4-Hydroxynonenal and PPARgamma ligands affect proliferation, differentiation, and apoptosis in colon cancer cells

PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.     

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.     

Differential modulation of cell cycle, apoptosis and PPARgamma2 gene expression by PPARgamma agonists ciglitazone and 9-hydroxyoctadecadienoic acid in monocytic cells

We sought to compare the effects of the thiazolidinedione ciglitazone with the endogenous fatty acid PPARgamma agonists 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), in U937 monocytic cells. Ciglitazone and 9-HODE inhibited cell proliferation and all three agonists increased cellular content of C18:0 fatty acids. Ciglitazone and 13-HODE resulted in an increased percentage of cells in S phase and ciglitazone reduced the percentage of cells in G2/M phase of cell cycle, whilst 9-HODE increased the percentage of cells in G0/1 and reduced the fraction in S and G2/M phases. 9-HODE selectively induced apoptosis in U937 cells, and increased PPARgamma2 gene expression. Induction of apoptosis by 9-HODE was not abrogated by the presence of the PPARgamma antagonist GW9662. Synthetic (TZD) and endogenous fatty acid ligands for PPARgamma, ciglitazone and 9- and 13-HODE, possess differential, ligand specific actions in monocytic cells to regulate cell cycle progression, apoptosis and PPARgamma2 gene expression.     

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.     

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.     

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.     

Apoptosis induced by activation of peroxisome-proliferator activated receptor-gamma is associated with Bcl-2 and NF-kappaB in human colon cancer

Peroxisome-proliferator activated receptor-gamma (PPARgamma) has been demonstrated to exert an inhibitory effect on cell growth in most cell types studied, but its role in colon cancer is still uncertain. The molecular mechanism between the activation of PPARgamma and its consequence is unknown. In the present report, we show that the expression of PPARgamma was significantly increased in tumor tissues from human colon cancer compared with non-tumor tissues and that PPARgamma ligands, 15-Deoxy-delta(12,14)prostaglandin J2 or ciglitizone, induced apoptosis in HT-29 cells, a human colon cancer cell line. The occurrence of apoptosis induced by PPARgamma ligands was sequentially accompanied by reduced levels of NF-kappaB and Bcl-2. Over-expression of Bcl-2 significantly protected the cells from apoptosis. This study suggested that a PPARgamma-Bcl-2 feedback loop may function to control the life-death continuum in colonic cells and that a deficiency in generation of PPARgamma ligands may precede the development of human colon cancer.     

Peroxisome proliferator-activated receptor gamma ligands improve the antitumor efficacy of thrombospondin peptide ABT510

An expanding capillary network is critical for several pathologic conditions. In cancer, the decrease of antiangiogenic thrombospondin-1 (TSP1) often enables an angiogenic switch, which can be reversed with exogenous TSP1 or its peptide derivative ABT510. TSP1 acts by inducing endothelial cell apoptosis via signaling cascade initiated at CD36, a TSP1 antiangiogenic receptor. Here, we show that the ligands of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), 15-deoxy-delta(12,14)-prostaglandin J2, troglitazone, and rosiglitazone increased PPARgamma and CD36 expression in endothelial cells and improved the efficacy of TSP1 and ABT510 in a CD36-dependent manner. The ABT510 and PPARgamma ligands cooperatively blocked angiogenic endothelial functions in vitro and neovascularization in vivo. In tumor xenografts, 15-deoxy-delta(12,14)-prostaglandin J2 and troglitazone synergistically improved antiangiogenic and antitumor effects of ABT510. Our data provide one mechanism for the in vivo angioinhibitory effect of PPARgamma ligands and show fine-tuning of the antiangiogenic efficacy via targeted up-regulation of the endothelial receptor.     

Suppression of prostaglandin E2 receptor subtype EP2 by PPARgamma ligands inhibits human lung carcinoma cell growth

Prostaglandin E(2) (PGE(2)), a major cyclooxygenase (COX-2) metabolite, plays important roles in tumor biology and its functions are mediated through one or more of its receptors EP1, EP2, EP3, and EP4. We have shown that the matrix glycoprotein fibronectin stimulates lung carcinoma cell proliferation via induction of COX-2 expression with subsequent PGE(2) protein biosynthesis. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibited this effect and induced cellular apoptosis. Here, we explore the role of the PGE(2) receptor EP2 in this process and whether the inhibition observed with PPARgamma ligands is related to effects on this receptor. We found that human non-small cell lung carcinoma cell lines (H1838 and H2106) express EP2 receptors, and that the inhibition of cell growth by PPARgamma ligands (GW1929, PGJ2, ciglitazone, troglitazone, and rosiglitazone [also known as BRL49653]) was associated with a significant decrease in EP2 mRNA and protein levels. The inhibitory effects of BRL49653 and ciglitazone, but not PGJ2, were reversed by a specific PPARgamma antagonist GW9662, suggesting the involvement of PPARgamma-dependent and -independent mechanisms. PPARgamma ligand treatment was associated with phosphorylation of extracellular regulated kinase (Erk), and inhibition of EP2 receptor expression by PPARgamma ligands was prevented by PD98095, an inhibitor of the MEK-1/Erk pathway. Butaprost, an EP2 agonist, like exogenous PGE(2) (dmPGE(2)), increased lung carcinoma cell growth, however, GW1929 and troglitazone blocked their effects. Our studies reveal a novel role for EP2 in mediating the proliferative effects of PGE(2) on lung carcinoma cells. PPARgamma ligands inhibit human lung carcinoma cell growth by decreasing the expression of EP2 receptors through Erk signaling and PPARgamma-dependent and -independent pathways.     

15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-1beta effects in human chondrocytes by inhibiting NF-kappaB and AP-1 activation pathways.

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin-1beta (IL-1beta) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARgamma ligands to modulate IL-1beta effects on human chondrocytes. RT-PCR and Western blot analysis revealed that PPARgamma expression was decreased by IL-1beta. 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), in contrast to troglitazone, was highly potent to counteract IL-1beta-induced cyclooxygenase-2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel-shift analyses demonstrated that 15d-PGJ2 inhibited NF-kappaB activation, while troglitazone was ineffective. Although 15d-PGJ2 attenuated activator protein-1 binding on the DNA, it potentiated c-jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d-PGJ2 action in human chondrocytes is mainly PPARgamma-independent.     

Effects of peripheral benzodiazepine receptor ligands on proliferation and differentiation of human mesenchymal stem cells

The peripheral benzodiazepine receptor (PBR) has been known to have many functions such as a role in cell proliferation, cell differentiation, steroidogenesis, calcium flow, cellular respiration, cellular immunity, malignancy, and apoptosis. However, the presence of PBR has not been examined in mesenchymal stem cells. In this study, we demonstrated the expression of PBR in human bone marrow stromal cells (hBMSCs) and human adipose stromal cells (hATSCs) by RT-PCR and immunocytochemistry. To determine the roles of PBR in cellular functions of human mesenchymal stem cells (hMSCs), effects of diazepam, PK11195, and Ro5-4864 were examined. Adipose differentiation of hMSCs was decreased by high concentration of PBR ligands (50 microM), whereas it was increased by low concentrations of PBR ligands (<10 microM). PBR ligands showed a biphasic effect on glycerol-3-phosphate dehydrogenase (GPDH) activity. High concentration of PBR ligands (from 25 to 75 microM) inhibited proliferation of hMSCs. However, clonazepam, which does not have an affinity to PBR, did not affect adipose differentiation and proliferation of hMSCs. The PBR ligands did not induce cell death in hMSCs. PK11195 (50 microM) and Ro5-5864 (50 microM) induced cell cycle arrest in the G(2)/M phase. These results indicate that PBR ligands play roles in adipose differentiation and proliferation of hMSCs.     

Evaluation of eicosanoids and NSAIDs as PPARgamma ligands in colorectal carcinoma cells

The activation of peroxisome proliferator activated receptor gamma (PPARgamma) may play a role in the control of colorectal carcinogenesis. The expression of PPARgamma was examined by Western blotting in human colorectal tumors and matched normal adjacent tissues, as well as in various colorectal carcinoma cell lines. In the tissues, the expression of PPARgamma was elevated in tumors relative to the adjacent normal tissues. Each colorectal carcinoma cell line expressed PPARgamma. The ability of various eicosanoids to bind PPARgamma in colorectal carcinoma cells was investigated using luciferase reporter assays. The well-known PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) strongly induced PPARgamma binding activity. Products of lipoxygenases displayed moderate binding activity, while other prostaglandins and fatty acids displayed little or no reporter activation. The activation of PPARgamma by 13(S)-HODE, the major metabolite of 15-lipoxygenase-1 from linoleic acid, was concentration dependent reaching maximum at 10 micro M (35-fold activation). The endogenous production of 13(S)-HODE by expression of 15-LO-1 did not activate PPARgamma. The ability of various nonsteroidal anti-inflammatory drugs (NSAIDs) to induce PPARgamma activation was also evaluated. The conventional NSAIDs that inhibit both cyclooxygenases (COX-1 and COX-2) also induced PPARgamma binding activity. In general, however, neither COX-1- nor COX-2-specific inhibitors induced the activation of PPARgamma. Taken together, the metabolites of 15-lipoxygenase and the conventional NSAIDs were confirmed as exogenous ligands for PPARgamma in colorectal carcinoma cells.     

Peroxisome proliferator-activated receptor gamma ligands regulate myeloperoxidase expression in macrophages by an estrogen-dependent mechanism involving the -463GA promoter polymorphism

A functional myeloperoxidase (MPO) promoter polymorphism, -463GA, has been associated with incidence or severity of inflammatory diseases, including atherosclerosis and Alzheimer's disease, and some cancers. The polymorphism is within an Alu element encoding four hexamer repeats recognized by nuclear receptors (AluRRE). Here we show that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists strongly regulate MPO gene expression through the AluRRE. Opposite effects were observed in granulocyte/macrophage colony-stimulating factor (GMCSF)- versus macrophage colony-stimulating factor (MCSF)-derived macrophages (Mphi): Expression was markedly up-regulated (mean 26-fold) in MCSF-Mphi and down-regulated (34-fold) in GMCSF-Mphi. This was observed with rosiglitazone and three other PPARgamma ligands of the thiazolidinedione class, as well as the natural prostaglandin metabolite 15-deoxy-Delta(12,14) prostaglandin J(2). The selective PPARgamma antagonist, GW9662, blocked both the positive and negative effects on MPO expression. Gel retardation assays showed PPARgamma bound hexamers 3/4, and estrogen receptor-alpha bound hexamers 1/2, with -463A in hexamer 1 enhancing binding. Estrogen blocked PPARgamma effects on MPO expression, especially for the A allele. Charcoal filtration of fetal calf serum eliminated the block of PPARgamma, whereas replenishing the medium with 17beta-estradiol reinstated the block. These findings suggest a model in which estrogen receptor binds the AluRRE, preventing PPARgamma binding to the adjacent site. The positive and negative regulation by PPARgamma ligands, and the block by estrogen, was also observed in transgenic mice expressing the G and A alleles. The mouse MPO gene, which lacks the primate-specific AluRRE, was unresponsive to PPARgamma ligands, suggesting the human MPO transgenes will enhance the utility of mouse models for diseases involving MPO, such as atherosclerosis and Alzheimer's.