling suggested that the nonyl group of 9 interacted with hydrophobic amino acid residues constructing the hydrophobic region of PPARgamma protein where the alkyl chain of 15d-PGJ2 is expected to be located.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.     

15-Deoxy-delta(12,14)-prostaglandin J(2) inhibits IL-1beta-induced IKK enzymatic activity and IkappaBalpha degradation in rat chondrocytes through a PPARgamma-independent pathway

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1beta (IL-1beta). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). We previously demonstrated that 15d-PGJ(2) was more potent than troglitazone to counteract IL-1beta effects on chondrocytes. Here, we studied the action of 15d-PGJ(2) on intracellular targets in nuclear factor-kappaB (NF-kappaB) signalling pathway in IL-1beta treated rat chondrocytes. We found that 15d-PGJ(2) decreased inhibitor kappaBalpha (IkappaBalpha) degradation but not its phosphorylation by specifically inhibiting IkappaB kinase beta (IKKbeta), but not IKKalpha, enzymatic activity. We further evaluated the involvement of PPARgamma in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARgamma protein, 15d-PGJ(2) pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE(2) production, p65 translocation and NF-kappaB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ(2) exhibited the same effect in chondrocytes overexpressing mutated PPARgamma protein. These results suggest that 15d-PGJ(2) exerts its anti-inflammatory effect in rat chondrocytes by a PPARgamma-independent mechanism, which can be conferred to a partial inhibition of IkappaBalpha degradation.     

Environmental and endogenous peroxisome proliferator-activated receptor gamma agonists induce bone marrow B cell growth arrest and apoptosis: interactions between mono(2-ethylhexyl)phthalate, 9-cis-retinoic acid, and 15-deoxy-Delta12,14-prostaglandin J2

The common commercial use of phthalate esters has resulted in significant human exposure to these bioactive compounds. The facts that phthalate ester metabolites, like endogenous PGs, are peroxisome proliferator-activated receptor (PPAR) agonists, and that PPARgamma agonists induce lymphocyte apoptosis suggest that phthalate esters are immunosuppressants that could act together with PGs to modulate early B cell development. In this study we examined the effects of a metabolite of one environmental phthalate, mono(2-ethylhexyl)phthalate (MEHP), and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), on developing B cells. MEHP inhibited [(3)H]thymidine incorporation by primary murine bone marrow B cells and a nontransformed murine pro/pre-B cell line (BU-11). Cotreatment with a retinoid X receptor alpha ligand, 9-cis-retinoic acid, decreased [(3)H]thymidine incorporation synergistically, thereby implicating activation of a PPARgamma-retinoid X receptor alpha complex. These results were similar to those obtained with the natural PPARgamma ligand 15d-PGJ(2). At moderate MEHP concentrations (25 or 100 microM for primary pro-B cells and a pro/pre-B cell line, respectively), inhibition of [(3)H]thymidine incorporation resulted primarily from apoptosis induction, whereas at lower concentrations, the inhibition probably reflected growth arrest without apoptosis. Cotreatment of bone marrow B cells with 15d-PGJ(2) and MEHP significantly enhanced the inhibition of [(3)H]thymidine incorporation seen with MEHP alone, potentially mimicking exposure in the bone marrow microenvironment where PG concentrations are high. Finally, MEHP- and 15d-PGJ(2)-induced death does not result from a decrease in NF-kappaB activation. These data demonstrate that environmental phthalates can cooperate with an endogenous ligand, 15d-PGJ(2), to inhibit proliferation of and induce apoptosis in developing bone marrow B cells, potentially via PPARgamma activation.     

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.     

The influence of 4-hydroxy-2-nonenal on proliferation, differentiation and apoptosis of human osteosarcoma cells

The product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE) is known to cause cell death at high concentrations, while at lower concentrations it can influence cell proliferation and differentiation. In our experiments we used human osteosarcoma cells (HOS), to test the influence of HNE on cell proliferation, differentiation and induction of apoptosis. Apoptosis induction was estimated by TiterTACS TUNEL test. The cells were in parallel counted and the DAPI staining method was used to distinguish between apoptotic and necrotic cells as well as to define the proportion of cells in mitosis. To test the influence of HNE on HOS cell differentiation, cells were treated every second day with HNE. After 10 days, the cells were stained for alkaline phosphatase, a marker for osteoblast differentiation. Cell growth inhibition was caused by supraphysiological concentrations of 10 or 100 microM HNE, while apoptosis was induced with supraphysiological as well as by the physiological amount of the aldehyde (1 microM). Necrosis appeared when cells were treated with 10 or 100 microM, but not with 1 microM HNE. The proportion of cells in mitosis gradually declined with increased HNE concentration. Multiple exposures of HOS cells to 10 microM HNE prevented HOS cell differentiation. These results indicated that HNE inhibits proliferation and differentiation of HOS cells in the same concentration dependent manner as it causes apoptosis. We thus assume that HNE might be one of the important signaling molecules regulating the growth of the human osteosarcoma cells.     

PPARgamma ligands suppress proliferation of human urothelial basal cells in vitro

Expression of peroxisome proliferator-activated receptor (PPAR) gamma in the human urinary tract through embryonic development suggests its possible roles in the development, proliferation, and differentiation of uroepithelium. Little is known, however, about physiological roles of PPARgamma in the urinary tract. We investigated effects of PPARgamma ligands on the proliferation of normal human urothelial cells and stromal cells cultivated from surgical specimens. Active proliferation in vitro as well as high molecular weight cytokeratin expression indicated that cultured urothelial cells possess basal cell phenotype. PPARgamma protein, expressed predominantly in the epithelial layer of the normal human urinary tract in vivo, was abundantly expressed in urothelial cells but barely detectable in stromal cells in vitro. Natural ligand for PPARgamma, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), as well as synthetic ones, troglitazone and pioglitazone, suppressed proliferation of the urothelial cells dose-dependently. These effects were PPARgamma specific because clofibrate or PGF(2alpha) did not affect proliferation of urothelial cells. Neither 9-cis retinoic acid or all-trans retinoic acid (ATRA) at 1 microM showed any synergism on the antiproliferative effects of PPARgamma ligands. Urothelial cells treated with PPARgamma ligands showed drastic morphologic changes and cell cycle arrest at G0/G1 phase accompanied with increased mRNA level of a cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Since 15d-PGJ(2) is present in vivo during the resolution phase of inflammation, these results indicated that PPARgamma might be involved in the terminal phase of urothelial re-epithelialization processes.