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CELLULAR AND MOLECULAR

The Synthetic Ligand of Peroxisome Proliferator-Activated Receptor- Ciglitazone Affects Human Glioblastoma Cell Lines

Department of Pathology, Laboratory of Molecular Pathology (N.S., J.E., J.B., Z.K.) and Laboratory of Experimental Medicine (P.D.), Faculty of Medicine, Palacky University, Olomouc, Czech Republic

Received November 28, 2003; accepted February 26, 2004.

	Abstract

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Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)- can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPAR ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPAR protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G₁ phase and partial block in G₂/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27^Kip1 and p21^Waf1/Cip1. It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPAR ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.

The peroxisome proliferator-activated receptors (PPARs) are a subgroup of ligand-activated transcription factors. They belong to the nuclear receptor family. Other members of this family include steroid and thyroid hormone receptors, retinoid receptors, and vitamin D receptors (Mangelsdorf et al., 1995). Peroxisome proliferators, like fatty acids, modulate tissue-specific responses; e.g., they stimulate the expression of enzymes involved in lipid catabolism, namely the peroxisomal -oxidation system (Heuvel, 1999; Cimini et al., 2000).

The PPAR family comprises three closely related gene products, PPAR, -/, and -. All have a highly conserved structure (Chattopadhyay et al., 2000).

PPAR, like other members of the nuclear receptor super-family, is characterized by three domains: the N-terminal domain (important for functional regulation by phosphorylation), the DNA binding domain (that binds receptor to specific DNA sequences: peroxisome proliferator response elements), and the ligand binding domain in the COOH-terminal region (Ehrmann et al., 2002).

Free PPAR is bound by corepressors that inactivate the transcription function of the receptor. This receptor is activated by a particular ligand into the ligand binding domain, resulting in conformational changes to PPAR, whereas the receptor is released from binding with the corepressor. PPAR then recruits coactivator proteins and RXR to form a heterodimeric complex of nuclear receptors. This complex (PPAR:RXR) binds to peroxisome proliferator response element on DNA (involved in the promoter of different genes) to regulate transcription. PPAR activation plays a role in diverse physiological and pathophysiological events including stimulation of adipocyte differentiation, activation of insulin, regulation of lipid metabolism, inhibition of tumor cell proliferation, and diverse effects on inflammatory processes (Houseknecht et al., 2002).

Endogenous PPAR ligands are polyunsaturated fatty acids and eicosanoids, such as 15-deoxy-delta 12,14-prostaglandin J2 and leukotriene B4. A number of synthetic PPAR ligands have been identified over the past 7 years, of which the most well known are the thiazolidinediones (TZDs) (pioglitazone, ciglitazone, rosiglitazone, etc.). TZDs are a class of antidiabetic agents that improve insulin sensitivity and reduce plasma glucose and blood pressure in patients with type 2 diabetes mellitus (Houseknecht et al., 2002).

Recent studies have shown that ligand activation of PPAR can induce differentiation and inhibit proliferation of prostate (Kubota et al., 1998; Hisatake et al., 2000), breast (Elstner et al., 1998; Mueller et al., 1998), colon (Sarraf et al., 1998), gastric (Takahashi et al., 1999), lung (Tsubouchi et al., 2000), bladder (Guan et al., 1999), thyroid (Ohta et al., 2001), and renal (Inoue et al., 2001) cancer cells, as well as liposarcomas (Tontonoz et al., 1997) in vitro and in vivo (Hisatake et al., 2000; Houseknecht et al., 2002; Han et al., 2003). These observations suggest that PPAR might be useful for cancer therapy. However, the role of PPAR ligands in the cell cycle control of human glioblastoma cells remains unknown.

The aim of our study was to highlight the antidiabetic drug ciglitazone and show that ciglitazone is able to inhibit cell viability and affect the cell cycle and apoptosis in four human glioblastoma cell lines.

	Materials and Methods

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Cell Lines and Culture Condition. The human glioblastoma cell lines, T98G, U-87 MG, A172, and U-118 MG, were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 1.7 mM L-glutamine, penicillin, and streptomycin. T98G and U-87 MG were cultured in humidified atmosphere with 5% CO₂ at 37°C and A172 and U-118 MG with 10% CO₂ at 37°C.

Chemicals. Ciglitazone {(±)-5-[4-(1-methylcyclohexylmethoxy) benzyl]-thiazolidine-2,4-dione} was obtained from Alexis Corporation (Läufelfingen, Switzerland). Ciglitazone was dissolved in dimethyl sulfoxide. The final concentration of vehicle in DMEM was 0.1%. 3,[4,4-dimethylthiazol-2-yl] 2,5 diphenyltetrazolium bromide (MTT) was purchased from Serva (Heidelberg, Germany).

Immunocytochemistry. Immunostaining was undertaken to establish the expression and localization of PPAR using fluorescent microscopy. The cells were seeded on small ring glasses into 60-mm culture dishes and incubated in 5 ml of DMEM with or without ciglitazone (IC₅₀) for 24 h. The dishes were washed in phosphate-buffered saline (PBS), fixed in methanol:acetone (1:1, v/v) for 10 min and stained with polyclonal rabbit primary antibody recognizing human PPAR (1:20; Santa Cruz Biochemicals, Santa Cruz, CA) for 1 h. After washing by PBS, the glasses were incubated for 30 min with secondary antibody anti-rabbit labeled with fluorescein isothiocyanate. Finally, DAPI staining was used for detection of the all cellular nucleus.

In Vitro Growth Rate: Cell Viability Assay. Cell survival was determined using a colorimetric MTT assay as described previously (Carmichael et al., 1987). In brief, assay was performed in quadruple in 96-well plates. Cells were plated out at a density of 2800 to 5000 cells per well. Following attachment for 24 h, the cells were treated for 12, 24, 48, or 72 h in presence or absence with different concentrations (5 x 10^-4-5 x 10^-8 M) of ciglitazone. The concentration leading to 50% inhibition of viability (IC₅₀) after 24 h was determined by measuring MTT reductase activity. The yellow solution, 10 µl of 0.5% MTT, was added to each well and incubated with the cells for 4 h at 37°C. Finally, the established blue crystals were solubilized in 100 µl of 10% SDS, and the absorbance was read at 540 nm using a microplate reader.

Fluorescence-Activated Cell Sorter. Flow cytometry was used to evaluate the number of cells in the particular phases of the cell cycle. Control and treated cells were washed twice with PBS and scraped from the tissue flask in EDTA, centrifuged at 1800 rpm for 10 min at 4°C, washed in cold PBS twice, and fixed with chilled -20°C ethanol (70%; v/v) by low-speed vortexing. For detection of DNA content analysis, we used propidium iodide staining. Finally, cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). All experiments were replicated three times.

Western Blot Analysis. The glioblastoma cell lines were seeded in 100-mm dishes. The cells were treated by IC₅₀ of ciglitazone. This concentration was evaluated by MTT analysis for 24 h (see above). To monitor changes over the 24-h treatment, we collected the cells after both 24 and 12 h.

The cell proteins were extracted with lysis buffer (10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol; 0.5 mM phenylmethylsulphonyl fluoride; 1 mM aprotinin; 1 mM pepstatin; 14 mM leupeptin; 50 mM NaF; 30 mM -glycerol-phosphate; 1 mM NA₃VO₄; and 20 mM p-nitrophenyl phosphate. Cells were incubated on ice for 1 h and vortexed every 10 min. After 30 min of centrifugation at 20,000 rpm, supernatants were collected and kept at -80°C. Protein concentrations were measured using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA) following the manufacturer's instruction. Twenty micrograms of total protein was separated on 10% polyacrylamide gels. Separated proteins were transferred onto nitrocellulose membrane by semidry electrotransfer. Nonspecific binding sites were blocked with blocking buffer (5% fat-free skimmer milk with 0.1% Tween 20 in PBS). Subsequently, the membrane was incubated with the particular primary antibody diluted in the blocking buffer overnight at 4°C. The membrane was then washed with the washing buffer (PBS and 0.1% Tween 20) for 1 h at room temperature. Then, membrane was subsequently incubated with diluted goat anti-mouse IgG-horseradish peroxidase conjugated antibody (1:6000 dilution; Santa Cruz Biochemicals) or goat anti-rabbit IgG-horseradish peroxidase conjugated antibody (1:2000 dilution; DakoCytomation Denmark A/S, Glostrup, Denmark) for 30 min at 4°C. The protein was detected by chemiluminescence reagent-ECL plus (Amersham Biosciences, Vienna, Austria).

The protein expression (after 12 or 24 h) of cell lysates in treated cells was compared with appropriate control (12 and 24 h). As an internal control, mouse monoclonal antibodies anti--tubulin (1:1000; Sigma-Aldrich, St. Louis, MO) were used.

Telomerase Activity. Telomerase activity was analyzed by the TRAPeze kit (Serologicals Corp., Norcross, GA) based on the telomeric repeat amplification protocol method. This kit is a highly sensitive in vitro assay system for detection of telomerase activity.

The concentration of proteins in cell lysates was measured by the Bradford method, and aliquots of the lysate containing 1 µg of protein were loaded for the primer elongation. In that step of the reaction, telomerase adds a number of telomeric repeats (GGTTAG) onto the 3' end of a substrate oligonucleotide. The extended products are amplified by the polymerase chain reaction (PCR) using the substrate oligonucleotide and reverse primer, generating a ladder of products with six base increments starting at 50 nucleotides: 50, 56, 62, 68, etc. Each reaction mixture contains a primers and template for amplification of a 36-bp internal standard that serves as PCR amplification internal control. After separation on 12% polyacrylamide gel, the telomerase ladder was visualized by Sybr Gold and CCD camera DIANA 2 (Raytest, Pittsburgh, PA). The analysis was replicated three times.

TdT-Mediated dUTP Nick Labeling (TUNEL). For detection of apoptotic cells, the TUNEL method was used. Staining was performed according to the protocol (In Situ Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany).

Statistics. The data of the MTT experiments are expressed as means ± S.E. for the four independent experiments (p < 0.05).

	Results

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PPAR in the Glioblastoma Cell Lines. Expression of PPAR protein by Western blot analysis and expression by immunocytochemistry were the first experiments performed in this study. All glioblastoma cell lines expressed PPAR protein, although the cell line U-118 MG expressed the highest level of proteins. The expression of PPAR protein was slightly lower in T98G and U-87 MG, whereas the lowest expression of PPAR protein was noticed in A172 cell lines (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Expression of PPAR. Human glioblastoma cell lines were treated by ciglitazone (IC50) for 24 h (CIG24) and compared with control cells (C24). The cell proteins were analyzed by Western blot analysis. For detection of expression of PPAR, the primary antibody mouse monoclonal anti-PPAR (clone E-8; Santa Cruz Biotechnology, Inc.) was used. The analysis has confirmed that all human glioblastoma cell lines expressed PPAR. In addition, the effect of ciglitazone was analyzed after 12 h of treatment (CIG12) and compared again with control untreated cells (C12). Western blot analysis showed that PPAR expression was unchanged.

These results were confirmed using immunocytochemistry. PPAR was located in the cytoplasm of glioblastoma cell lines (Fig. 2a).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Localization of PPAR following ciglitazone treatment. Glioblastoma cell line U-87 MG was treated by ciglitazone (IC50) for 24 h and compared with control cells. a to d, cells were incubated with primary antibody monoclonal mouse anti-PPAR antibody (clone E-8; Santa Cruz Biotechnology, Inc.) for 2 h. After that, the cells were stained by anti-mouse fluorescein isothiocyanate secondary antibody (a, c, and e) and by DAPI (b, d, and f). DAPI staining was used for detection of all cellular nuclear. a and b, untreated control cells. c and d, cells treated by ciglitazone. e and f, control staining without primary antibody anti-PPAR. Immunocytochemistry staining showed higher positivity of PPAR in treated cells than in control cells. The localization of PPAR was unchanged. The original magnification was 100 x 10.

Ciglitazone Reduced the Cell Viability of Glioblastoma Cells. To evaluate the effect of the PPAR ligand, ciglitazone, on the viability of human glioblastoma cell lines, we analyzed cell viability by MTT analysis. As shown in Fig. 3, ciglitazone inhibited viability in a dose-dependent manner in all four glioblastoma cells. IC₅₀ was reached in T98G in a concentration of 1.8 x 10^-4 M, in U-87 MG, 1.7 x 10^-4 M; in A172, 1.5 x 10^-4 M; and in U-118 MG, 1.5 x 10^-4 M after 24 h. We found that ciglitazone inhibited cell viability after 48 and 72 h as well (Fig. 3).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of ciglitazone on cell viability in four human glioblastoma cell lines. The following concentrations of ciglitazone (1.0 x 10-5, 1.0 x 10-4, 1.2 x 10-4, 1.4 x 10-4, 1.6 x 10-4, 1.8 x 10-4, 2.0 x 10-4, and 2.2 x 10-4 M) were applied to T98G, U-87 MG, A172, and U-118 MG cell lines for 24, 48, and 72 h. The concentration capable of reducing the viability of human glioblastoma cell lines by 50% after 24 h (IC50) was used for following the effect of ciglitazone on cell cycle, apoptosis, differentiation, and telomerase activity on glioblastoma cell lines in all experiments.

Expression of PPAR following Ciglitazone Treatment. Immunocytochemistry showed that after treatment of the cells by ciglitazone, the localization remained unchanged, but we observed stronger expression of PPAR in our cell lines (Fig. 2). Subsequently, we failed to observe any changes of protein expression by Western blot analysis (Fig. 1).

Cell Cycle Arrest. The flow cytometry analysis showed a drop in cell number in S phase and G₂/M phases of the cell cycle in glioblastoma cell lines after treatment by ciglitazone. Ciglitazone caused a decrease in percentage of the cell line T98G in S phase of the cell cycle from 37% in control, untreated cells to 19% in treated cells. A similar decrease of the cells in the S phase of the cell cycle was observed in other cell lines: a reduction in the S phase in U-87 MG from 29% in control cell line to 4% in treated cells, in A172 from 22 to 8% in treated cells, and in U-118 MG from 20 to 9% in treated cells (Fig. 4). These results indicate that ciglitazone suppressed viability of T98G, U-87 MG, A172, and U-118 MG by inducing a block in G₁ phase and partial block in G₂/M phases of the cell cycle. The arrest of the cell cycle in phase G₂/M in U-118 MG is debatable.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Cell cycle distribution of T98G, U-87 MG, A172, and U-118 MG cells after flow cytometry analysis. The cell lines were treated by ciglitazone (IC50) for 24 h, harvested, stained with propidium iodide, and analyzed using the flow cytometer. Histograms of the treated cells were compared with control untreated cells and with cells grown with adequate concentration of vehicle (dimethyl sulfoxide). Horizontal and vertical axes indicate relative nuclear DNA content and number of events (cells), respectively.

The effect of ciglitazone on the spectrum of the cell cycle after treatment by ciglitazone IC₅₀ for 24 h was compared using histograms of the cell cycle for control cells (cells cultured in DMEM) and with histograms of the cell cycle of cells cultured in an appropriate concentration of vehicle in DMEM. The two control histograms were basically equal in all glioblastoma cell lines (Fig. 4).

Expression of Cell Cycle-Related Proteins. Western blot analysis was used to detect changes in expression of cell cycle regulatory proteins. The cell lines differ from each other in basic level of cell cycle related proteins. For example, the cell line U-87 MG had mutated tumor suppressors p53 and PTEN. The expression of GFAP (marker of differentiation for astrocytoma cells) was very low in this line. In contrast, in the cell line U-118 MG, we observed only mutated PTEN tumor suppressor, not p53. T98G had mutated p53 and un-expressed p21^Waf1/Cip1. It is suspected that the T98G cell line had mutation of cell cycle and apoptosis of several pathways. We failed to detect p27^Kip1 in the A172 cell line. In addition, PTEN was completely deleted in A172. U-118 MG failed to express androgen receptor.

Ciglitazone had different effects on the expression of the proteins in each cell line (Fig. 5). After treatment of glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG by ciglitazone, the expression of cyclin D1 was decreased. This effect was observed after 12 and 24 h. The phosphorylation of Rb protein was diminished after 12 and 24 h in all cell lines. The level of total Rb was not markedly reduced as phosphorylation of Rb protein. In addition, the expression of inhibitors of cyclin-dependent kinase, p21^Waf1/Cip1 and p27^Kip1, was increased after 12 and 24 h as well. Apart from A172, we failed to observe the expression of p27^Kip1 after treatment of the cells by ciglitazone. The expression of protein p16^Ink4a was very low. This protein was down-regulated after treatment of U-118 MG cell line by ciglitazone. In another cell line, we were unable to detect any changes in the level of this protein because of its low level. Analysis of the expression of mdm-2 protein revealed the lowest level of this protein in the T98G cell line. Ciglitazone caused an increase in level of mdm-2 protein in U-87 MG and in A172. The level of mdm-2 in T98G and U-118 MG was unchanged. Ciglitazone resulted in a decrease in level cyclin B1 in U-87 MG, A172, and U-118 MG. The expression of cyclin B1 was unaltered in the T98G cell line.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Western blot analysis of p53, mdm-2, p21, p27, p16, cyclin D1, phosphorylated protein Rb, protein Rb, cyclin B, and androgen receptor (AR) in U-87 MG, A172, U-118 MG, and T98G cells treated by ciglitazone. Cells were incubated in medium with ciglitazone (IC50) for 24 h. The expressions of proteins of treated cells (CIG12, ciglitazone, 12 h, IC50; CIG24, ciglitazone, 24 h, IC50) were confronted with the protein expression of control, untreated cells (C12, control, 12 h; C24, control, 24 h). mcm-7 expression was used as a loading marker.

Interestingly, ciglitazone led to reduced expression of androgen receptor in T98G, U-87 MG, and A172. U-118 MG showed no expression of androgen receptor.

Apoptosis. To determine whether ciglitazone was able to induce apoptosis, we focused on small particles in the flow cytometry analysis. The analysis showed that ciglitazone increased the proportion of subG₁ fraction in treated cells in comparison with untreated control cells. The number of apoptotic cells was increased from 8% in the control to about 25% in cells treated with ciglitazone in U-87 MG. Ciglitazone induced elevation of the subG₁ fraction in all four human glioblastoma cell lines (Table 1).

In addition, apoptosis was evaluated using expression of apoptosis-related proteins. The level of pro-caspase 3 remained almost unchanged. We found no activation of procaspase 3. No cleavage of poly ADP/ribose-polymerase was observed. Expression of antiapoptotic protein bcl-2 was significantly decreased in U-118 MG and U-87 MG. Expression of this protein was slightly diminished in A172 as well. In T98G, the level of bcl-2 was unchanged. The apoptotic protein bax was increased after 12 h in T98G (Fig. 6). The expression of PTEN was decreased in U-87 MG and U-118 MG after 24 h. The level of c-myc was increased after both 12 and 24 h in A172 and U-87 MG. The expression of this protein was decreased in U-118 MG cell lines after treatment of the cells by ciglitazone for 24 h. The expression of c-myc was not changed in T98G.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Western blot analysis of bcl-2, bax, bid, caspase-3, poly ADP/ribose-polymerase, c-myc, and PTEN in U-87 MG, A172, U-118 MG, and T98G cells treated by ciglitazone. Cells were incubated in medium with ciglitazone (IC50) for 24 h. We checked the effect of ciglitazone (IC50) on the protein level after 12 h. The protein expressions of treated cells (CIG12, ciglitazone, 12 h, IC50; CIG24, ciglitazone, 24 h, IC50) were compared with the protein expression of control, untreated cells (C12, control, 12 h; C24, control, 24 h). The expression of mcm-7 was used as a loading marker.

TUNEL staining failed to confirm apoptosis. Ciglitazone slightly but not significantly increased the number of TUNEL positive cells (data not shown).

Differentiation. The glioblastoma cell line U-87 MG expressed very low levels of glial fibriall acid protein (GFAP). GFAP is a marker for the differentiation of astrocytoma cells. The level of GFAP was increased after treatment of U-87 MG and A172 for 12 and 24 h. The level of GFAP was decreased after 12 h in U-118 MG.

We were able to observe a slight decrease of vitamin D receptor (VDR) after 12 h in U-87 MG. The drop was much higher after 24 h. In the A172 cell line, the expression of VDR after 12 h was decreased, and after 24 h, it increased. A similar situation was found for U-118 MG. The protein expression of VDR in T98G was unchanged (Fig. 7).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Western blot analysis of GFAP and VDR in U-87 MG, A172, U-118 MG, and T98G cells treated by ciglitazone. Cells were incubated in medium with ciglitazone (IC50) for 24 h. We checked the effect of ciglitazone (IC50) on the protein level after 12 h. The protein expressions of treated cells (CIG12, ciglitazone, 12 h, IC50; CIG24, ciglitazone, 24 h, IC50) were compared with the protein expression of control, untreated cells (C12, control, 12 h; C24, control, 24 h). The expression of mcm-7 was used as a loading marker.

Decrease in Telomerase Activity. Telomerase activity was decreased after treatment of the glioblastoma cell lines U-87 MG and U-118 MG treated by ciglitazone (CIG) for 24 h (IC₅₀) compared with activity of control cells (Fig. 8).

View larger version (165K): [in this window] [in a new window]   Fig. 8. Analysis of telomerase activity. Telomerase activity was analyzed by TRAPeze kit in U-87 MG, U-118 MG, A172, and T98G cells treated by ciglitazone (IC50) for 24 h (IC, internal control for PCR amplification). Telomerase activity in control samples did not change over time. We detected decreased telomerase activity in U-87 MG and U-118 MG treated by CIG for 24 h (IC50) compared with control cell activity (C).

	Discussion

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Novel therapies for the treatment of central nervous system neoplasm are needed. The present study focused on the cell cycle, differentiation, telomerase activity, and apoptosis in human glioblastoma cell lines. Cell lines were characterized using protein expression. Two cell lines (U-87 MG and A172) had mutated p53. Another two (T98G and U-118 MG) had wild-type p53. One cell line (A172) had a completely deleted gene of the tumor suppressor PTEN. One cell line had the intact inhibitor of CDK, p27^Kip1. T98G had high resistance to different drugs (Mohri et al., 2000). In summary, we found a spectrum of signaling pathways that are involved in regulation of the cell cycle, cell survival, and apoptosis in the cells derived from human astrocytoma.

Studies of humans, animals, and cultured cells support the suggestion that the modulation of PPAR activation may have therapeutic use in the future (Bull, 2003). PPARs are versatile and potent regulators of cellular function. They play an important role in rodent hepatocarcinogenesis, inflammation, atherosclerosis development, lipid metabolism, diabetes, and cancer (Everett et al., 2000; Kersten et al., 2000; Miller et al., 2000; Murphy and Holder, 2000; Peters et al., 2000). There are several lines of evidence to show that ligands of PPAR are able to reduce growth of several carcinoma cell lines (Tsubouchi et al., 2000; Yang and Frucht, 2001; Kato et al., 2002; James et al., 2003).

We have shown that ciglitazone is able to reduce viability of four human glioblastoma cell lines in a dose-dependent manner. This is in agreement with the results of James et al. (2003), who showed that ciglitazone and RXR ligand SR11237 inhibited the growth of breast and lung cancer cells. Kim et al. (2003) reported similar results in neuroblastoma cell lines.

Contrary to Kato et al. (2002), we detected expression of PPAR in T98G, U-87 MG, A172, and U-118 MG. However, the localization of PPAR in human glioblastoma cell lines treated by ciglitazone remained unchanged. The greater expression of PPAR that was detected by immunocytochemistry may suggest that the receptor was activated by its own specific ligand.

We have found that ciglitazone affects the cell cycle in the case of glioblastoma cells. It caused a decrease in the expression of cyclin D1 in all human glioblastoma cell lines. Cyclin D1 is responsible for passage of the cell through the G₁ phase of the cell cycle. Cyclin D1, together with cyclin-dependent kinase (Cdk4 and Cdk6), phosphorylates the Rb protein that binds transcriptional factors E2F and blocks their function. Following phosphorylation, the E2Fs are released from their complex with the Rb protein. Transcriptional factors E2F are necessary for entrance of the cell to the S phase of cell cycle. Thus, cyclin D1 governs phase G₁. Cyclin D1 overexpression is required for oncogene-induced tumorigenesis. There is a signal transduction cross talk between PPAR ligands and mitogenic signals that induce cyclin D1. Reduction of cyclin D1 abundance in vivo using antisense transgenic mice increased expression of PPAR in vivo (Wang et al., 2003).

Ciglitazone caused an increase in the cyclin-dependent kinase inhibitors p21^Waf1/Cip1 and p27^Kip1, together with decreased expression of cyclin D1 with consequent decrease in phosphorylation of Rb protein. This leads to arrest in the G₁ of the cell cycle. According to our results from the Western blot analysis together with those from flow cytometry analysis, we presume that ciglitazone blocked the cell cycle of the glioblastoma cell lines in the G₁ phase.

This is supported by the observation of Yang and Frucht (2001), who demonstrated that ciglitazone inhibited growth in colon cancer cells by means of G₁ cell cycle arrest. Takashima et al. (2001) showed that PPAR ligands inhibited growth of esophageal adenocarcinoma cells; thus, this may be due to G₁ arrest and induction of apoptosis. Wakino et al. (2002) confirmed G₁ arrest. They showed that TZDs block events critical for reentry of quiescent vascular smooth muscle cells into cell cycle, significantly the G₀/G₁ to the S phase. In addition, they found that PPAR ligands attenuated the mitogen-induced degradation of p27^Kip1. Fuse et al. (2000) found a relationship between increasing malignancy of human gliomas and lowered level of p27^Kip1. Their results demonstrate that low-grade astrocytomas were p27-positive, whereas p27^Kip1 protein was rarely detected in high-grade astrocytoma. An increasing mortality risk was significantly associated with low level of p27^Kip1 expression, suggesting that a decreased p27^Kip1 level might be a useful prognostic indicator of the clinical behavior of a malignant glioma (Alleyne et al., 1999). In the present study, we demonstrated that ciglitazone is able to induce the level of p27^Kip1 in T98G, U-87 MG, and U-118 MG. This could influence the malignancy of tumors and may have therapeutic benefit. Decreased levels of cyclin B and analysis of cell cycle by flow cytometry revealed the partial block of G₂/M phases of the cell cycle in ciglitazone-treated cells. Arrest of the cell cycle in G₂/M phase in U-118 MG is speculative. We suspect that massive induction of apoptosis in U-118 MG may affect the proportion of the cells in different phases of the cell cycle; therefore, the percentage of the cells did not change in G₂/M phase. We concluded that ciglitazone is able to arrest the cell cycle in G₂/M because of decreased S phase and decrease of level of cyclin B. It seems that the block in phase G₁ is larger than the block in phases G₂/M.

According to Hisatake et al. (2000), the PPAR ligand suppressed expression of androgen receptor, and this caused suppression of androgen-responsive genes in human prostate cancer cell lines. We confirmed that ciglitazone decreased expression of androgen receptor in glioblastoma cell lines as well, although the consequences of this down-regulation are unknown in brain cells as yet.

We showed that ciglitazone inhibited cell viability and caused cell cycle arrest. The crucial question is whether this PPAR ligand is able to stimulate apoptosis. We demonstrated that the expression of pro-apoptotic protein bax was increased after treatment of the cell line T98G by ciglitazone. In this cell line, expression of the antiapoptotic protein bcl-2 was not changed. In contrast, the rest of our cell lines, U-87 MG, A172, and U-118 MG, showed decreased levels of anti-apoptotic protein bcl-2, whereas the level of bax was unchanged. In addition, flow cytometry revealed that the number of the cells in the subG₁ fraction rose in treated cells compared with control cell lines. These results confirm that ciglitazone supports apoptosis despite negative TUNEL staining. The principle of TUNEL staining in detection of later apoptosis changes may explain the TUNEL negative results.

Moreover, we found increased level of the differentiation-related protein GFAP in U-87 MG and A172, and this may be in accord with the suggested effects of ciglitazone in the differentiation process in various tumors (Sarraf et al., 1998; Kubota et al., 1998).

Another aspect of our work was focused on analysis of telomerase activity in human glioblastoma cell lines. Telomerase is RNA-dependent DNA polymerase. It comprises the RNA unit (human telomeric repeat) and the catalytic unit human telomerase reverse transcriptase. Most somatic cells in adults have very low telomerase activity or human telomerase reverse transcriptase transcript. The telomerase is constitutively active in most tumor cells. The rise in telomerase activity is a marker of tumor progression.

Telomerase is fine marker of cancer since it is observed in almost all tumor tissue with a high degree of positivity. Telomerase activity is a useful marker of tumor grade in astrocytoma, where it is impossible to distinguish benign from malignant tumors, e.g., brain tumors, according to morphology.

Telomerase activity has been detected in over 85% of all tumors tested, spanning more than 20 different types of cancers. Therefore, cancer cells are characterized by high telomerase activity. GBM in particular express an active form of telomerase, and this leads to the uncontrolled proliferation characteristic of GBM cells. We showed that all studied cell lines, T98G, U-87 MG, A172, and U-118 MG, possess telomerase active. Our human glioblastoma cell lines U-87 MG and U-118 MG treated by IC₅₀ ciglitazone showed a reduction in telomerase activity. This provides us with new possibilities for control of cell proliferation in the case of glioblastoma cells.

In conclusion, the thiazolidinedione class of drug are powerful PPAR ligands capable of not only modifying the energy metabolism of cells but also of causing arrest of the cell cycle resulting in apoptotic changes in cancer cells' origin GBM. This study reveals a new way for modifying cell viability of brain tumor cells.

The treatment of cancer is undergoing a shift. Traditionally, cancer patients are treated with radiation therapy and cytotoxic agents that aim to have a greater effect on proliferating cancer cells than they do on noncancerous cells. Unfortunately, their effects on GBM patients have been modest, and median survival has remained largely unchanged over the past decade. Major advances in molecular and cellular biology have substantially improved our understanding of the genetic and proteomic changes involved in tumorigenesis. Our finding has potential application to new treatment of cancer patients.

	Acknowledgements

 
We thank Dr. J. Bartek (CCC Danish Cancer Society, Copenhagen, Denmark) and Dr. B. Vojtesek (Masaryk Memorial Cancer Institute, Brno, Czech Republic) for providing some primary antibodies.

	Footnotes

 
This research was supported by Internal Grant Agency of the Ministry of Health of the Czech Republic Grant NC 6726-3/2001.

DOI: 10.1124/jpet.103.063438.

ABBREVIATIONS: GBM, glioblastoma multiforme; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; TZD, thiazolidinedione; DMEM, Dulbecco's modified Eagle's medium; MTT, (3,[4,4-dimethylthiazol-2-yl]2,5 diphenyltetrazolium bromide); PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; PCR, polymerase chain reaction; TUNEL, TdT-mediated dUTP nick labeling; GFAP, glial fibriall acid protein; VDR, vitamin D receptor; CIG, ciglitazone; CDK, cyclin-dependent kinase.

Address correspondence to: Nicol Strakova, Department of Pathology and Laboratory of Molecular Pathology, Faculty of Medicine, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic. E-mail: Nicol.Strakova{at}seznam.cz

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