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NEUROPHARMACOLOGY

The Nonthiazolidinedione Tyrosine-Based Peroxisome Proliferator-Activated Receptor Ligand GW7845 Induces Apoptosis and Limits Migration and Invasion of Rat and Human Glioma Cells

Department of Neurosciences, Alzheimer Research Laboratory, Case Western Reserve University, Cleveland, Ohio (C.G., G.E.L.); Department of Neurology, University of Bochum, Bochum, Germany (U.S.); and Department of Neurology, University of Muenster, Muenster, Germany (M.T.H.)

Received October 24, 2004; accepted January 19, 2005.

	Abstract

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Despite new approaches, treatment options for malignant gliomas are still limited, calling for further development of therapeutic strategies. The peroxisome proliferator-activated receptor (PPAR), a member of the nuclear hormone receptor family, represents a possible new target for neoplastic therapies. Synthetic PPAR agonists were developed and are already in clinical use for the treatment of type II diabetes, since PPAR plays a crucial role in lipid metabolism and regulation of insulin sensitivity. Beyond these metabolic effects, PPAR agonists exhibit antineoplastic effects in various malignant tumor cells. Here, we investigated the antineoplastic effects of the nonthiazolidinedione tyrosine-based PPAR ligand (S)-2-(1-carboxy-2-{4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phenyl}ethylamino)benzoic acid methyl ester (GW7845) in rat and human glioma cells. GW7845 reduced cellular viability of rat C6 glioma and human glioma cells in a time-dependent manner. Analysis of GW7845-treated tumor cells revealed induction of apoptotic cell death as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and cleaved caspase-3 activation. Furthermore, GW7845 reduced proliferation of C6 glioma cells as measured by Ki-67 immunore-activity. There was also a reduction of migration and invasion, assessed by Boyden chamber and spheroid experiments. Together, these data indicate that the PPAR agonist GW7845 may be of potential use in treatment of malignant gliomas.

A new antineoplastic treatment approach may lie in targeting the peroxisome proliferator-activated receptor (PPAR) with specific agonists (Grommes et al., 2004). PPAR is a member of the PPAR family, a subclass of nuclear hormone receptors, enabling the cell to respond to extracellular stimuli by transcriptionally regulating gene expression. Three isoforms of PPARs have been identified and designated as , /, and , all encoded by different genes. PPARs form heterodimers with the retinoic acid receptor and exhibit ligand-induced transcriptional regulatory activity through sequence-specific PPAR-responsive elements in its target genes (Willson et al., 2000). For more than a decade, work on PPARs was driven by their important role for regulation of cellular metabolism, especially in tissues known for high rates of -oxidation such as liver, heart, muscle, and kidney. Activation of the PPAR subtype results in reduced serum glucose (Lemberger et al., 1996); therefore, recently developed synthetic PPAR agonists, which are members of the thiazolidinedione family, are already in clinical use as antidiabetic drugs [pioglitazone (Actos); rosiglitazone (Avandia)].

PPAR agonists exhibit antineoplastic effects in a number of different cancer entities (Grommes et al., 2004). We recently reported that several PPAR agonists induce apoptosis in rat and human glioma cell lines, which can be blocked by a PPAR antagonist and BAX antisense oligonucleotides (Zander et al., 2002). In keeping with this, it was shown that PPAR agonists also inhibited the growth of BT4Cn rat glioma cells, an effect that was abolished by the PPAR antagonist GW9662 (Berge et al., 2001). Beside thiazolidinediones, nonthiazolidinedione tyrosine-based PPAR ligands have been described as potent and selective PPAR agonists (Willson et al., 2000). Because the nonthiazolidinedione tyrosine-based PPAR ligand GW7845 (Cobb et al., 1998) showed antineoplastic effects in human breast cancer (Liu et al., 2003) and rat mammalian carcinogenesis (Suh et al., 1999), we determined the antineoplastic effects of GW7845 in several glioma cells.

We demonstrate that GW7845 reduces cellular viability of C6 rat glioma and human glioma cell lines (A172 and U87) and inhibits C6 glioma cell proliferation, measured by Ki-67 expression in vitro. Moreover, we demonstrate that GW7845 induces apoptotic cell death and cell cycle arrest. As a further antineoplastic mechanism, we found that GW7845 decreased the migration and invasion of glioma cells, thus affecting one of the key properties defining malignancy.

	Materials and Methods

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Materials. GW7845 (GlaxoSmithKline, Uxbridge, Middlesex, UK), prostaglandin-J2 (BIOMOL Research Laboratories, Hamburg, Germany), and GW9662 (Calbiochem, Darmstadt, Germany) were dissolved in dimethyl sulfoxide (DMSO) obtained from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, trypan blue solution, penicillin, streptomycin, fetal calf serum, phosphate-buffered saline (PBS), trypsin-EDTA, and proteinase K were purchased from Invitrogen (Karlsruhe, Germany). Ki-67 antibody was purchased from NeoMarkers (Fremont, CA), and cleaved caspase-3 and BAX antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA). For Western blot analysis, the secondary anti-rabbit antibody was obtained from Amersham Biosciences Inc. (Piscataway, NJ). Secondary antibody for immunohistochemistry (Alexa Fluor 488-conjugated goat anti-rabbit IgG) was purchased from Molecular Probes (Eugene, OR).

Cell Culture. Rat C6 glioma cells were grown in DMEM and human glioma cells (U87 and A172) in RPMI 1640 medium, supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin in a 5% CO₂ atmosphere. Primary astrocyte cultures were prepared as described previously (Zander et al., 2002) and grown in DMEM, supplemented with 2.5% (v/v) fetal calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin in a 5% CO₂ atmosphere.

Viability Assay. Cellular viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT; Sigma-Aldrich) and trypan blue staining. Briefly, C6 cells, U87 cells and A172 cells (5 x 10³/well) or primary astrocytes (5 x 10³/well) were seeded in a 96-well plate and exposed to different concentrations of GW7845 or prostaglandin J₂ (1, 10, and 30 µM; n = 10). DMSO served as vehicle control (0.1% of final concentration). At 1, 3, 5, and 7 days, 10 µl of MTT (5 mg/ml PBS) was added to each well, and plates were incubated at 37°C for 2 h. Medium was then removed, and cells were resuspended in 100 µl of DMSO. Cellular viability was assessed by colorimetric change using Spectramax 340PC plate reader (Molecular Devices, Sunnyvale, CA) at = 550 nm. For blocking experiments, C6 cells were pretreated with 2 µM GW9662 for 30 min and subsequently with or without 30 µM GW7845. After 5 days, cellular viability was evaluated using MTT assay. For trypan blue staining, cells were seeded at a concentration of 50,000 cells/well in a six-well plate and exposed to GW7845 (30 µM) or DMSO. At 1, 3, 5, and 7 days after initial treatment, cells were washed, trypsinized, and briefly centrifuged at 2000g for 5 min. Cells were then stained and counted. Experiments were performed in triplicates.

Life/Death Assay. Cell survival was further assessed using calcein AM and ethidium homodimer (Molecular Probes) as described previously (Spencer et al., 2003). Calcein AM is taken up into living cells and is metabolized in the cytoplasm by esterases into the green fluorescent product calcein so that viable cells show uniform green fluorescence (emission 530 nm) under appropriate excitation (488 nm). Ethidium homodimer is excluded from living cells but can cross the compromised plasma membrane of dying cells and interact with nucleic acids to give a strong red fluorescence (emission 585 nm) under appropriate excitation (488 nm). GW7845 untreated and treated C6 cells were washed, calcein AM (4 µM) and ethidium homodimer (2 µM) were added, and the cells were incubated for 30 min at 37°C. The fluorescence was assessed using a fluorescent microscope (Leica, Wetzlar, Germany). Numbers of live and dead cells were determined.

Flow Cytometry Studies. For flow cytometry analyses of GW7845-treated and untreated samples, cells were collected, and the cell cycle profile of propidium iodide-stained cells was determined. For cell cycle analyses, cells were pelleted at 1000g for 3 min and washed twice in 2 ml of cold PBS. After centrifugation, cells were resuspended in 2 ml of cold PBS and fixed by the gradual addition of 6 ml of 95% cold ethanol with gentle vortexing. After at least 1 h at 4°C, cells were collected by centrifugation and cell pellets were treated with 1 mg/ml RNase type I-A (Sigma-Aldrich) at 37°C for 30 min. Propidium iodide (PI) was added to a final concentration of 0.05 mg/ml and incubated at 4°C for 30 min. Samples were analyzed using the Case Western Reserve Core Facility for fluorescence activated cell sorting analysis. For cell cycle analyses, the PI was excited at 488 nm, and its fluorescence was collected through a 630/22-nm band pass (BP) filter. To detect apoptosis or cells with sub-G₁ DNA levels, PI fluorescence was collected through 530/30 BP and 630/22 BP filters, respectively.

Detection of DNA Fragmentation. C6 cells were harvested and collected by centrifugation (3000g; 5 min) and then lysed with 400 µl of SE buffer (75 mM NaCl and 25 mM EDTA) containing 1% (w/v) SDS and 2 U of proteinase K/ml and incubated for 2 h at 55°C. Proteins were precipitated by addition of 140 µl of 5 M NaCl and centrifugation (11,000g; 15 min). Subsequently, DNA in the supernatant was precipitated by addition of 1000 µl of ethanol and centrifugation (11,000g; 15 min). After washing with 70% (v/v) ethanol, DNA was resuspended in H₂O, separated on an agarose gel, and stained with ethidium bromide.

Western Blot. For Western blot analysis, C6 cells were treated with GW7845 for 6, 12, 24, 48, and 72 h or with DMSO for 6 and 72 h. Cells then were washed with PBS, harvested with PBS/phenylmethylsulfonyl fluoride (PMSF) [20 mM PMSF (Sigma Chemie, Taufkirchen, Germany) in PBS] containing aprotinin and leupeptin, collected by centrifugation, lysed in Tris-HCl [50 mM Tris-HCl, pH 8, 120 mM NaCl, 5 mM EDTA, 0.5% (v/v) Nonidet P-40, and 160 mM PMSF], and sonicated. Homogenates were collected by centrifugation (15 min; 11,000g; 4°C), and the protein concentration in the supernatant was determined using the Bio-Rad protein assay (Bio-Rad, Munich, Germany). Lysates (20–40 µg) were separated on a 10% (w/v) SDS-polyacrylamide gel under reducing conditions and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA). Nonspecific binding was blocked by incubation with 5% (w/v) skimmed milk in TBS for 2 h. After incubation with the primary antibody overnight at 4°C [rabbit anti-BAX 1:1000, rabbit anti-cleaved caspase-3 1:1000 in TBS containing 0.1% (v/v) Tween 20], membranes were washed three times in TBS/Tween for 5 min. Membranes were subsequently incubated for 120 min in TBS/Tween containing secondary peroxidase-conjugated antibody at room temperature (anti-rabbit 1:1000). Signals were visualized by chemoluminescence (Pierce Chemical, Rockford, IL), and band intensities were quantified using NIH Image 1.62 software (National Institutes of Health, Bethesda, MD).

Immunocytochemistry. For immunocytochemistry, C6 cells were treated with GW7845 for 48 and 72 h or with DMSO for 6 and 72 h. Cells were fixed in 4% paraformaldehyde, rinsed in TBS, and blocked with 5% normal goat or horse serum and subsequently incubated with rabbit anti-Ki-67 (1:200 dilution) or rabbit anti-cleaved caspase-3 (1:200 dilution) at 4°C overnight. Sections were then washed extensively with PBS before incubation with secondary antibodies. Incubation was carried out at room temperature for 1 h. After washing with PBS three times, stained slides were mounted with PBS/glycerol (1:1) and viewed under a fluorescent microscope (Leica). Counterstaining was carried out with 4,6-diamidino-2-phenylindole (DAPI) staining (1:500 in PBS).

To determine the proliferation-index, Ki-67-positive cells in GW7845 and vehicle-treated cells were counted, and the percentage of Ki-67-positive cells in 1 x 10³ tumor cells (DAPI stain) was calculated and statistically compared. The percentage of cleaved caspase-3-positive cells was assessed by counting the immunopositive cells in 1 x 10³ tumor (DAPI stain) cells and statistically compared.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling Assay for Apoptosis. Frozen brain tissue sections were examined for apoptosis using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Apoptosis was evaluated using the DeadEnd fluorometric TUNEL system (Promega, Madison, WI) according to the manufacturer's instructions. The percentage of TUNEL-positive cells was assessed by counting the immunopositive cells in 1 x 10³ C6 cells (DAPI stain) after 72 h of GW7845 or DMSO treatment and statistically compared.

Boyden Chamber Assay. The migration of C6 cells in the presence or absence of GW7845 was assessed using a Boyden chamber (AP48; Neuro Probe, Gaithersburg, MD) with an 8-µm polycarbonate polyvinylidene difluoride filter (Osmonics, Minnetonka, MN). Cells were suspended in DMEM containing 2.5% fetal calf serum and GW7845 at 30 µM or vehicle (DMSO). C6 glioma cells were pretreated for 24 h in the presence or absence of 30 µM GW7845. Cells (5 x 10³ in 50 µl of GW7845/medium or vehicle/medium) were then plated in the wells of the upper compartment of the chamber (six-well/condition), and the wells of the lower compartment were filled with DMEM. Incubation was performed at 37°C in 5% CO₂ for 4 h. After incubation, cells on the upper surface of the filter, which had not migrated, were gently scraped off, and the filters were then fixed in methanol and subsequently stained with DAPI (1:500 in PBS; Sigma-Aldrich). The number of cells that migrated to the lower surface of the filter was counted using public domain software NIH Image 1.62. Total numbers of migrating cells under GW7845-treated and vehicle-treated condition were compared statistically. Experiments were performed in duplicates.

Glioma Spheroids. C6 glioma spheroids were cultured in 10-mm Petri dishes coated with 0.75% Noble Agar (Difco, Detroit, MI) prepared in DMEM (Pedersen et al., 1993). Briefly, 3 x 10⁶ cells were suspended in 10 ml of medium, seeded onto 0.75% agar plates, and cultured until spheroids had formed. Spheroids of about 200-µm diameter were selected for the experiment, treated with 30 µM GW7845 or DMSO, and spheroid growth was assessed daily.

Statistical Evaluation. Cellular viability data, tumor volumes, immunopositive cells, clinical scores, proliferation index, and densitometric results of Bax and cleaved caspase-3 Western blots were analyzed by Student's t test using Prism version 3.00 software (GraphPad Software Inc., San Diego, CA).

	Results

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Reduced Cellular Viability of Rat C6 Glioma and Human Glioma Cells. Viability of rat C6 glioma cells and human glioma cells (U87 and A172) was assessed after incubation with increasing concentrations (C6: 1, 10, and 30 µM; U87/A172: 30 µM) of the PPAR agonist GW7845 at days 1, 3, 5, and 7 (Fig. 1, A and B; Fig. 2A). Ten and 30 µM GW7845 significantly reduced the cellular viability of C6 rat glioma cells (Fig. 1A) in a time-dependent manner. A significant reduction of cellular viability of C6 cells was observed at 3, 5, and 7 days after treatment with 10 µM and 30 µM GW7845 (Fig. 1A). In the human glioma cell lines U87 and A172, 30 µM GW7845 significantly reduced cellular viability at 3, 5, and 7 days (Fig. 1B). Trypan blue staining confirmed this finding and showed a reduction in cellular viability after treatment with 30 µM GW7845 in C6 cells (Fig. 2A) and human glioma cells (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 1. GW7845 reduced cellular viability of rat and human glioma cells. Viability assay of rat C6 glioma cells (A), human U87 and A172 glioma cells (B), or primary astrocytes (C) incubated with the PPAR agonist GW7845 (GW). Cells were treated with different concentrations of GW7845 (1, 10, and 30 µM for C6 and primary astrocytes; 30 µM for U87 and A172), and cellular viability was measured at 1, 3, 5, and 7 days using the MTT assay. The data are expressed as percentage viable cells relative to untreated control cultures. Asterisks indicate significant level in Student's t test: ***, p < 0.001 (n = 10); **, p < 0.05. D, life/death assay at 72 and 96 h of drug treatment. E, numbers of live and dead cells in GW7845- and DMSO-treated C6 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 2. GW7845-induced cellular viability is PPAR-dependent. Trypan blue staining of rat C6 glioma cells (A), incubated with the PPAR agonist GW7845 (GW). Cells were treated with 30 µM GW7845, and trypan blue staining was assessed at 1, 3, 5, and 7 days. The data are expressed as percentage viable cells relative to untreated control cultures (n = 3). B, viability assay of rat C6 glioma cells incubated with the natural PPAR agonist prostaglandin J2 (PGJ2). Cells were treated with different concentrations of PGJ2 (10, 30, and 100 µM), and cellular viability was assessed at 1, 3, 5, and 7 days using the MTT assay. The data are expressed as percentage viable cells relative to untreated control cultures (n = 10). C, MTT assay of C6 cells coincubated with 30 µM GW7845 and 2 µM GW9662. The data are expressed as percentage viable cells relative to untreated control cultures (n = 10). Asterisks indicate significant level in Student's t test: ***, p < 0.001.

At all time points and concentrations evaluated, viability of primary astrocytes was not affected by GW7845 treatment (Fig. 1C), indicating that PPAR ligand-mediated cell death may be restricted to neoplastic cells. These results were further confirmed by the determination of living and dying cells using the above-described live/death assay. GW7845 treatment resulted in increased numbers of dead cells, with a corresponding reduction in the number of viable glioma cells compared with the solvent controls (Fig. 1, D and E).

To further investigate the PPAR dependence of GW7845 effects on cellular viability of the C6 cells, the influence of the natural PPAR agonist prostaglandin J₂ and of the PPAR antagonist GW9662 was analyzed. Prostaglandin J₂ reduced cellular viability of C6 cells in a time- and dose-dependent manner (Fig. 2B). Furthermore, the PPAR antagonist GW9662 reversed the GW7845-dependent reduction of cellular viability in C6 cells, whereas GW9662 alone has no influence on cellular viability (Fig. 2C).

Induction of Apoptotic Cell Death in Vitro. To assess whether GW7845 induces apoptotic cell death in the same way as other PPAR agonists, Bax- and cleaved caspase-3 expression as well as TUNEL staining was investigated in vitro. The expression of Bax and cleaved caspase-3 was detected in lysates of C6 cells treated with 30 µM GW7845 or DMSO using a rabbit-anti Bax or cleaved caspase-3 antibody. Both proteins were induced in the GW7845-treated cells (Fig. 3, A, B, and F). Extracellular signal-regulated kinase 2 expression served as a loading control. Immunocytochemistry revealed elevated cleaved caspase-3 expression at 48 h of GW7845 treatment compared with vehicle controls (Fig. 3, A and B). In parallel, up-regulated TUNEL staining was detected at 72 h after initiation of GW7845 treatment compared with only a few positive cells in the vehicle-treated group (Fig. 3, C and D). Additionally, GW7845-induced DNA fragmentation was detectable at 48 and 72 h, indicating the apoptotic nature of the observed cell death (Fig. 3E).

View larger version (54K): [in this window] [in a new window]   Fig. 3. GW7845 induced apoptotic cell death in vitro. C6 rat glioma cells were treated with 30 µM GW7845. At 48 h (A and B), the proapoptotic protein cleaved caspase-3 is induced by GW7845 treatment (A) compared with DMSO-treated cells (B). After 72 h of treatment, an up-regulation TUNEL signal can be observed in the GW7845 treated cells (C) compared with DMSO treatment (D). Scale bars, 200 µM (A–D). E, DNA fragmentation in GW7845-treated cells after 48- and 72-h treatment compared with DMSO treatment. F, protein level of the proapoptotic proteins Bax and cleaved caspase-3. C6 cells were treated with 30 µM GW7845, and protein lysates were generated after 6, 12, 24, 48, and 72 h. C6 treated with DMSO at 6 and 72 h served as controls. DMSO, vehicle-treated cells; GW, GW7845-treated cells.

Flow Cytometry Analysis of Cell Cycle and Apoptosis in C6 Glioma Cells after GW7845 Treatment. Cell cycle analysis showed induction of apoptotic cell death demonstrated by an increase in numbers of cells with sub-G₁ levels of DNA after 48 and 72 h of GW7845 treatment compared with control treatment (Fig. 4, A and C). Furthermore, GW7845 reduced the percentage of C6-cells in S phase and raised the number of cells in the G₁ phase of the cell cycle (Fig. 4, A and B).

View larger version (31K): [in this window] [in a new window]   Fig. 4. GW7845-induced apoptotic cell death and cell cycle arrest. GW7845-treated and untreated C6 cells were analyzed for apoptotic cell death and cell cycle arrest at 24, 48, and 72 h using flow cytometry (A). B, comparison of the percentages of cells in G1, G2/M, or S phase at 24, 48, and 72 h of treatment (n = 3). C, comparison of apoptotic cells in the sub-G1 group at 24, 48, and 72 h of treatment (n = 3). DMSO, vehicle-treated cells; GW, GW7845-treated cells.

Ki-67 Immunoreactivity and Proliferation Index Is Reduced after GW7845 Treatment in Vitro. To further characterize the GW7845-induced effects, Ki-67 expression, a marker for tumor proliferation and malignancy, was evaluated. C6 cells were treated with 30 µM GW7845 or vehicle, and Ki-67 immunoreactivity was assessed at 48 and 72 h. GW7845 reduced the number of Ki-67 immunopositive cells (Fig. 5A) compared with vehicle treatment (Fig. 5B) at both time points. Thus, the percentage of Ki-67-positive, proliferating cells among total tumor cells (PI staining; Fig. 5, D and E), expressed as proliferation index, was significantly reduced at 48 and 72 h of GW7845 treatment (Fig. 5C).

View larger version (47K): [in this window] [in a new window]   Fig. 5. GW7845 reduced Ki-67 immunoreactivity and proliferation index in vitro. C6 rat glioma cells were treated with 30 µM GW7845. At 48 and 72 h (A), Ki-67 immunoreactivity is reduced by GW7845 treatment compared with vehicle-treated cells (B). The proliferation index assessed by counting Ki-67-positive cells and calculating the percentage of Ki-67-positive cells in 1000 tumor cells (propidium iodide staining; D and E) is significantly reduced at 48 and 72 h of GW8745 treatment (C). Scale bars, 200 µM (A–D). E, black columns, vehicle treatment; gray columns, GW7845 treatment. Asterisks indicate significant level in Student's t test: ***, p < 0.001; *, p < 0.05 (n = 5).

Invasion of C6 Rat Glioma Cells after GW7845 Treatment in Vitro. In addition to proliferation, the ability of tumor cells to invade the surrounding and intact tissue characterizes the malignancy state of gliomas. To assess whether GW7845 also affects the ability of glioma cells to migrate and invade, we used tumor spheroids and the Boyden chamber assay, respectively. Tumor spheroids treated with GW7845 (Fig. 6) showed reduced invasion compared with control treatment with a significant difference in tumor spreading at 4 days of treatment. GW7845-pretreated (30 µM) (Fig. 7E) and untreated (Fig. 7, A, B, and D) C6 glioma cells were suspended in the upper chamber of a Boyden chamber containing GW7845 at 30 µM (Fig. 7, B and E) or vehicle (Fig. 7, A and D). At 4 h, cells that migrated to the other side of an 8-µm filter were stained with DAPI and counted. GW7845 treatment during Boyden chamber incubation reduced the invasiveness of C6 cells significantly (Fig. 7, A–C). Pretreatment of C6 cells for 24 h with GW7845 further enhanced this inhibitory action (Fig. 7, D–F).

View larger version (61K): [in this window] [in a new window]   Fig. 6. Reduced C6 spheroid outgrowth after GW7845 treatment. C6 spheroids were treated with 30 µM GW7845 or DMSO and tumor outgrowth was measured in square millimeters. GW7845 reduced tumor outgrowth significantly after 4 and 5 days of treatment compared with control treatment. Scale bar, 200 µM. Asterisks indicate significant level in Student's t test: **, p < 0.01; *, p < 0.05 (n = 4).

View larger version (51K): [in this window] [in a new window]   Fig. 7. GW7845 reduces invasion of C6 rat glioma cells. Boyden chamber assay in vitro. A and B, untreated C6 glioma cells incubated with vehicle (A) or 30 µM GW7845 (B) stained with DAPI. Scale bars, 200 µm. C, number of migrated cells. Black columns, vehicle treatment; gray columns, GW7845 treatment. Data are presented as mean ± S.E. of six chamber filters from a single experiment. Experiments were performed in duplicates. Asterisks indicate significant level in Student's t test: **, p < 0.01 (n = 6). D and E, pretreated C6 glioma cells (24 h GW7845 30 µM or 24 h vehicle) incubated with 30 µM GW7845 (E) or vehicle (D) stained with DAPI. Scale bars, 200 µm. C, number of migrated cells. Black columns, vehicle treatment; gray columns, GW7845 treatment. Data are presented as mean ± S.E. of six chamber filters from a single experiment. Experiments were performed in duplicates. Asterisks indicate significant level in Student's t test: ***, p = 0.001 (n = 6).

	Discussion

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Because gliomas still have a poor prognosis, the need for further development of new treatment strategies is urgent. The antineoplastic effects of different PPAR agonists on human and rat glioma cell lines (Zander et al., 2002) led us to investigate synthetic PPAR agonist. Nonthiazolidinedione tyrosine-based PPAR ligands have been described as potent and selective PPAR agonists (Willson et al., 2000) and GW7845 as one of the nonthiazolidinedione tyrosine-based PPAR ligands that showed antineoplastic effects in human breast cancer (Liu et al., 2003) and rat mammalian carcinogenesis (Suh et al., 1999). Therefore, we wanted to characterize its antineoplastic efficiency on rat and human glioma cell in vitro. In the present study, GW7845 induced a significant decrease of cellular viability of rat and human glioma cells, demonstrating its antineoplastic potency in vitro. Importantly, we demonstrated that the reduction of cellular viability by GW7845 is restricted to neoplastic cell types, because primary astrocytes were unaffected by GW7845 treatment. Although concentrations of GW7845 higher than 1 µM can influence differentiation, even in primary astrocytic cultures, the morphological appearance of GW7845-treated primary astrocytes did not change over the time course, indicating that only malignant glioma cells are affected by the drug treatment.

To characterize GW7845-induced cell death, markers for apoptotic cell death were evaluated. We investigated Bax protein expression because initial in vitro data indicate that other PPAR-agonists mediate their antineoplastic effects in rat and human glioma cell lines through Bax up-regulation and subsequent induction of apoptosis (Zander et al., 2002). Bax protein as well as the cleavage of its downstream target, the effector caspase-3, were up-regulated by GW7845 over time. The effect peaked at 24 h of GW7845 treatment, a time point which is consistent with earlier studies carried out on C6 cells and other PPAR agonists (Zander et al., 2002). Correlating with this finding, a significant increase in cleaved caspase-3 immunohistochemistry and TUNEL labeling could be detected at 48 h and at 72 h of GW7845 treatment, respectively. Furthermore, GW7845 treatment induced DNA fragmentation at 48 and 72 h. Together, these findings indicate that GW7845 induced apoptotic cell death of rat C6 gliomas.

To verify the induction of apoptotic cell death and to examine the effects of GW7845 on the cell cycle of C6 cells, flow cytometry analysis was performed. Flow cytometry analysis indicated the induction of apoptosis after GW7845 treatment and the induction of a G₁ phase arrest. To further assess whether this cell cycle arrest leads to a reduced proliferation, Ki-67 immunohistochemistry was evaluated. Ki-67 is a nuclear protein expressed in proliferating cells and serves as an important neuropathological marker for diagnosis of human gliomas (Brown and Gatter, 2002). Cells in G₀ (quiescent) phase are negative for Ki-67 (Torp, 2002), and its expression in the normal brain is very low. In astrocytomas, Ki-67 expression is up-regulated and correlates well with the malignancy grade of the tumor and the clinical prognosis of the respective patient (Parkins et al., 1991; Torp, 2002). GW7845 treatment reduced Ki-67 expression and diminished the proliferation index in vitro, suggesting that a reduction of viability, decreased proliferation, and induction of apoptosis in concert reduced tumor cell number in vitro.

Next, to the reduction of the overall number of tumor cells, it seems a favorable goal for an antineoplastic treatment strategy to prevent the migration and invasion of neoplastic cells into adjacent tissue. The cell cycle arrest induced by GW7845 also might influence the invasiveness of C6 cells, as described in human breast cancer cells where GW7845 inhibits invasion (Liu et al., 2003). Using tumor spheroids and the Boyden chamber assay, we tested possible GW7845 effects on tumor cell migration and invasion. Both assays were described to reliably assess C6 cell migration and invasion and are therefore used to test drug effects (Sottocornola et al., 1998). A dramatic reduction of cell migration and invasion was observed in the tumor spheroids as well as in the Boyden chamber assay after GW7845 treatment. These results indicate for the first time that PPAR agonist treatment inhibits key functions and properties of glioma malignancy, such as the ability to migrate and invade.

The concentrations of GW7845 required to affect cellular viability are higher than expected according to its known in vitro receptor binding affinity (Sakamoto et al., 2000). Although GW7845 is a synthetic PPAR agonist and produces its receptor-dependent effects on adipocytes, PPAR-independent mechanisms also may account for the observed antineoplastic action as it has been reported for thiazolidinediones (Chawla et al., 2001). Other nonthiazolidinedione tyrosine-based PPAR ligands expressed their antineoplastic effects in the same micromolar range (data not shown), indicating that this finding is not only limited to GW7845 but a characteristic to the entire nonthiazolidinedione tyrosine-based PPAR-ligand drug family. Therefore, the described effects of GW7845 on gliomas may be the result of both PPAR-dependent and -independent mechanisms. The natural PPAR-ligand prostaglandin J₂ showed antineoplastic effects similar to that of GW7845 on glioma cells in vitro. Furthermore, the PPAR-antagonist GW9662 blocks the GW7845-induced effects on cellular viability of glioma cells. These data suggest that PPAR activation is likely to be a major contributor to the antineoplastic effects induced by GW7845.

Drugs that reduce cell proliferation and induce tumor cell death are of possible therapeutic potential in human tumors. Although the precise molecular basis of the antineoplastic mechanisms of PPAR agonists is yet not fully understood, the clinically used thiazolidinediones may already offer a new therapeutic approach in human glioma therapy because of their dual actions of reducing proliferation and induction of apoptosis. The optimal mode and time of application as well as the efficiency of the combination with other treatment have to be further investigated.

	Footnotes

 
C.G. is supported by a grant of the Deutsche Forschungsgemeinschaft (GR 2018/1-1). U.S. and M.T.H. are supported by a grant from the German Cancer Research Council (10-1795). G.E.L. received financial support for research projects on anti-inflammatory actions of PPAR agonists by GlaxoSmithKline. Case Western Reserve University holds a U.S. patent on the use of PPAR agonists in inflammatory indications in the nervous system. The intellectual property and research support do not relate to the antineoplastic actions of the drugs.

doi:10.1124/jpet.104.078972.

ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; GW7845, (S)-2-(1-carboxy-2-{4-[2-(5-methyl-2-phenyloxazol-4-yl)-ethoxy]phenyl}ethylamino)benzoic acid methyl ester; GW9662, 2-chloro-5-nitro-N-phenylbenzamide; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; BP, band pass; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Address correspondence to: Dr. Michael T. Heneka, Department of Neurology, University of Muenster, Albert-Schweitzer-Str. 33, 48149 Muenster, Germany. E-mail: heneka{at}uni-muenster.de

	References

Top Abstract Materials and Methods Results Discussion References

 

Berge K, Tronstad KJ, Flindt EN, Rasmussen TH, Madsen L, Kristiansen K, and Berge RK (2001) Tetradecylthioacetic acid inhibits growth of rat glioma cells ex vivo and in vivo via PPAR-dependent and PPAR-independent pathways. Carcinogenesis 22: 1747–1755.[Abstract/Free Full Text]

Brown DC and Gatter KC (2002) Ki67 protein: the immaculate deception? Histopathology 40: 2–11.[CrossRef][Medline]

Chawla A, Barak Y, Nagy L, Liao D, Tontonoz P, and Evans RM (2001) PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat Med 7: 48–52.[CrossRef][Medline]

Cobb JE, Blanchard SG, Boswell EG, Brown KK, Charifson PS, Cooper JP, Collins JL, Dezube M, Henke BR, Hull-Ryde EA, et al. (1998) N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 3. Structure-activity relationship and optimization of the N-aryl substituent. J Med Chem 41: 5055–5069.[CrossRef][Medline]

DeAngelis LM (2001) Brain tumors. N Engl J Med 344: 114–123.[Free Full Text]

Grommes C, Landreth GE, and Heneka MT (2004) Antineoplastic effects of peroxisome proliferator-activated receptor gamma agonists. Lancet Oncol 5: 419–429.[CrossRef][Medline]

Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, and Cavenee WK (2002) The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 61: 215–225; discussion, 226–219.[Medline]

Lemberger T, Desvergne B, and Wahli W (1996) Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology. Annu Rev Cell Dev Biol 12: 335–363.[CrossRef][Medline]

Liu H, Zang C, Fenner MH, Possinger K, and Elstner E (2003) PPARgamma ligands and ATRA inhibit the invasion of human breast cancer cells in vitro. Breast Cancer Res Treat 79: 63–74.[CrossRef][Medline]

Parkins CS, Darling JL, Gill SS, Revesz T, and Thomas DG (1991) Cell proliferation in serial biopsies through human malignant brain tumours: measurement using Ki67 antibody labelling. Br J Neurosurg 5: 289–298.[Medline]

Pedersen PH, Marienhagen K, Mork S, and Bjerkvig R (1993) Migratory pattern of fetal rat brain cells and human glioma cells in the adult rat brain. Cancer Res 53: 5158–5165.[Abstract/Free Full Text]

Sakamoto J, Kimura H, Moriyama S, Odaka H, Momose Y, Sugiyama Y, and Sawada H (2000) Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone. Biochem Biophys Res Commun 278: 704–711.[CrossRef][Medline]

Sottocornola E, Colombo I, Vergani V, Taraboletti G, and Berra B (1998) Increased tumorigenicity and invasiveness of C6 rat glioma cells transfected with the human alpha-2,8 sialyl transferase cDNA. Invasion Metastasis 18: 142–154.[CrossRef][Medline]

Spencer JP, Rice-Evans C, and Williams RJ (2003) Modulation of pro-survival Akt/protein kinase B and ERK1/2 signaling cascades by quercetin and its in vivo metabolites underlie their action on neuronal viability. J Biol Chem 278: 34783–34793.[Abstract/Free Full Text]

Suh N, Wang Y, Williams CR, Risingsong R, Gilmer T, Willson TM, and Sporn MB (1999) A new ligand for the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW7845, inhibits rat mammary carcinogenesis. Cancer Res 59: 5671–5673.[Abstract/Free Full Text]

Torp SH (2002) Diagnostic and prognostic role of Ki67 immunostaining in human astrocytomas using four different antibodies. Clin Neuropathol 21: 252–257.[Medline]

Willson TM, Brown PJ, Sternbach DD, and Henke BR (2000) The PPARs: from orphan receptors to drug discovery. J Med Chem 43: 527–550.[CrossRef][Medline]

Zander T, Kraus JA, Grommes C, Schlegel U, Feinstein D, Klockgether T, Landreth G, Koenigsknecht J, and Heneka MT (2002) Induction of apoptosis in human and rat glioma by agonists of the nuclear receptor PPARgamma. J Neurochem 81: 1052–1060.[CrossRef][Medline]

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