Peroxisome proliferator-activated receptor-γ ligands as cell-cycle modulators

CANCER TREATMENT REVIEWS (2004) 30, 545–554

www.elsevierhealth.com/journals/ctrv

LABORATORY–CLINIC INTERFACE

Peroxisome proliferator-activated receptor-c ligands as cell-cycle modulators Stamos Theocharisa,b,*, Alexandra Margelic, Philippe Vielhb, Gregory Kouraklisd a

Department of Forensic Medicine and Toxicology, Medical School, University of Athens, 75, Mikras Asias Street, GR 11527 Athens, Greece b Unit of Cytology and Cytometry, Department of Tumor Biology, Medical Section, Institut Curie, 26, rue d’Ulm, 75231 Paris Cedex 05, France c Department of Clinical Biochemistry, “Aghia Sophia” Children’s Hospital, Thivon and Papadiamandopoulou Street, GR 11527 Athens, Greece d Department of Surgery, Medical School, “Laikon” Hospital, University of Athens, 17, Aghiou Thoma Street, GR 11527 Athens, Greece

KEYWORDS

Summary The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, initially described as molecular targets for compounds which induce peroxisomal proliferation. PPAR-c, the best characterized of the PPARs, is a ligand-activated transcription factor and a key regulator of adipogenic differentiation and glucose homeostasis. PPAR-c ligands have recently been demonstrated to affect proliferation, differentiation and apoptosis of different cell types. Recent in vitro and in vivo studies suggest the importance of specific PPAR-c ligands as cellcycle modulators, establishing their antineoplastic properties. In this review, the latest knowledge on the role of PPAR-c ligands as cell-cycle modulators is presented, discussing also their role in cell proliferation, apoptosis and cancer. c 2004 Elsevier Ltd. All rights reserved.

PPAR-c ligands; Cell cycle; Apoptosis; Cancer; Treatment




Introduction The discovery of a subgroup of ligand-attached nuclear receptors, the peroxisome proliferator-activated receptors (PPARs), acting as modulators of peroxisome proliferation triggered much work at both molecular and biochemical levels.1;2 Peroxisome proliferation occurs only in rodents as a consequence of the activation of PPARs by specific

* Corresponding author. Present address: 21 Thessalias Street, Zografou, Athens 15772, Greece. Tel./fax: +30-210-778-0114. E-mail address: [email protected] (S. Theocharis).




molecules, called peroxisome proliferators (PPs).3 PPARs are also present in humans, presenting different functions, able to be activated by ligands of physiological or pharmacological origin.3 PPARs have been implicated in cell proliferation, differentiation and apoptosis.4 The PPARs were cloned and firstly characterized as orphan members of the nuclear receptor gene family that includes the receptors for the steroid, retinoid and thyroid hormones.5;6 Thyroid hormones and PPARs have overlapping metabolic effects regulating a similar subset of genes involved in maintaining lipid homeostasis. To date, three subtypes of PPAR exist, being the products of distinct genes, commonly designated as PPAR-a,

0305-7372/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctrv.2004.04.004

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PPAR-c and PPAR-b (also called -d or NUC-1), localized on chromosomes 22, 3 and 6, respectively, with distinct tissue distribution and biological activities.1;7–11 At first, it was shown that PPARs were activated by substances that could induce peroxisomal proliferation1;7 and by natural fatty acids.11;12 The potency of various chemicals to activate PPARs is subtype specific and PPAR-a, -b, and -c pattern of expression varies widely in a tissue-specific manner. PPAR-a is expressed in liver, intestine, pancreas, kidney, muscle, heart, skeletal muscle, adrenals and cells from the vascular wall and it was initially discovered in rodents as the mediator of the response to PPs.13 PPAR-c is mainly expressed in adipose tissues where it plays a role in lipid metabolism, being also expressed in intestine, mammary gland, endothelium, liver, skeletal muscle, prostate, colon and in cell types throughout the body, including monocytes, macrophages/foam cells and type2 alveolar pneumocytes.3;13;14 PPAR-b is expressed in a wide range of tissues, as human embryonic kidney, small intestine, heart, adipose tissue, skeletal muscle and developing brain, with a less-defined function.3 It has been implicated in keratinocyte differentiation, wound healing and more recently, in mediating very low-density lipoprotein (VLDL) signaling of the macrophage.15 In common with other members of the nuclear receptor gene family, the PPARs function as ligand-activated transcription factors (Fig. 1), forming heterodimer with the retinoid X receptor (RXR).14 Upon ligand binding, the complex of PPAR and RXR binds to specific recognition sites on DNA, the peroxisome proliferator response elements (PPREs) and regulates transcription of specific genes (Fig. 1).5;8;16–19 A PPRE, consisting of an almost per-

fect direct repeat of the sequence TGACCT (Fig. 1) spaced by a single base pair, has been identified in the upstream regulatory sequences of genes related to metabolic pathways.20 The PPAR-c and RXR complex, along with other co-activators for transcription, activate transcription of target genes.21 The activated PPAR/RXR heterodimer could interact with heat-shock protein-72 (HSP-72)22 , liver X receptor (LXR)23 , PPAR-binding protein (PBP)24 , steroid receptor coactivator-1 (SRC-1)25 , integrator protein p30026 , thyroid hormone receptor-associated protein (TRAP)/vitamin D3 receptor-interacting protein (DRIP) complex27 , cAMP response element-binding protein (CBP) and growth regulatory genes such as c-jun28 , modulating gene transcription.14 PPAR-c is the most extensively studied of the three PPAR subtypes to date, showing a remarkable conservation across all the different species from which it has been cloned.6 A number of investigators have shown that PPAR-c expression is not restricted to adipose tissue, being also expressed in several other tissues. Work reported over the past years provided insights into the mechanisms whereby ligand activation of PPAR-c regulates not only systemic glucose and lipid homeostasis, but also has implications in the biology of monocytes, cell-cycle regulation, cancer development and treatment. Differential promoter usage and alternate splicing of the gene generate three mRNA isoforms: PPAR-c 1 and PPAR-c 3 mRNA that both encode the same protein product expressed in most tissues, whereas the PPAR-c 2 mRNA encodes the PPAR-c 2 protein, that contains an additional 28 amino acids and is specific to adipocytes.14;29

RXR

Receptors

PPAR

Ligands

Transcription

TGACCTn TGACCTn PPRE

Figure 1 Basic mechanism of PPARs as ligand-activated transcription factors. Upon ligand binding, PPARs form heterodimeric complex with RXR, that binds to the PPRE and drives the transcription of target genes.

PPAR-c and cell-cycle

547

PPAR-c ligands

different ligands depends on the experimental setting. It has been shown that activation of PPARc by specific ligands, e.g., troglitazone (TGZ) and rosiglitazone depends on the experimental setting. TGZ can behave as a partial agonist for PPAR-c in certain cellular settings, compared with the more potent ligand rosiglitazone. In addition, TGZ antagonized rosiglitazone-induced PPAR-c transcriptional activity. The different transcriptional activities of TGZ and rosiglitazone could be attributed to differential cofactor recruitment caused by these two ligands, to the conformational change in the receptor that increases its affinity for transcriptional coactivator proteins and to the

The unsaturated fatty acids bind all three PPARs, with PPAR-a exhibiting the highest affinity, while saturated fatty acids are poor PPAR ligands in general.30 The ligands of PPAR-c include several prostanoids as 15-deoxy-D12;14 -prostaglandin J2 (15d-PGJ2) and 15-hydroxy-eicosatetrenoic acid (HETE), metabolites of arachidonic acid.3 The pharmacological ligands for PPAR-c are the thiazolidinediones (TZDs), a class of oral antidiabetic agents and a variety of non-steroidal anti-inflammatory drugs (NSAIDs), as shown in Table 1.3;31;32 It has been referred that the activation of PPAR-c by

Table 1 Chemical structures of commonly used PPAR-c ligands O

O O

N

O

S O

O

O 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2)

troglitazone (TGZ)

O

O NH

S

N

O

N

N

O

S

O

O

pioglitazone

rosiglitazone (BRL49653)

S OH O

O O

O

N

N N

H

N N

ciglitazone (CIG)

O

LY 171883

S O N

O

O

O S N HOOC

GW 0072

englitazone

COOH

O N

NH O O

GI 262570

N

H

O

N

548 presence of different PPAR-c isoforms.33 PPAR-c ligands exert anti-tumoral effects through inhibiting cell growth and inducing cell differentiation in several types of human malignant neoplasms.

PPAR-c ligands and gene expression PPAR target genes play a key role in systemic lipid metabolism and energy homeostasis with preferential expression of PPAR-a in the liver and PPAR-c in the adipose tissue.30 PPAR-c upregulate genes encoding fatty acid metabolism, such as lipoprotein lipase, fatty acid transport proteins, malic enzyme and phosphoenolpyruvate carboxykinase (PEPCK), considered as PPAR-c target genes with identified PPREs.30 The gene encoding the insulindependent glucose transporter (GLUT4) and signaling molecules as c-Cbl protooncogene product and c-Cbl-associated protein (CAP) identified as principal genes involved in glucose homeostasis and insulin-signaling pathway, are also upregulated by PPAR-c activation.29;30 TZDs increase adiponectin gene expression and circulating adiponectin levels, suggesting a direct correlation between PPAR-c activity and adiponectin expression.29;34;35 The nuclear receptor LXR-a that enhances expression of the ATP-binding cassette transporter A1 (ABCA1) has been shown to be a PPAR-c target gene in human and mouse macrophages.36;37 CD36 was also confirmed as a positively regulated PPAR-c target gene.29 Ligands of PPAR-c modulate the growth of epithelial cells derived from diverse organs. The promoter and enhancer region of the PSA gene containing androgen receptor response elements (AREs) has been extensively analyzed and has been shown that expression of PSA is regulated by binding of the androgen/androgen receptor (AR) complex to AREs. PPAR-c ligands, TGZ, pioglitazone and 15d-PGJ2 downregulated androgen-stimulated reporter gene activity in the prostate cancer cell line LNCaP. Reporter gene studies revealed that TGZ inhibited androgen activation of the AREs in the PSA regulatory region. Incubation of LNCaP cells with TGZ dramatically suppressed PSA protein expression without suppressing that of AR, suggesting that TGZ inhibited ARE activation by a mechanism other than downregulation of AR expression, implying also antiprostate cancer effect of TGZ in vitro and in vivo.38;39 PPAR-c mRNA levels in the colon are nearly equivalent to that found in adipocytes. Microarray technology was used to identify PPAR-c gene targets in intestinal epithelial cells, highlighting some

S. Theocharis et al. mechanisms by which PPAR-c regulates intestinal epithelial cell biology.40 The induction or repression observed for each gene, was confirmed by using two structurally distinct PPAR-c agonists (rosiglitazone and GW7845), and the alterations in the expression were blocked by co-treatment with a specific PPAR-c antagonist (GW9662). In that study, it was revealed that selective targets of PPAR-c included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen (CEA) family were induced by PPAR-c. Cultured cells treated with PPAR-c ligands demonstrated an increase in Ca2þ independent, CEA-dependent homotypic aggregation, suggesting a potential role for PPAR-c in regulating intercellular adhesion.40 In human non-small cell lung carcinoma (NSCLC) cells, TGZ and pioglitazone treatment inhibited cellular growth and induced apoptosis in a timeand dose-dependent manner. Subtraction cloning analysis identified that TGZ stimulated expression of the growth arrest and DNA-damage inducible (GADD) 153 gene, and the increased expression of GADD153 mRNA was confirmed by an array analysis of the 160 apoptosis-related genes. The GADD153 protein levels were also increased by TGZ treatment. In cells lacking PPAR-c expression (3T3-L1 cell line), TGZ treatment did not stimulate GADD153 mRNA expression, while when an antisense phosphorothionate oligonucleotide that attenuated the TGZ-induced growth inhibition was applied, GADD153 gene expression was inhibited. Such data proposed the GADD153 gene as a candidate factor implicated in growth inhibition and apoptosis induced by TGZ through PPAR-c activation.41 The phosphatase and tensin homologue mutated on chromosome ten (PTEN) tumor suppressor gene modulates several cellular functions, including cell migration, survival and proliferation by antagonizing phosphatidylinositol-3 kinase (PI3K)-mediated signaling cascades.42 In different tumor cell lines, including the Caco2 colorectal cancer cells, PTEN expression was upregulated by rosiglitazone that activated PPAR-c. This upregulation was correlated with decreased PI3K activity, resulting in reduced proliferation rate of Caco2 cells.43 Antisensemediated disruption of PPAR-c expression prevented the PTEN upregulation, suggesting a PPAR-c role in regulating PI3K signaling by modulating PTEN expression in tumor-derived cells.43 Oxidized low-density lipoprotein (oxLDL) has been shown to play an important role in cellular

PPAR-c and cell-cycle differentiation and atherosclerotic lesion formation. Genes induced or suppressed by oxLDL in human monocytic THP-1 cells were searched and among them one was dramatically stimulated by the oxLDL, found to contain sequences corresponding to ferritin light chain (L-ferritin), a ubiquitous protein regulating cellular differentiation. An increase of L-ferritin mRNA was observed when the cells were treated with different lipid components in the oxLDL (9-HODE, 13-HODE, 25-hydroxycholesterol) and with the endogenous PPAR-c ligand 15d-PGJ2, in a time- and dose-dependent manner. Consequently, oxLDL or its constituents are related to the stimulation of L-ferritin expression via PPAR-c mRNA in this monocytic cell line.44 The signal transducer and activator of transcription (STAT) family mediates the cascade of transcriptional events leading to adipogenesis. The differentiation-dependent upregulation of STAT protein expression proved to be downstream regulated by PPAR-c in a ligand-dependent manner.45 It becomes evident that PPAR-c ligands through different pathways activate genes implicated in metabolism and in growth modulation of different cell types.

PPAR-c ligands and cell-cycle progression Studies based on tumoral cell lines have implicated PPAR-c in cell cycle withdrawal. Ligand activation of PPAR-c induces cell cycle withdrawal of preadipocytes via suppression of the transcriptional activity of E2F/DP DNA-binding complex.46 When preadipocytes reenter the cell cycle, PPARc expression was induced, coincident with an increase of DNA synthesis, suggesting involvement of the E2F family of cell-cycle regulators. E2Fs represent the link between proliferative signaling pathways, triggering clonal expansion, and terminal adipocyte differentiation through regulation of PPAR-c expression, reinforcing the complicated role of the E2F protein family in the control of both cell proliferation and differentiation.47 PPAR-c activation decreased the binding of E2F/ DP heterodimers to its target genes, partly mediated through the downregulation of the protein phosphatase 2A (PP2A).48 Inhibition of E2F/DP activity could also being achieved via retinoblastoma protein (pRb) activation. PPAR-c ligands inhibited pRb phosphorylation in vascular smooth muscle cells, maintaining pRb that controls cellcycle progression, in its active form, abrogading the G1 to S phase transition of these cells.49 TZDs and non-TZDs inhibited vascular smooth muscle

549 cell growth through inhibition of pRb phosphorylation.49–51 Another suggested mechanism involving PPAR-c in the mediation of cell-cycle arrest, was provided by the study of Morrison and Farmer,52 who suggested a role of PPAR-c in upregulating the cyclin dependent kinase inhibitors (CDKIs) p18 and p21 during adipogenesis. Similar inhibition was proved using pancreatic cancer cell lines. TGZ treatment inhibited the growth of six out of nine pancreatic cancer cell lines used, by G1 phase cell-cycle arrest through the upregulation of p21 mRNA and protein expression.53 TZDs treatment resulted in both cellular and clonogenic growth inhibition and G1 cell-cycle arrest of human pancreatic cancer cell lines resulting in p21 induction and increased expression of differentiation markers.54 In the pancreatic cell line Panc-1, growth inhibition was found post-TGZ treatment by induction of G1 phase accumulation accompanied by increased expression of p27 but not of p21.53;55 In different hepatoma cell lines (HLF, HuH-7, HAK-1B, HAK-5), a dose-dependent cytostatic effect of TGZ was found. The G0 to G1 cell-cycle arrest was related to alterations of p21 protein expression. HLF, which was deficient in pRb, responded most profoundly to TGZ, showing an increased expression not only in p21 but also in p27 and p18, suggesting that these proteins might be involved in TGZ-induced cell-cycle arrest in human hepatoma cell lines.56 Recent work has shown that PPAR-b was upregulated after loss of adenomatous polyposis coli (APC) tumor suppressor gene function and that transcriptional activation of the PPAR-c inhibited tumor growth.57 In conditionally K-Ras-transformed rat intestinal epithelial cells (IEC-iK-Ras), PPAR-b levels and its activity were increased. PPAR-b upregulation occurred due to increased mitogen-activated protein kinase (MAPK) activity and receptor activation required the endogenous production of prostacyclin via the cyclooxygenase2 (COX-2) pathway.57 It was also demonstrated that PPAR-c activation exerted antineoplastic effects in IEC-iK-Ras transformed cells. PPAR-c activation resulted in a delay in transit through the G1 phase of the cell cycle that was associated with inhibition of PI3K/Akt activity and a reduction of cyclin D1 expression. Two structurally related nuclear receptors the PPAR-c and PPAR-b, seem to have different modes of action during neoplastic transformation. PPAR-c appeared to modulate differentiation and growth signal inhibition, whereas PPAR-b was upregulated by oncogenic Ras and activated by COX-2-derived prostaglandins pathway.57 It was also shown that PPAR-b activation

550 could be involved in keratinocyte proliferation and differentiation.58;59 Hupfeld and Weiss60 reported that TZDs inhibited vascular smooth muscle cell growth by inhibiting cyclin D1 and cyclin E levels, supporting another possible mechanism for TZD action, except of the already known inhibition of p21 and p27. TGZ treatment also inhibited the growth of MCF-7 breast carcinoma cells by accumulating cells in the G1 phase, targeting G1 cell-cycle regulators such as pRb and cyclin D1.61 TGZ inhibited MCF-7 cell proliferation by blocking critical events for G1 to S phase progression. Accumulation of cells in G1 was accompanied by an attenuation of pRb phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TGZ correlated with decreased protein levels for several G1 regulators of pRb phosphorylation (cyclin D1, CDK2, CDK4, CDK6). Induction of cyclin D1 overexpression partially rescued MCF-7 cells from TGZmediated G1 cell-cycle arrest.61 TGZ treatment of T24 bladder cancer cells dramatically inhibited cell proliferation and induced cell death.62 This was accompanied by increased expression of two CDKIs the p21 and p16, and by reduced cyclin D1 expression, consistent with G1 arrest.62 Myelomonocytic U937 cells, when cultured with TGZ, were arrested in the G1 phase of the cell cycle. Simultaneous treatment of myeloid leukemia cell lines with both TGZ and the ligand LG100268 that binds either RXR or the retinoic acids receptors, resulted in additive suppression of clonal growth. These data verified that TGZ when combined with retinoids became moderately potent inhibitor of clonogenic growth of acute myeloid leukemia cells.63 TGZ or pioglitazone treatment markedly suppressed cell proliferation of the promyelocytic leukemia cell line HL60, exerting a G0 to G1 cellcycle arrest as well as an apoptotic effect.64;65 The human gastric cancer cell line MKN45, expressed PPAR-c mRNA and protein. In MKN45 cells, treatment with TGZ transactivated the transcription of PPRE-driven promoter. TGZ or pioglitazone treatment inhibited the growth of MKN45 cells in a dose-dependent manner. Co-incubation of MKN45 cells with high TGZ dose, induced apoptosis evident by DNA ladder formation, consequently PPAR-c activation inhibits cell growth and induces apoptosis.66

PPAR-c ligands and apoptosis Apoptotic cell death was reported in a wide variety of experimental cancer models, in vitro and in vivo post treatment with PPAR-c ligands.67–70 TNF-re-

S. Theocharis et al. lated apoptosis inducing ligand (TRAIL) is a member of the TNF family of cytokines that induces apoptosis. As TRAIL preferentially kills tumor cells, sparing normal tissues, interest has emerged in applying this biological factor for anticancer therapy in humans. A variety of natural and synthetic PPAR-c ligands sensitize tumor but not normal cells to apoptosis induction by TRAIL. PPAR-c ligands selectively reduced levels of FLICE-inhibitory protein (FLIP), an apoptosis-suppressing protein that blocks early events in TRAIL/TNF family death receptor signaling. PPAR-c modulators induced ubiquitination and proteasome-dependent degradation of FLIP, without concomitant reductions in FLIP mRNA.71 Incubation of cortical neurons with 15d-PGJ2 induced morphological changes including neurite degeneration and nuclear condensation that were consistent with apoptotic cell death. All these changes were prevented by pretreatment of neurons with Z-VAD, a general caspase inhibitor.72 Modulation of apoptosis related genes expression by PPAR-c ligands was examined in HT-29 colon cancer cells and screened with cDNA arrays, while the results were confirmed by quantitative RT-PCR analysis. The PPAR-c ligands 15d-PGJ2 and TGZ, suppressed DNA synthesis of HT-29 cells whereas ligands for PPAR-a and PPAR-b had no significant effects. Both PPAR-c ligands used, induced HT-29 cell death in a dose-dependent manner which was associated with an increase in fragmented DNA and was sensitive to a caspase inhibitor. Among several genes selected by cDNA array screening, quantitative RT-PCR analysis confirmed downregulation of c-myc expression and upregulation of c-jun and GADD153 expression by the PPAR-c ligands 15d-PGJ2 and TGZ. As c-myc is an important target gene of the APC/b-catenin and/or APC/c-catenin pathway, activation of PPAR-c signaling appeared to compensate for deregulated c-myc expression caused by mutated APC.73 The APC tumor suppressor gene is involved in programmed cell death, regulating apoptosis. The c-myc oncogene that controls cell proliferation, may be also regulated by APC. The APC protein binding to b-catenin, a component of cell–cell adherent junctions, acts as negative signal to proliferation. c-jun and GADD153 gene protein expression lead to growth arrest and apoptosis. Apoptosis was also induced in HT-29 human colon cancer cells post ciglitazone treatment.74 TGZ inhibited the growth of liver cancer cell lines PLC/PRF/5, HepG2 and HuH-7, by inducing apoptosis through caspase 3 activation supporting evidence that TGZ could be potentially useful as an

PPAR-c and cell-cycle apoptosis inducer for the treatment of hepatocellular carcinoma (HCC).75 TGZ and 15d-PGJ2 treatment inhibited the growth of human lung cancer cells through the induction of apoptosis, result that did not occur when the PPAR-a agonist bezafibrate was applied.76 Combined treatment of NSCLC cell lines with histone deacetylase (HDAC) inhibitors and PPAR-c ligands resulted in enhanced growth inhibitory capacity in adenocarcinomas compared to single treatment, suggesting that both drugs could be used in combination, for the future management of NSCLC77 and possibly for the treatment of other cancer types.78 Treatment of MCF-7 breast cancer cell line with TGZ for 4 days reversibly inhibited cancer cell growth. Combination treatment of the cells with TGZ and 9 cis-retinoic acid irreversibly inhibited growth and induced apoptosis, associated with a dramatic decrease of their bcl-2 protein levels.79 Similar effects were noted with in vitro cultured breast cancer tissues from patients, but not with normal breast epithelial cells. TGZ significantly inhibited MCF-7 tumor growth in triple immunodeficient mice. Combined administration of TGZ and 9 cis-retinoic acid exerted apoptosis and fibrosis of these tumors without signs of toxicity.79 Activation of PPAR-c with either 15d-PGJ2 or TGZ attenuated cellular proliferation of the estrogen receptor (ER)negative breast cancer cell line MDA-MB-231, as well as of the ER-positive cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell-cycle progression and apoptosis, suggesting that induction of apoptosis may be the primary biological response resulting from PPAR-c activation in some breast cancer cells and further emphasize a potential role for PPAR-c ligands for treatment of breast cancer.80 In breast cancer cells, 15d-PGJ2-induced potent and irreversible S-phase arrest that was correlated with expression of genes critical to cell-cycle arrest and apoptosis, including those encoding the p21 and p27 proteins. Inhibition of RNA or protein synthesis abrogates apoptosis induced by the 15d-PGJ2 in breast cancer cells but potentiates apoptosis induced by TNF-a or CD95/Fas ligand. Additionally, 15d-PGJ2-induced caspase activation, blocked by peptide caspase inhibitors, showing that de novo gene transcription was necessary for 15d-PGJ2-induced apoptosis in breast cancer cells.81 Clay et al.82 in a more recent study reported that while 15d-PGJ2 activates PPRE-mediated transcription, PPAR-c is not required for apoptosis induction in breast cancer cells, proposing cyclopentenone prostaglandins as the inducers of apoptosis. TGZ treatment induced growth inhibition in cells derived from malignant glioma (SK-MG-1) or neu-

551 roblastoma (NB-1) cell lines. Further analyses revealed that this growth inhibition was caused by a PPAR-c mediated induction of apoptosis.83 PPAR-c protein was abundantly expressed in human primary astrocytes and in human malignant astrocytoma cell line T98G. Treatment of cells with either 15d-PGJ2 or ciglitazone resulted in apoptotic cell death.84 The PPAR-c ligands 15d-PGJ2, GW1929 and phenylacetate (PA) induced differentiation of neuroblastoma cell line LA-N-5 cells to a similar phenotype as evident by inhibition of cell proliferation, neurite outgrowth, increased acetylcholinesterase activity, and decreased N-myc gene expression.85 All these functional and molecular effects of PPAR-c ligands, were inhibited by cotreatment with specific PPAR-c antagonists (GW9662 and/or GW0072). The PPAR-c ligands ciglitazone, LY171 833 and 15d-PGJ2 inhibited proliferation and induced cell death that was characterized by DNA fragmentation and nuclear condensation, in human (U87MG and A172) and rat (C6) glioma cell lines. In contrast, primary murine astrocytes were not affected by PPAR-c ligand treatment.86 The apoptotic cell death in the glioma cell lines treated with PPAR-c ligands was correlated with the transient upregulation of Bax and Bad protein levels. Furthermore inhibition of Bax expression by specific antisense oligonucleotides protected glioma cells against PPAR-c-mediated apoptosis, indicating an essential role of Bax in this process. PPAR-c ligands not only induced apoptosis but additionally caused re-differentiation, indicated by the increased expression of the re-differentiation marker N-cadherin.86 Pioglitazone inhibited also dose-dependently the proliferation of human chondrosarcoma cell line OUMS-27 by apoptosis induction.87 Although anti-proliferative properties have also been associated with PPAR-c activation, it seems that the action of PPAR-c on cell cycle, proliferation, differentiation and apoptosis depends on the cell type and/or the mutational events that predispose tissues to cancer development. Nevertheless, most of the data concerning PPAR-c participation in cellular function has been generated in vitro in cell culture conditions, whereas results are often difficult to extrapolate to in vivo situations.

Conclusions Since their discovery at previous decade, it became clear that PPARs are important modulators in the regulation of complex pathways of mammalian

552 cells’ metabolism, recently implicated in cancer. It has been suggested that PPAR-c in in vitro and in vivo model systems may behave as a tumor suppressor gene. In contrast, data in murine models suggest that PPAR-c might enhance tumor formation. PPAR-c, proved to be a regulator of the expression of many genes relevant to carcinogenesis. PPAR-c ligands induce growth arrest, apoptosis and differentiation in a variety of transformed cells. Selective PPAR-c modulators (SPARMs), proved to have desired effects on specific genes and target tissues without undesirable effects on others. In vitro and in vivo studies in adipocyte tissue and in different cell lines have substantiated the importance of PPAR-c ligands in mediating cell cyclerelated events, through different mechanisms of action. The genomic response to PPAR-c activation remains complex and the understanding of the interactions between PPARs and other factors will modulate the restricted range of the PPAR target genes, participating in cell-cycle modulation. The final PPAR-c action appears to be dependent on a dynamic balance between RXR, TR-alpha, other cellular factors and nuclear hormone receptors. The pleiotropic effects mediated by PPARs on cellcycle progression and apoptosis, will be elucidated by understanding the underlying pathways of their action. At fundamental level, effort focused on the molecular basis of known PPAR-c ligands action is needed in order to discover-synthesize novel agents. The anticancer efficacy of PPAR-c ligands and their very low toxicity makes them candidates for use in the adjuvant setting in combination with other drugs and for chemoprevention of selected cancers. At clinical level further studies are important in order to delineate optimal in vivo dosage, duration of treatment and possibly efficacy of PPAR-c ligands alone or in combination with other agents in the fight against cancer. It becomes so far evident that PPAR-c ligands acting as cell-cycle and apoptosis modulators and as differentiation inducing agents, could be used as promising antineoplastic drugs for cancer chemotherapy and chemoprevention in the future.

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