Materials and methods

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

Materials

Human NB cell lines SH-SY5Y and SK-N-AS (American Type Culture Collection, Manassas, VA, USA) were made available by the laboratory of Dr CJ Thiele. All the reagents for cell cultures were from Sigma Chemical Co. (St Louis, MO, USA). Tissue plastic-ware was from Bibby Sterilin (Staffordshire, UK). Rosiglitazone was purchased from Alexis (San Diego, CA, USA).

Cell proliferation

DNA synthesis in NB cells was evaluated by ³[H]-thymidine incorporation assay. The cells were seeded in 24-well plates and treated with RGZ for 24 and 48 h. During the last 4 h, the cells were pulsed with 1 Ci well^{-1 3}[H]-thymidine (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Finally, the cells were washed with PBS and fixed by cold trichloroacetic acid (5% in distilled water). A 600 l well^-1 portion of extraction buffer (NaOH 0.25 N/SDS 0.1%) was added for 30 min at 37°C for DNA extraction. Successively, after pH neutralisation with 6 N HCl, the samples were analysed by a -counter (Betamatic V, Kontron Instruments Ltd, Bletchley, UK). The PPAR antagonist 2,2-bis[4-(glycidyloxy)phenyl]propane (BADGE, Sigma, Milan, Italy) was used at the concentration of 20 M and it was added to the cultured cells 30 min before RGZ exposure.

For cell proliferation assay, the cells were seeded in six-well plates. After 24 h, the cells were washed with PBS and different doses of RGZ were added in RPMI medium 1% FBS for 24 or 48 h. Then, the cells were detached (trypsin 0.25%/EDTA) and the cell number was determined by a haemocytometer.

Cell viability

Cell viability following exposure to different RGZ doses (0.1–20 M) was determined by MTS assay (Promega Corporation, Madison, WI, USA), as described previously (Benvenuti et al, 2005). Briefly, the cells were seeded in 96-well plates and exposed to RGZ for 48 h. The assay was then performed according to the manufacturer's instructions. The samples were analysed by an ELISA plate reader (Seac-Radim, Moncalieri, Italy) at 490 nm wavelength. The PPAR antagonist BADGE (Sigma) was used at the concentration of 20 M and it was added to the cultured cells 30 min before RGZ exposure.

Cell adhesion assay

The cells were plated in 96-well plates and different doses of RGZ (0.1–20 M) were added for 24 and 48 h. After washing with PBS, 100 l of Bengal Rose stain (0.25% in PBS) was added to each well for 5 min at room temperature (Gamble and Vadas, 1988). After aspiration of the stain and washing with PBS, the stain was released by adding 200 l well^-1 of an ethanol/PBS (1 : 1) solution for 30 min. The samples were analysed by an ELISA plate reader (Seac-Radim) at 570 nm wavelength.

Invasion assay

Cell invasion was evaluated using the Chemicon Cell Invasion Assay (Temecula, CA, USA). The assay is performed in a 24-well tissue culture plate with 12 cell culture inserts and each Invasion Chamber insert contains an 8 m pore size membrane over which a thin layer of extracellular matrix is dried. The cells were suspended in serum-free medium and RGZ (20 M) was added. Successively, after rehydration of the insert, the cells were plated in each Invasion Chamber insert. After 24 h, the noninvading cells on the upper surface of the filter were removed, whereas the invasive cells were stained with the Cell Stain Solution provided with the kit, eluted with 150 l insert^-1 of 10% acetic acid, transferred onto a microplate and analysed by an ELISA plate reader (Seac-Radim) at 540 nm wavelength.

Quantitation of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) mRNA

The measurement of MMP-9 and TIMP-1 mRNA was performed by using a multiplex quantitative real-time RT–PCR method, based on TaqMan technology, as described previously (Cioppi et al, 2004). Standard curves for MMP-9 and TIMP-1 mRNAs consisted of serial 1 : 10 dilutions from 2.5 10⁷ to 2.5 10² fg total RNA from the fibrosarcoma cell line HT1080 (American Type Culture Collection).

ELISA assay for MMP-9

Matrix metalloproteinase-9 was measured in conditioned medium using an ELISA-based commercial kit (Pharmacia, Uppsala, Sweden), following the manufacturer's instructions. Matrix metalloproteinase-9 can be measured in a range of 1–32 ng ml^-1, and the sensitivity of the assay is 0.6 ng ml^-1.

Immunocytochemistry and flow cytometry analysis for cleaved caspase 3

The amount of caspase 3 immunoreactive cells was determined as described previously (Benvenuti et al, 2005). Briefly, the cells were seeded in chamber slides and exposed to RGZ (20 M). After 48 h, the cells were fixed in paraformaldehyde, then incubated with a rabbit polyclonal antibody against human cleaved caspase 3 (Asp 175) (Cell Signalling Technology Inc., Beverly, MA, USA) and subsequently with a biotinylated secondary antibody. The reaction product was visualised by ABC peroxidase-based detection protocol and AEC kit (Vectastain ABC kit, Vector Laboratories Inc., Burlingame, CA, USA). Finally, the cells were counterstained with haematoxylin and apoptotic cells/field were counted in 10 fields ( 40).

Moreover, cleaved caspase 3 was evaluated by flow cytometry, which was performed as described previously (Carloni et al, 1998). Briefly, the cells were seeded onto 100 mm Petri dishes at the density of 3 10⁶ cells and incubated for 24 h in complete medium. Then, RGZ (20 M) was added and the cells were incubated at 37°C in humidified atmosphere (5% CO₂/95% air). After 48 h, the cells were detached (trypsin 0.25%/EDTA) and fixed with 3% paraformaldehyde in distilled H₂O for 10 min at 37°C. After permeabilisation in ice-cold 90% methanol, the cells were incubated with a rabbit polyclonal antibody against cleaved caspase 3 (Asp 175) (Cell Signalling Technology) or with a nonspecific IgG as control at the appropriate dilution (1 : 25) in Incubation Buffer (BSA 0.5% in PBS 1 ). Subsequently, the cells were washed with Incubation Buffer and incubated with a FITC-conjugated goat anti-rabbit secondary antibody (1 : 100) (Southern Biotechnology Associates Inc., Birmingham, AL, USA) for 30 min. FITC green fluorescence was detected at 515–555 nm using a FL-1 detector of a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) equipped with a 15 mW argon-ion laser for excitation. Debris were gated out by establishing a region around the population of interest on the Forward Scatter vs Side Scatter dot plot. For each sample, 10 000 events in the region of interest were recorded at a flow rate of 200–300 cells/s^-1. Data were processed with analysis software LYSYS II (Becton Dickinson).

Transient transfection of NB cells

In order to determine the activity of PPAR in NB cells, the cells were transfected at the density of 4 10⁶ cells (SK-N-AS) and 2 10⁶ cells (SH-SY5Y) in 100-mm dishes with 2 g peroxisome proliferator response element (adipocyte response element)-7₃-tk-luciferase reporter plasmid (containing three copies of the peroxisome proliferator response element from the adipocyte lipid binding protein gene ligated to a herpes simplex thymidine kinase promoter upstream from a luciferase gene), 0.15 g of human PPAR expression plasmid, and 1 g pSV₂CAT (a vector containing Simian virus 40 early promoter and enhancer sequences that drives a chimeric chloramphenicol acetyl transferase gene) as an internal control by calcium phosphate precipitation, as described previously (Ferruzzi et al, 2005). Twenty-four hours after transfection, the cells were treated with RGZ. Twenty-four hours later, the cells were harvested, washed twice with PBS and lysed. Fifty microlitres of cell extract was incubated with luciferase assay reagent based on the original protocol by de Wet et al (1987). The number of relative light units with a 3-s delay and a 30-s incubation was measured using a Sirius1 luminometer (Berthold Detection System, Pforzheim, Germany). Chloramphenicol acetyl transferase activity was measured as described previously (Crabb and Dixon, 1987). The conversion of chloramphenicol to its acetylated products was quantified on a -scanner (Ambis System, San Diego, CA, USA).

Sequence analysis of the PPAR gene

Genomic DNA was extracted from cell cultures using the automated DNA extraction system Biorobot EZ1 Workstation (Qiagen, Crawley, W Sussex, UK). The entire coding region of the PPAR gene and the exon–intron boundaries were amplified by PCR, using primers sequences and PCR conditions as described previously (Costa-Guda et al, 2005). Amplicons were purified using the QIAquick purification kit (Qiagen) and direct sequencing was carried out using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Warrington, Cheshire, UK), according to the manufacturer's instructions. Sequencing reactions were purified using the DyeEx 2.0 Spin kit (Qiagen) and samples were run on an ABI 310 capillary sequencer (Applied Biosystems).

Western blot analysis of PPAR expression

The NB cells were washed and resuspended in lysis buffer. In addition, the human adenocarcinoma cell line PANC-1 and HPAC (American Type Culture Collection) were used as the positive and negative control for PPAR expression, respectively (Galli et al, 2004). After protein measurement (Coomassie kit, Bio-Rad Laboratories, Hercules, CA, USA), 30 g of protein was diluted in 2 Laemmli's reducing sample buffer, incubated at 95°C for 5 min and loaded onto a 10% polyacrylamide-bisacrylamide gel. After SDS–PAGE, proteins were electroblotted into nitrocellulose (Sigma Chemical Co.). Equivalent protein loading was verified by staining parallel gels with Coomassie R. After blocking in 5% skimmed milk for 2 h, nitrocellulose membranes were washed and then immunostained with a rabbit anti-human PPAR antibody (1 : 1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by a secondary anti-mouse IgG antibody (1 : 2000) (New England Biolabs, Beverly, MA, USA). The antibody-reacted proteins were revealed by LumiGLO chemiluminescent reagent and peroxide (New England Biolabs).

PPAR phosphorylation assay

Protein extraction was performed by boiling the cell pellet in 10% glycerol, 2% SDS and 50 mM Tris-HCl (pH 7.5) for 10 min. The lysate was diluted 1 : 10 with RIPA-like buffer containing 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 5 mM EDTA, 25 mM -glycerophosphate, 1 mM Na₃VO₄, 100 M PMSF, supplemented with Complete Protease Inhibitor Cocktail (Roche Applied Science, Milan, Italy). SDS was then added to achieve a final concentration of 1% and immunoprecipitation was performed with a rabbit anti-human PPAR antibody (1 : 100) (Santa Cruz Biotechnology) at 4°C, for 3 h. Immunocomplexes were recovered by incubation with Protein-A Sepharose (Sigma) for additional 16 h at 4°C. The immunocomplexes were then dissociated by boiling for 5 min in Laemmli's buffer, the beads were collected by centrifugation and SDS–PAGE was performed with the supernatant. The protein were electroblotted to nitrocellulose transfer membrane Protean (Whatman, VWR International, Milan, Italy) and were detected by incubating the filter with the anti-PPAR- antibody or with an anti-phosphoserine mouse monoclonal antibody (1 : 400) (clone PSR-45, Sigma) followed by a secondary anti-rabbit IgG (1 : 2000) or anti-mouse IgG (1 : 2000) antibody, respectively (Amersham Biosciences). Detection of the protein bands was performed using the Amersham ECL plus Kit (Amersham Biosciences).

Statistical analysis

All the experiments were carried out in triplicate or esaplicate and were repeated at least three times. Data were expressed as means.e. Statistical differences were analysed using one-way analysis of variance. Significance was adjusted for multiple comparisons of means using Bonferroni's approximation.

Results

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

Effects of RGZ treatment on cell proliferation

The effects of RGZ on ³[H]-thymidine incorporation in the two NB cell lines SK-N-AS and SH-SY5Y are reported in Figure 1. In SK-N-AS (Figure 1A), a significant reduction of DNA synthesis was determined by exposure to RGZ (1–20 M) for 24 and 48 h, whereas in SH-SY5Y (Figure 1B), this effect was observed only after a 48-h exposure.

Figure 1.

(A and B) Effect of a 24- or a 48-h treatment with RGZ on ³[H]thymidine incorporation in SK-N-AS (A) and SH-SY5Y (B) cells. ^*P<0.05 vs control untreated cells (C). (C and D) Effect of a 24- or a 48-h treatment with RGZ on SK-N-AS (C) and SH-SY5Y (D) cell proliferation. ^*P<0.05 vs control untreated cells (C).

Full figure and legend (146K)

With regard to cell counts, a significant antiproliferative effect was observed following a 24- or 48-h treatment with RGZ (10 and 20 M) in SK-N-AS (Figure 1C). In SH-SY5Y, a significant reduction of the cell number was observed only after a 48-h treatment with 20 M RGZ (Figure 1D), although to a lower extent than in SK-N-AS. Only a higher concentration of RGZ (80 M) was able to determine in SH-SY5Y a reduction of the cell number (54.41.3%, means.e. vs untreated cells, considered as 100%) similar to the decrease determined by the same dose of RZG in SK-N-AS (57.91.4%) (data not shown).

Effects of RGZ on cell viability and adhesion

The effect of RGZ on NB cell viability is shown in Figure 2A and B. In SK-N-AS, cell viability was significantly reduced after a 48-h exposure to RGZ (0.1–20 M) and 20 M RGZ determined the maximal inhibition (54%) (Figure 2A). In SH-SY5Y cells, RGZ (0.1–20 M) was also able to reduce cell viability, but the decrease was limited to 13% (20 M RGZ) (Figure 2B).

Figure 2.

(A and B) Role of RGZ on SK-N-AS (A) and SH-SY5Y (B) cell viability, as assessed by MTS assay (see Materials and Methods). ^*P<0.05 vs control untreated cells (C). (C and D) Effect of different concentrations and time of exposure to RGZ (24 or 48 h) on SK-N-AS (C) and SH-SY5Y (D) cell adhesion, as assessed by Bengal Rose assay (see Materials and Methods). ^*P<0.05 vs control untreated cells (C).

Full figure and legend (167K)

Moreover, the effect of RGZ on cell adhesion was determined. Interestingly, in SK-N-AS cells RGZ significantly induced an inhibition of cell adhesion, which was already present after 24 h of incubation at the concentration of 10 and 20 M (Figure 2C). After a 48-h treatment, a significant inhibitory effect was already present at a lower dose (5 M). Conversely, in SH-SY5Y cells no inhibitory effect was observed for all the tested RGZ concentrations, neither after a 24-h nor after a 48-h exposure (Figure 2D).

Effects of the PPAR antagonist BADGE on cell proliferation and viability

In order to determine whether the effects of RGZ on cell proliferation and viability were truly mediated by PPAR, additional experiments were performed in SK-N-AS using the PPAR antagonist BADGE. The exposure to 20 M BADGE did not determine any effect both on ³[H]-thymidine incorporation and on cell viability, thus indicating that at this concentration this compound had no toxic effect. The same concentration of BADGE was able to revert the inhibitory effect of 20 M RGZ on ³[H]-thymidine incorporation (Figure 3A) as well as on cell viability (Figure 3B).

Figure 3.

Role of the PPAR antagonist BADGE (indicated as B) (20 M) in counteracting the effect on ³[H]thymidine incorporation (A) and on cell viability (B) induced by 20 M RGZ in SK-N-AS cells. ^*P<0.05 vs control untreated cells (C).

Full figure and legend (55K)

Effect of RGZ on cell invasiveness and on the expression of MMP-9/TIMP-1

The effect of RGZ (20 M) on the invasiveness ability of NB cells was determined. When SK-N-AS cells were tested, the number of invasive cells markedly decreased after RGZ exposure. A representative example is shown in Figure 4A and B. At variance with SK-N-AS, no effect was observed in SH-SY5Y (not shown).

Figure 4.

Role of RGZ on SK-N-AS cell invasiveness. (A) Untreated control cells. (B) Cells treated with 20 M RGZ for 24 h. The invasive cells on the lower surface of the membrane were stained with crystal violet (see Materials and Methods). (C) Spectrophotometric analysis on extracts from stained cells eluted from the membrane invasion chamber. ^*P<0.05 vs control untreated cells (C).

Full figure and legend (210K)

These results were quantitatively confirmed by spectrophotometric analysis on extracts from stained cells eluted from the membrane invasion chamber (Figure 4C).

Moreover, in both cell lines the effect of different doses of RGZ (1, 10 and 20 M) on the expression of MMP-9 and of its specific tissue inhibitor TIMP-1 was investigated. In untreated SK-N-AS cells, the amount of MMP-9 mRNA, as assessed by real-time RT–PCR, was 12.031.97 10³ fg 25 ng total RNA^-1 (means.e.). Rosiglitazone (10 and 20 M) significantly decreased the level of MMP-9 transcript (Figure 5A). Accordingly, the amount of protein was significantly reduced by RZG in a dose-dependent manner (Figure 5B). In SH-SY5Y cells, no MMP-9 transcript was detectable (data not shown). As per TIMP-1 expression, the amount of mRNA in untreated cells was 6.930.67 10⁷ fg 25 ng total RNA^-1 in SK-N-AS and 6.40.2 10⁶ fg 25 ng total RNA^-1 in SH-SY5Y cells. Rosiglitazone did not determine any significant variation, although a trend towards an increase was observed in SK-N-AS (Figure 5C). Altogether, these results suggest that RGZ effectively counteracts cell invasiveness in SK-N-AS, but not in SH-SY5Y cells.

Figure 5.

Amount of MMP-9 mRNA (A) and protein (B) and of TIMP-1 mRNA (C) in SK-N-AS cells in the absence or in the presence of different concentrations of RGZ. ^*P<0.05 vs control (indicated as C).

Full figure and legend (76K)

Effects of RGZ on apoptosis

In order to determine whether RGZ is also able to induce apoptosis in the two NB cell lines used in this study, the number of cells showing positivity for cleaved caspase 3 was evaluated by immunocytochemistry. In SK-N-AS, the amount of apoptotic cells in untreated samples was 0.440.23% (means.e.), whereas RGZ treatment (20 M) significantly increased the number of caspase 3-positive cells (2.120.24%, P<0.05). In SH-SY5Y, there was no difference in the amount of cleaved caspase 3-positive cells in untreated vs RGZ-treated samples (0.430.13 vs 0.490.11%, respectively, P>0.05). Representative examples are shown in Figure 6. Several immunostained SK-N-AS cells were present after exposure to RGZ (Figure 6B), compared to the absence of staining in untreated cells (Figure 6A). In contrast, only a very few SH-SY5Y cells showed positivity for cleaved caspase 3 either before (Figure 6C) or after (Figure 6D) RGZ treatment. The effect of treatment with RGZ (20 M) on caspase 3 activation in SK-N-AS was confirmed by flow cytometry analysis. After labelling activated caspase 3, treated cells showed a shift to higher values of fluorescence, when compared to control cells (9.73, treated vs 8.28, control, median values of fluorescence histograms). Conversely, no effect was observed in SH-SY5Y (8.51 treated, vs 8.48, control, median values of fluorescence histogram) (data not shown).

Figure 6.

Immunocytochemistry for the detection of cleaved caspase 3-positive cells. (A and C) Control untreated cells (SK-N-AS and SH-SY5Y, respectively). (B and D) RGZ-treated cells (SK-N-AS and SH-SY5Y, respectively). Magnification 40.

Full figure and legend (318K)

PPAR activity in NB cells

Because of the different response to RGZ, PPAR activity was determined in SK-N-AS and SH-SY5Y cells. Peroxisome proliferator-activated receptor activity was investigated by transient transfection assay and PPAR transactivation was monitored by the activity of transfected adipocyte response element-7₃-tk-luciferase reporter cells. In SK-N-AS, RGZ treatment (20 M) induced a near three-fold increase of the reporter activity compared to untreated control cells (Figure 7). Conversely, no effect was observed in SH-SY5Y after RGZ exposure. Transfection of NB cell lines with a human PPAR expression plasmid stimulated the reporter activity about 1.5- and eight-fold over the control in SK-N-AS and SH-SY5Y, respectively. Furthermore, the treatment of PPAR-transfected cells with RGZ caused an additional five- and 17-fold increase of the reporter activity in the two cell lines, respectively.

Figure 7.

PPAR transcriptional activity in control untreated NB cells (C), in cells treated with RGZ (20 M) and in cells transfected with PPAR in the absence or in the presence of RGZ. L/C: peroxisome proliferator response element-n7₃-tk-luciferase reporter activity, normalised for CAT activity. ^*P<0.05 vs C. ^**P<0.05 vs PPAR transfected cells in the absence of RGZ.

Full figure and legend (33K)

PPAR gene sequencing and expression in NB cells

In order to determine whether the different function of PPAR as a transcriptional activator in the two NB cell lines was due to gene mutations, sequence analysis of the entire coding region of the PPAR gene was performed. However, no mutation was found in either SH-SY5Y or SK-N-AS. Similarly, no evident difference in the amount of expression of PPAR protein was observed between the two cell lines, as assessed by Western blot analysis (Figure 8A). Equivalent protein loading was verified by staining parallel gels with Coomassie R, as shown in Figure 8B. Because there is evidence that PPAR phosphorylation reduces the activity of the receptor (Shao and Lazar, 1999), PPAR immunoprecipitation was performed, followed by SDS–PAGE and Western blot analysis using either an anti-PPAR or an anti-phosphoserine monoclonal antibody. Interestingly, the amount of phosphorylated PPAR was found to be markedly lower in SK-N-AS, thus suggesting that the higher PPAR activity in this cell line compared to SH-SY5Y may be due to a different phosphorylation status of the receptor (Figure 8C).

Figure 8.

(A) Western blot analysis of the amount of PPAR in SK-N-AS and SH-SY5Y. PANC-1 and HPAC cells (human pancreatic adenocarcinomas) were used as the positive and negative control, respectively. (B) Coomassie R-stained gel, showing equal protein loading of the same samples indicated in A. (C) Detection of total (anti-PPAR antibody) and phosphorylated (anti-P-Ser antibody) PPAR, after PPAR immunoprecipitation.

Full figure and legend (52K)

Discussion

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

In the present study, the effect of the PPAR ligand RGZ on two NB cell lines, which have a stromal (S) (SK-N-AS) and a neuroblast (N) (SH-SY5Y) phenotype (Ciccarone et al, 1989; Ross et al, 2003; Servidei et al, 2004), was addressed. We first demonstrated that cell proliferation was readily inhibited by RGZ in the S-type cell line, whereas this effect was less evident in the N-type cells. Cell viability was significantly reduced by RGZ in both cell lines, although a much stronger maximal inhibition was observed in SK-N-AS. The effects of RGZ on cell proliferation and viability were readily counteracted by the PPAR antagonist BADGE, thus indicating that PPAR activation has an important role in eliciting these biological effects. Furthermore, the capability of cultured cells to adhere to a solid substrate in response to RGZ was assessed. In fact, cell adhesion assay is an indicator of cell spreading and adhesion molecules play a critical role in promoting the processes leading to tumour invasion and metastasis (Cavallaro and Christofori, 2004). Again, we found that RGZ readily decreased SK-N-AS cells adhesion starting from a 24-h exposure (10 and 20 M), whereas it had no effect on SH-SY5Y cells, even after a 48-h treatment. As an additional feature of malignant behaviour, cell invasiveness was determined. Rosiglitazone effectively reduced the passage of the S-type cells, but not of the N-type cells, through the extracellular matrix-coated membranes. Therefore, altogether these results provide evidence that RGZ acts as an effective inhibitor of cell proliferation, adhesion and invasiveness in SK-N-AS, but not in the SH-SY5Y NB cell line.

It has to be mentioned that the inhibitory effect of RGZ on SK-N-AS cell invasiveness might be partially PPAR-independent. With regard to this point, the activity of MMPs, which promote the invasion of the extracellular matrix by tumoral cells, has been related to the progression of a variety of tumours, including NB (Sugiura et al, 1998; Chantrain et al, 2004). Accordingly, MMPs have been addressed as a target for cancer therapy (Overall and Kleifeld, 2006). RZG has been shown for instance to reduce the expression/activity of MMPs in a PPAR-independent manner in adrenal (Ferruzzi et al, 2005) and pancreatic tumour cells (Farrow et al, 2003; Galli et al, 2004) and to increase the expression of TIMP-1 in breast cancer cells (Liu et al, 2003). In SK-N-AS, but not in SH-SY5Y, RGZ significantly reduced the amount of MMP-9, whereas the expression of the MMP inhibitor TIMP-1 showed a small but not statistically significant increase at higher levels. The demonstration that the proliferation of embryonic stem cells with a null mutation for PPAR could be inhibited by glitazones (Palakurthi et al, 2001), which were also able to differentiate these cells into macrophages (Chawla et al, 2001), further supports the presence of PPAR-independent mechanisms of agonist action. Furthermore, in our study the role of RGZ in inducing apoptosis in NB cells was addressed. Interestingly, very recently apoptosis has been indicated as a surveillance mechanism against metastases in NB (Stupack et al, 2006). In fact, it has been shown that suppression of caspase 8 expression occurs during the development of NB metastases in vivo, whereas reconstitution of caspase 8 expression potentiated apoptosis and counteracted the spreading of tumour cells. Peroxisome proliferator-activated receptor agonists induce apoptosis in different cell types and in most studies it has been shown that caspase 3 plays an important role in mediating apoptotic death (Grommes et al, 2004). We found that RGZ (20 M) increased the amount of cleaved (i.e. activated) caspase 3 only in SK-N-AS cells. However, both immunocytochemistry and flow cytometry experiments did not show a very strong apoptotic effect of RZG, thus suggesting that RGZ does not appear to play a major role in stimulating apoptosis in these NB cells. These results are partially in agreement with a previous report, in which RGZ (up to 50 M) was not able to induce apoptosis in different NB cell lines, although SK-N-AS cells were not evaluated (Servidei et al, 2004).

The different response of SK-N-AS and SH-SY5Y cells to RGZ appeared to be due to the function of PPAR as a transcriptional activator, which was readily demonstrated in SK-N-AS but not in SH-SY5Y, as assessed by transient transfection with a PPAR responsive element. This difference might be due to a mutation of the PPAR gene. No mutation in exons 3 and 5, the biologically important regions for DNA and ligand binding of PPAR, respectively, has been detected previously in the two NB cell lines, which were used in our study (Servidei et al, 2004). A K422Q mutation has been described previously in exon 6 of PPAR in colon cancer cell lines, but it did not alter ligand-induced transactivation (Gupta et al, 2003). Nevertheless, we sequenced the entire coding region of the PPAR gene in SK-N-AS and in SH-SY5Y and no mutation was found. Only when SH-SY5Y cells were transfected with a human PPAR expression plasmid, the response to RGZ was effectively elicited, thus confirming that the lack of response was truly due to the very low transactivation potential of the endogenous PPAR. It has to be mentioned that the phosphorylated status of PPAR is important in determining its transcriptional activity. In fact, there is evidence that PPAR phosphorylation reduces the activity of the receptor (Shao and Lazar, 1999). We performed Western blot analysis of total and phosphorylated PPAR and we found that the amount of the phosphorylated form was markedly lower in SK-N-AS than in SH-SY5Y. This finding indicates that the lower activity of PPAR in the latter NB cell line may be likely due to a different phosphorylation status of the receptor. In addition, it might be hypothesised that PPAR overexpression in transfected SH-SY5Y cells might increase the level of unphosphorylated PPAR beyond a critical threshold, thus eliciting transcriptional activity. Other recent studies have shown variable antineoplastic effects of PPAR agonists in NB cell lines expressing PPAR (Han et al, 2001, Servidei et al, 2004, Valentiner et al, 2005), but the activity of PPAR was addressed only in one study, in which PPAR was found to be functionally active in different cell lines and particularly in SK-N-AS (Servidei et al, 2004). However, the relationship between PPAR transactivation and the response to TZDs was not addressed. Thus, our study extends the work of other authors, because the different response of NB cells to TZDs was related for the first time to differences in PPAR transactivation, which, in turn, appear to be due to a different phosphorylation status of the receptor.

Our original results, if confirmed in different cell models, might also open a window on potential prognostic and therapeutic applications, based on PPAR and PPAR ligands. The presence in the tumour tissue of a transcriptionally active PPAR might for instance be useful to select those patients, for whom TZDs might be beneficial. In fact, although some of the effects of TZDs may be PPAR-independent, as mentioned previously, admittedly most of them are dependent on the presence of a transcriptionally active PPAR. The low toxicity demonstrated by RGZ as an antidiabetic agent (Yki-Järvinen, 2004) and the fact that the inhibitory effects on cell proliferation, adhesion and invasiveness were present in our study at concentrations similar to those achieved in vivo (Cox et al, 2000) are additional important points to be raised in favour of the possible use of this drug in NB.

References

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

1. Benvenuti S, Luciani P, Vannelli GB, Gelmini S, Franceschi E, Serio M, Peri A (2005) Estrogen and selective estrogen receptor modulators exert neuroprotective effects and stimulate the expression of selective Alzheimer's disease indicator-1, a recently discovered antiapoptotic gene, in human neuroblast long-term cell cultures. J Clin Endocrinol Metab 90: 1775–1782 | PubMed | ChemPort |
2. Burstein HJ, Demetri GD, Mueller E, Sarraf P, Spiegelman BM, Winer EP (2003) Use of the peroxisome proliferator-activated receptor (PPAR) gamma ligand troglitazone as treatment for refractory breast cancer: a phase II study. Breast Cancer Res Treat 79: 391–397 | Article | PubMed | ISI | ChemPort |
3. Carloni V, Romanelli RG, Mercurio AM, Pinzani M, Laffi G, Cotrozzi G, Gentilini P (1998) Knockout of alpha6 beta1-integrin expression reverses the transformed phenotype of hepatocarcinoma cells. Gastroenterology 115: 433–442 | Article | PubMed | ChemPort |
4. Cavallaro U, Christofori G (2004) Multitasking in tumor progression: signaling functions of cell adhesion molecules. Ann NY Acad Sci 1014: 58–66 | Article | PubMed | ChemPort |
5. Chantrain CF, Shimada H, Jodele S, Groshen S, Ye W, Shalinsky DR, Werb Z, Coussens LM, DeClerck YA (2004) Stromal matrix metalloproteinase-9 regulates the vascular architecture in neuroblastoma by promoting pericyte recruitment. Cancer Res 64: 1675–1686 | Article | PubMed | ISI | ChemPort |
6. Chawla A, Barak Y, Nagy L, Liao D, Tontonoz P, Evans RM (2001) PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat Med 7: 48–52 | Article | PubMed | ISI | ChemPort |
7. Ciccarone V, Spengler BA, Meyers MB, Biedler JL, Ross RA (1989) Phenotypic diversification in human neuroblastoma cells: expression of distinct neural crest lineages. Cancer Res 49: 219–225 | PubMed | ISI | ChemPort |
8. Cioppi F, Simi L, Luciani P, Petraglia F, Susini T, Cobellis L, Serio M, Maggi M, Peri A (2004) Expression of uteroglobin and matrix metalloproteinase-9 genes in endometrial cancer: relationship to estrogen and progesterone receptor status. Oncol Rep 11: 427–433 | PubMed | ChemPort |
9. Costa-Guda J, Rosen ED, Jensen RT, Chung DC, Arnold A (2005) Mutational analysis of PPARG as a candidate tumour suppressor gene in enteropancreatic endocrine tumours. Clin Endocrinol 62: 603–606 | Article | ChemPort |
10. Cox PJ, Ryan DA, Hollis FJ, Harris AM, Miller AK, Vousden M, Cowley H (2000) Absorption, disposition, and metabolism of rosiglitazone, a potent thiazolidinedione insulin sensitizer, in humans. Drug Metab Dispos 28: 772–780 | PubMed | ChemPort |
11. Crabb DW, Dixon JE (1987) A method for increasing the sensitivity of chloramphenicol acetyltransferase assay in extracts of transfected cultured cells. Anal Biochem 63: 88–92 | Article |
12. de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7: 725–737 | PubMed | ISI | ChemPort |
13. Debrock G, Vanhentenrijk V, Sciot R, Debiec-Rychter M, Oyen R, Van Oosterom A (2003) A phase II trial with rosiglitazone in liposarcoma patients. Br J Cancer 89: 1409–1412 | Article | PubMed | ChemPort |
14. Demetri GD, Fletcher CD, Mueller E, Sarraf P, Naujoks R, Campbell N, Spiegelman BM, Singer S (1999) Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor- ligand troglitazone in patients with liposarcoma. Proc Natl Acad Sci USA 96: 3951–3956 | Article | PubMed | ChemPort |
15. Desvergne B, Wahli W (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20: 649–658 | Article | PubMed | ISI | ChemPort |
16. Farrow B, O'Connor KL, Hashimoto K, Iwamura T, Evers BM (2003) Selective activation of PPARgamma inhibits pancreatic cancer invasion and decreases expression of tissue plasminogen activator. Surgery 134: 206–212 | Article | PubMed |
17. Ferruzzi P, Ceni E, Tarocchi M, Grappone C, Milani S, Galli A, Fiorelli G, Serio M, Mannelli M (2005) Thiazolidinediones inhibit growth and invasiveness of the human adrenocortical cancer cell line H295R. J Clin Endocrinol Metab 90: 1332–1339 | PubMed | ChemPort |
18. Galli A, Ceni E, Crabb DW, Mello T, Salzano R, Grappone C, Milani S, Surrenti E, Surrenti C, Casini A (2004) Antidiabetic thiazolidinediones inhibit invasiveness of pancreatic cancer cells via PPARgamma independent mechanisms. Gut 53: 1688–1697 | Article | PubMed | ISI | ChemPort |
19. Gamble JR, Vadas MA (1988) A new assay for the measurement of the attachment of neutrophils and other cell types to endothelial cells. J Immunol Methods 109: 175–184 | Article | PubMed | ChemPort |
20. Garaventa A, Luksch R, Lo Piccolo MS, Cavadini E, Montaldo PG, Pizzitola MR, Boni L, Ponzoni M, Decensi A, De Bernardi B, Bellani FF, Formelli F (2003) Phase I trial and pharmacokinetics of fenretinide in children with neuroblastoma. Clin Cancer Res 39: 2032–2039
21. Grommes C, Landreth GE, Heneka MT (2004) Antineoplastic effects of peroxisome proliferator-activated receptor agonists. Lancet Oncol 5: 419–429 | Article | PubMed | ChemPort |
22. Gupta RA, Sarraf P, Mueller E, Brockman JA, Prusakiewicz JJ, Eng C, Willson TM, DuBois RN (2003) Peroxisome proliferator-activated receptor-mediated differentiation. J Biol Chem 278: 22669–22677 | Article | PubMed | ChemPort |
23. Han SW, Greene ME, Pitts J, Wada RK, Sidell N (2001) Novel expression and function of peroxisome proliferator-activated receptor gamma (PPAR) in human neuroblastoma cells. Clin Cancer Res 7: 98–104 | PubMed | ISI | ChemPort |
24. Hisatake J, Ikezoe T, Carey M, Holden S, Tomoyasu S, Koeffler HP (2000) Down-regulation of prostate-specific antigen expression by ligands for peroxisome proliferator-activated receptor gamma in human prostate cancer. Cancer Res 60: 5494–5498 | PubMed | ISI | ChemPort |
25. Jiang C, Ting AT, Seed B (1998) PPAR agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 82–86 | Article | PubMed | ISI | ChemPort |
26. Jude-Aubry C, Pernin A, Favez T, Burger AG, Wahli W, Meier CA, Desvergne B (1997) DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. J Biol Chem 272: 25252–25259 | Article | PubMed |
27. Koeffler HP (2003) Peroxisome proliferator-activated receptor and cancers. Clin Cancer Res 9: 1–9 | PubMed | ISI | ChemPort |
28. Kroll TG, Sarraf P, Pecciarini L, Chen CJ, Mueller E, Spiegelman BM, Fletcher JA (2000) PAX8-PPAR1 fusion oncogene in human thyroid carcinoma. Science 289: 1357–1360 | Article | PubMed | ISI | ChemPort |
29. Kulke MH, Demetri GH, Sharpless NE, Ryan DP, Shivdasani R, Clark JS, Spiegelman BM, Kim H, Mayer RJ, Fuchs CS (2002) A phase II study of troglitazone, an activator of the PPARgamma receptor, in patients with chemotherapy-resistant metastatic colorectal cancer. Cancer J 8: 395–399 | PubMed | ISI |
30. Liu H, Zhang C, Fenner MH, Possinger K, Elstner E (2003) PPARgamma ligands and ATRA inhibit the invasion of human breast cancer cells in vitro. Breast Cancer Res Treat 79: 63–74 | Article | PubMed | ChemPort |
31. Marx N, Mach F, Sauty A, Leung JH, Sarafi MN, Ransohoff RM, Libby P, Plutzky J, Luster AD (2000) Peroxisome proliferator-activated receptor- activators inhibit IFN--induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. J Immunol 64: 6503–6508
32. Melino G, Thiele CJ, Knight RA, Piacentini M (1997) Retinoids and the control of growth/death decisions in human neuroblastoma cell lines. J Neurooncol 31: 65–83 | Article | PubMed | ChemPort |
33. Mueller E, Smith M, Sarraf P, Kroll T, Aiyer A, Kaufman DS, Oh W, Demetri G, Figg WD, Zhou XP, Eng C, Spiegelman BM, Kantoff PW (2000) Effects of ligand activation of peroxisome proliferator-activated receptor in human prostate cancer. Proc Natl Acad Sci USA 97: 10990–10995 | Article | PubMed | ChemPort |
34. Nwako JO, Robbins ME (2001) Peroxisome proliferator-activated receptor-gamma expression in human malignant and normal brain, breast and prostate-derived cells. Prostaglandins Leukot Essent Fatty Acids 64: 241–245 | PubMed |
35. Overall CM, Kleifeld O (2006) Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer 94: 941–946 | Article | PubMed | ChemPort |
36. Palakurthi SS, Aktas H, Grubissich LM, Mortensen RM, Halperin JA (2001) Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation. Cancer Res 61: 6213–6218 | PubMed | ISI | ChemPort |
37. Reginato MJ, Bailey ST, Krakow SL, Minami C, Ishii S, Tanaka H, Lazar MA (1998) A potent antidiabetic thiazolidinedione with unique peroxisome proliferator-activated receptor -activating properties. J Biol Chem 273: 32679–32684 | Article | PubMed | ISI | ChemPort |
38. Reynolds CP, Matthay KK, Villablanca JG, Maurer BJ (2003) Retinoid therapy of high-risk neuroblastoma. Cancer Lett 197: 185–192 | Article | PubMed | ISI | ChemPort |
39. Ross RA, Biedler JL, Spengler BA (2003) A role for distinct cell types in determining malignancy in human neuroblastoma cell lines and tumors. Cancer Lett 197: 35–39 | Article | PubMed | ChemPort |
40. Sarraf P, Mueller E, Smith WM, Wright HM, Kum JB, Aaltonen LA, de la Chapelle A, Spiegelman BM, Eng C (1999) Loss-of-function mutations in PPAR associated with human colon cancer. Mol Cell 3: 799–804 | Article | PubMed | ISI | ChemPort |
41. Servidei T, Morosetti R, Ferlini C, Cusano G, Scambia G, Mastrangelo R, Koeffler HP (2004) The cellular response to PPARgamma ligands is related to the phenotype of neuroblastoma cell lines. Oncol Res 14: 345–354 | PubMed | ChemPort |
42. Shao D, Lazar MA (1999) Modulating nuclear receptor function: may the phos be with you. J Clin Invest 12: 1617–1618
43. Shimada H, Joshi VV (1997) Pathology of neuroblastoma. In Principles and Practice of Genitourinary Oncology Raghavan D, Scher HI, Leibel SA, Lange H (eds). pp 1069–1076. New York: Lippincott-Raven Publishers
44. Strakova N, Ehrmann J, Dzubak P, Bouchal J, Kolar Z (2004) The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines. J Pharmacol Exp Ther 309: 1239–1247 | Article | PubMed | ChemPort |
45. Stupack DG, Teitz T, Potter MD, Mikolon D, Houghton PJ, Kidd VJ, Lahti JM, Cheresh DA (2006) Potentiation of neuroblastoma metastasis by loss of caspase-8. Nature 439: 95–99 | Article | PubMed | ISI | ChemPort |
46. Sugiura Y, Shimada H, Seeger RC, Laug WE, DeClerck YA (1998) Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis. Cancer Res 58: 2209–2216 | PubMed | ISI | ChemPort |
47. Valentiner U, Carlsson M, Erttmann R, Hildebrandt H, Schumacher U (2005) Ligands for the peroxisome proliferator-activated receptor- have inhibitory effects on growth of human neuroblastoma cells in vitro. Toxicology 213: 157–168 | Article | PubMed | ChemPort |
48. Voigt A, Zintl F (2003) Effects of retinoic acid on proliferation, apoptosis, citotoxicity migration, and invasion of neuroblastoma cells. Med Pediatr Oncol 40: 205–213 | Article | PubMed | ISI | ChemPort |
49. Yki-Järvinen H (2004) Thiazolidinediones. N Engl J Med 351: 11016–11018

Acknowledgements

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

This manuscript is dedicated to the memory of Deborah C, a young mother and an extraordinary person, who passed away after a courageous struggle against cancer. This study was partially supported by a grant from Ente Cassa di Risparmio di Firenze and from regione Toscana (Tresor project, principal investigator Prof. Mario Serio).