QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.053876v1 307/2/505    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, E. J. Articles by Hong, J. T. Search for Related Content PubMed PubMed Citation Articles by Kim, E. J. Articles by Hong, J. T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

CELLULAR AND MOLECULAR

Peroxisome Proliferator-Activated Receptor- Activator 15-Deoxy-^12,14-Prostaglandin J₂ Inhibits Neuroblastoma Cell Growth through Induction of Apoptosis: Association with Extracellular Signal-Regulated Kinase Signal Pathway

National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul, Korea (E.J.K., K.S.P., S.Y.C.); College of Pharmacy, Ewha Woman's University, Seoul, Korea (Y.Y.S.); College of Pharmacy, Chungbuk National University, Chungbuk, Korea (D.C.M., Y.S.S., K.S.K., S.S., Y.P.Y., M.K.L., K.W.O., J.T.H.); and Laboratory of Cellular Biology, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea (D.Y.Y.)

Received May 6, 2003; accepted July 29, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptor- (PPAR-) ligands have been demonstrated to inhibit growth of several cancer cells. Here, we investigated whether one of the PPAR- ligands, 15-deoxy-^12,14-prostaglandin J2 (15-deoxy-PGJ₂) inhibits cell growth of two human neuroblastoma cells (SK-N-SH and SK-N-MC) in a PPAR--dependent manner. PPAR- was expressed in these cells, and 15-deoxy-PGJ₂ increased expression, DNA binding activity, and transcriptional activity of PPAR-. 15-Deoxy-PGJ₂ also inhibited cell growth in time- and dose-dependent manners in both cells. Cells were arrested in G₂/M phase after 15-deoxy-PGJ₂ treatment with concomitant increase in the expression of G₂/M phase regulatory protein cyclin B1 but decrease in the expression of cdk2, cdk4, cyclin A, cyclin D1, cyclin E, and cdc25C. Conversely, related to the growth inhibitory effect, 15-deoxy-PGJ₂ increased the induction of apoptosis in a dose-dependent manner. Consistent with the induction of apoptosis, 15-deoxy-PGJ₂ increased the expression of proapoptotic proteins caspase 3, caspase 9, and Bax but down-regulated antiapoptotic protein Bcl-2. 15-Deoxy-PGJ₂ also activated extracellular signal-regulated kinase (ERK) 2. In addition, mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor PD98059 (2'-amino-3'-methoxyflavone) decreased 15-deoxy-PGJ₂-induced ERK2 activation, and expression of PPAR-, capase-3, and cyclin B1. Moreover, MEK1/2 inhibitor PD98059 significantly prevented against the 15-deoxy-PGJ₂-induced cell growth inhibition. We also found that PPAR- antagonist GW9662 (2-chloro-5-nitro-N-phenylbenzamide) reversed the 15-deoxy-PGJ₂-induced cell growth inhibition, PPAR- expression, and activation of ERK2. These results demonstrate that 15-deoxy-PGJ₂ inhibits growth of human neuroblastoma cells via the induction of apoptosis in a PPAR--dependent manner through activation of ERK pathway and suggest that 15-deoxy-PGJ₂ may have promising application as a therapeutic agent for neuroblastoma.

A variety of anticancer drugs have been shown to exert their inhibitory action on cancer cell growth by eliciting cell cycle arrest and apoptosis and by modulating the expression of regulatory proteins (Cullingford et al., 1998; Koniaras et al., 2001; Schwartz and Waxman, 2001). Although inhibitory effect of 15-deoxy-PGJ₂ on cancer cell growth through interference of cell cycle progression and induction of apoptosis have been reported as partial possible mechanisms in many cells, complete molecular mechanisms have been not demonstrated. Therefore, underlying molecular mechanisms representing the 15-deoxy-PGJ₂-induced interferences of cell cycle progression and induction of apoptosis were further studied. It has been known that the MAP kinase family of proteins, known as c-Jun NH₂-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAP kinase signals, play important roles in cell survival and apoptosis. The activation of the JNK and p38 MAP kinase pathways has been implicated in the phosphorylation of a variety of proapoptotic downstream effectors, whereas the ERK MAP kinase pathway is more often associated with cell survival and cell proliferation (Mackeigan et al., 2000; Dai et al., 2001; Yu et al., 2001). However, activation of ERK signal pathway in the apoptosis of neuronal cell has been reported (Blazquez et al., 2000; Seo et al., 2001). The Fas-triggered apoptosis in SHEP, a human neuroblastoma cells (Goillot et al., 1997), and peroxynitrite-induced apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells was reported to be involved in the activation of ERK pathway (Oh-hashi et al., 1999). We therefore further examined involvement of ERK signaling pathway in the 15-deoxy-PGJ₂-caused cell growth inhibition. These results show that 15-deoxy-PGJ₂ inhibits growth of human neuroblastoma cells via the induction of apoptosis in a PPAR--dependent manner through ERK signaling pathway and suggests that 15-deoxy-PGJ₂ may have promising application as a therapeutic agent for neuroblastoma.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Culture and Viability Assay. Human neuroblastoma cells (SK-N-SH, a dopaminergic cell; SK-N-MC, a nondopaminergic cell) were cultured in Dulbecco's modified Eagle's medium and F-12 nutrient supplemented with 10% heat inactivated fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 units/ml) at 37°C under an atmosphere of 5% CO₂ and 95% air. The cells were treated with 15-deoxy-PGJ₂ (0-16 µM). Cell viability was determined by direct cell counting after trypan blue staining or by tetrazolium salt 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and was presented as percentage of control. For the assay, 10 µl of MTT dye was directly added to the cell cultures. After 2 h, media were removed and then the cells were lysed with 100 µl of dimethyl sulfoxide. The absorbance at 540 nm was read with a microplate reader.

Transfection and Assay of Luciferase Activity. Neuroblastoma cells were transiently transfected using LipofectAMINE at a LipofectAMINE/DNA ratio of 3:1, the method described in the specifications of the manufacturer. Cells (4 x 10⁵ cells/60-mm dish) were exposed to a mixture containing 1 µg/dish of expression plasmid pFA-GAL4-PPAR-, which contains human PPAR- (Hwang et al., 2002; from D.Y. Yoon, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea), pFR-Luc (Gal4-UAS-luciferase; from Stratagene, La Jolla, CA), and pSV--galactosidase (Promega, Madison, WI). Transfection mixture was replaced after 5 h with 10% charcoal-stripped fetal serum-containing media (Hyclone Laboratories, Logan, UT). Twenty-four hours after treatment of 15-deoxy-PGJ₂, cells were harvested in 1x luciferase lysis buffer. Relative light units from firefly luciferase activity were determined using a luminometer (MGM Instruments, Hamden, CT) and normalized to the relative light units from Renilla luciferase using the dual luciferase kit (Promega).

Detection of Apoptosis. Apoptotic cells were determined by the morphological changes after 4,6-diamidino-2-phenylindole (DAPI) staining under fluorescence microscopic observation (DAS microscope, 100 or 200x; Leica Microsystems, Inc., Deerfield, IL). For each determination, three separate 100-cell counts were scored. Apoptosis was expressed as a percentage calculated from the number of cells with apoptotic nuclear morphology divided by the total number of cells counted.

DNA Flow Cytometric Analysis. Cells were harvested by trypsin-EDTA release and fixed in ice-cold 70% ethanol. At least 1 to 2 h before flow cytometric analysis, the cells was resuspended in a 1-ml aliquot of modified Vindelov's DNA staining solution (10 µg/ml RNase A and 5 µg/ml propidium iodide in phosphate-buffered saline). Flow cytometric analysis was performed with flow cytometer (EPICS XL, Miami, FL). Cells in the G₁, S, and G₂/M phase of the cell cycle were determined with Modfit LT (Verity House Software, Top-sham, ME).

Western Blotting. Cells were homogenized with lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 1 mM phenylmethylsulfonyl fluoride (PMFS), 10 µl/ml aprotinin, 1% igapel 630 (Sigma-Aldrich, St. Louis, MO), 10 mM NaF, 0.5 mM EDTA, 0.1 mM EGTA, and 0.5% sodium deoxycholate], and centrifuged at 23,000g for 1 h. Equal amount of proteins (50 µg) were separated on a SDS/12% polyacrylamide gel and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Biosciences Inc., Piscataway, NJ). Blots were blocked for 2 h at room temperature with 5% (w/v) nonfat dried milk in Tris-buffered saline [10 mM Tris-HCl (pH 8.0) and 150 mM NaCl] solution containing 0.05% Tween 20. The membrane was then incubated for 3 h at room temperature with specific antibodies at dilutions specified by the manufacturer. Rabbit polyclonal antibodies against PPAR-, ERK2, p38 MAP kinase, and JNK1 (1:500) as well as caspase-3, caspase-9, p53, Bax, Bcl-2 (1:1000), and mouse monoclonal antibodies against their phosphorylated forms (1:500) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used. The blot was then incubated with the corresponding conjugated anti-rabbit immunoglobulin G-horseradish peroxidase (Santa Cruz Biotechnology Inc.). Immunoreactive proteins were detected with the enhanced chemiluminescence Western blotting detection system. The relative density of the protein bands against control -actin band was quantified by densitometry using Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, Rochester, NY).

Gel Mobility Shift Assay. Gel mobility shift assay was done using a slight modification of a previously described method (Jung et al., 2003). Briefly, the cultured cells were washed three times with ice-cold phosphate-buffered saline (pH, 7.6) and pelleted. The pellets were resuspended in 400 µl of cold buffer containing 10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM PMSF, and then centrifuged at 11,000g for 4 min to remove everything except the nuclei. The pellets were resuspended in a second buffer containing 20 mM HEPES, 20% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM PMSF. After centrifugation at 11,000g for 6 min, the supernatant contained the nuclear proteins. The protein level was determined by a microplate modification of the Bradford method (Bio-Rad bulletin 1177; Bio-Rad, Hercules, CA). A double-stranded 23-base pair oligonucleotide probe (5'-GGACCAGGACAAAGGTCACGTTC-3') was synthesized in Bioneer Inc. (Cheongju, Korea) according to the sequence of rat acyl-CoA oxidase for oligonucleotide of PPAR- binding site. A double-stranded oligonucleotide of the NF-B binding site was obtained from Promega. The DNA-protein binding activity against PPAR- and NF-B was assayed by gel mobility shift assay as described elsewhere (Jung et al., 2003). In brief, 10 µg of nuclear protein was incubated in 25-µl total volume of incubation buffer [10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 4% glycerol, and 0.08 mg/ml salmon sperm DNA] at 4°C for 15 min followed by another 2-min incubation with 100 µCi of [-³²P]ATP-labeled oligonucleotide containing PPAR- or NF-B binding site oligonucleotides. The DNA-protein binding complex was run on a 6% nondenatured polyacrylamide gel at 150 V for 2 h. Gels were dried and autoradiographed using Kodak MR film at -80°C overnight.

Immunocytochemical Staining. The cells treated with different dose of 15-deoxy-PGJ₂ were cultured in LabTek chamber slides (Nalge Nunc International, Naperville, IL) and then the cells were fixed with 4.5% glutaraldehyde for 30 min. Immunocytochemical staining was performed with Vectastatin avidin-biotin peroxidase complex kit (Vector Laboratories, Burlingame, CA). The primary antibodies against human PPAR- (2.5 µg/ml; Santa Cruz Biotechnology Inc.) were used. The color of the cells was developed by immersion in a peroxidase substrate solution containing 0.05% diaminobenzidine and 0.01% hydrogen peroxide in 0.05 M Tris (pH 7. 4) for 5 min. Positive staining was indicated as dark green or brownish black deposits.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Expression of PPAR-. To investigate whether the inhibitory effect of 15-deoxy-PGJ₂ on cell growth is dependent of PPAR-, expression of PPAR- was first determined in two neuroblastoma cells. Expression of PPAR- was only detectable in the nontreated cells; however, the expression was gradually increased by 15-deoxy-PGJ₂ treatment in dose- (0-16 µM) and time-dependent (maximum induction was seen at 24 h after treatment) manners in both cells (Fig. 1, A and B). PPAR- immunoreactivity against anti-PPAR- was also more intense in the treated (12 µM 15-deoxy-PGJ₂) cells than in untreated cells. Moreover, 15-deoxy-PGJ₂ caused nuclear translocation of PPAR-, which is localized predominantly in the perinuclear region and cytoplasms in the untreated cells (Fig. 2A). To investigate whether the ability of 15-deoxy-PGJ₂ to actually activate PPAR--dependent transcription in these cells, the functional status of endogenous PPAR- was determined using luciferase reporter assay system. By treatment with 15-deoxy-PGJ₂, concentration-dependent significant increase of PPAR- transcription activity was found in the cells transiently transfected with PPAR- construct, and this increase in transcription activity was inhibited by cotreatment with PPAR- antagonists in SK-N-SH cells (Fig. 2B). However, the basal level of PPAR- transcription activity was not inhibited by treatment with PPAR- antagonists in both cells. Similar response was found in SK-N-MC cells (data not shown). We also performed a gel mobility shift assay to assess whether 15-deoxy-PGJ₂ increased the DNA binding activity of PPAR-. DNA binding activity of PPAR- in untreated cells was slight, but a dose-dependent increase of binding activity was observed in the cells treated with 15-deoxy-PGJ₂ (Fig. 2C, a). This binding activity was inhibited in the presence of cold PPAR- oligonucleotides, but not in unrelated labeled NF-B oligonucleotides in SK-N-SH cells (Fig. 2C, b), and the DNA binding complex was supershifted by anti-PPAR-1 (Fig. 2C, c). Similar extent of the binding activity was also found in the SK-N-MC (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Dose-related expression of PPAR- in SK-N-SH and SK-N-MC cells. Cells were treated with 12 µM 15-deoxy-PGJ2 for different times (A) or treated with various dose of 15-deoxy-PGJ2 for 24 h (B). Whole cell extracts were prepared and PPAR- expression was examined by Western blot analysis as described under Materials and Methods. These data were visualized with enhanced chemiluminescence (a) and densitometry analysis (b). Related density (density of PPAR- band against -actin) is mean ± S.D. of three experiments, with triplicate of each experiment.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Immunoreactive for PPAR- (A) and transcriptional activation determined by luciferase activity (B) and DNA binding activity of PPAR- (C). A, immunoreactive for PPAR- in SK-N-SH cells (a and c) and in SK-N-MC cells (b and d) treated with 12 µM 15-deoxy-PGJ2. Untreated cells (a and b) showed that PPAR- was localized predominantly in the perinuclear region and cytoplasm. Cells treated with 15-deoxy-PGJ2 demonstrated nuclear translocation of PPAR- (c and d). Cells were plated at a density of 2 x 104 on poly-D-lysine-coated chamber slide, and the peroxidase visualized protocol using diaminbenzidine was done according to the Vectastain ABC kit instructions. Magnification, 200x by light photomicroscope. B, transcriptional activation of PPAR-. Luciferase activity was determined in the cells transfected with PPAR- plasmid construct after treatment of agents for 24 h as described under Materials and Methods. Lane 1, control; lane 2, cells treated with 15-deoxy-PGJ2 (4 µM); lane 3, cells treated with15-deoxy-PGJ2 (12 µM); lane 4, cells treated with PPAR- antagonist GW9662 (50 µM); lane 5, cells treated with PPAR- antagonist bisphenol A diglycidyl ester (BADGE, 50 µM); lane 6, cells treated with 15-deoxy-PGJ2 (12 µM) in the presence of GW9662 (50 µM); and lane 7, cells treated with 15-deoxy-PGJ2 (12 µM) in the presence of BADGE (50 µM). Values are mean ± S.D. of three experiments, with triplicate of each experiment. Similar patterns of results were obtained from SK-N-MC cells (data not shown). C, effect of 15-deoxy-PGJ2 on the activation of PPAR-. Cells were treated with various doses of 15-deoxy-PGJ2 for 24 h. DNA binding activity of PPAR- was determined by gel mobility shift assay as described under Materials and Methods. Lane a1, control; lane a2, cells treated with 15-deoxy-PGJ2 (4 µM); lane a3, cells treated with 15-deoxy-PGJ2 (8 µM); lane a4, cells treated with 15-deoxy-PGJ2 (12 µM); and lane a5, cells treated with 15-deoxy-PGJ2 (16 µM). Lane b1, cells treated with 15-deoxy-PGJ2 (12 µM); lane b2, competition assay with unlabeled oligonucleotides of PPRE (50x excess); lane b3, competition assay with unlabeled oligonucleotides of PPRE (100x excess); lane b4, competition assay with unlabeled oligonucleotides of PPRE (200x excess); lane b5, competition assay with unlabeled oligonucleotides of PPRE (400x excess) and lane b6, competition assay with labeled irrelevant oligonucleotides of NF-B (200x). Lane c1, control untreated; lane c2, cells treated with 15-deoxy-PGJ2 (12 µM); and lane c3, supershift band by PPAR-1 (3 µg). Similar patterns of results were obtained from three experiments in the SK-N-SH cells, and in the SK-N-MC cells (data not shown).

Inhibition of Cell Growth and G₂/M Phase Arrest. To evaluate a possible effect of specific PPAR- ligand 15-deoxy-PGJ₂ on the cell growth of human neuroblastoma cells, we analyzed cell viability using MTT assay and direct cell counting. 15-Deoxy-PGJ₂ inhibited neuroblastoma cell growth in dose- (0-16 µM) and time-dependent manners in SK-N-SH cells (Fig. 3A). Similar response to 15-deoxy-PGJ₂ was found in SK-N-MC cells (Fig. 3B). Accordingly, 15-deoxy-PGJ₂ similarly decreased cell number in dose- and time-dependent manners. 15-Deoxy-PGJ₂ also arrested the cell in the G₂/M phase of cell cycle accompanied by a reduction of cells in the G₀/G₁ phase. The percentage of the cells in the G₂/M phase was about 10% in the untreated both cells, but the number was gradually increased up to about 50% by the treatment of 15-deoxy-PGJ₂ (Fig. 4, A and B).

View larger version (61K): [in this window] [in a new window]   Fig. 3. Morphological changes and cell viability of SK-N-SH cells (A) and SK-N-MC cells (B) by 15-deoxy-PGJ2. Morphological changes were observed under microscope (magnification, 200x), and cell viability was determined by MTT assay or by directly counting as described under Materials and Methods. Values are mean ± S.D. of three experiments, with triplicate of each experiment.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Cell cycle analysis of SK-N-SH cells (A) and SK-N-MC cells (B) treated with various doses of 15-deoxy-PGJ2. DNA contents were analyzed by flow cytometry as described under Materials and Methods. a, control; b, 2 µM; c, 4 µM; d, 8 µM; e, 12 µM; and f, 16 µM 15-deoxy-PGJ2 treatment for 24 h. Similar results were obtained from three experiments.

Induction of Apoptosis. To delineate whether the inhibition of cell growth by 15-deoxy-PGJ₂ was due to increase in the induction of apoptosis, we evaluated changes in the chromatin morphology of human neuroblastoma cells using DAPI staining. Consistent with the loss of viability, apoptosis determined after 24-h treatment was also increased in a dose-dependent manner in both cells. The number of apoptotic cells from 2% in the control was increased up to about 50 or 40% in the cells treated with 15-deoxy-PGJ₂ (16 µM) in the SK-N-MC and SK-N-SH cells, respectively (Fig. 5, A and B).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Effects of 15-deoxy-PGJ2 on induction of apoptosis of SK-N-SH cells (A) and SK-N-MC cells (B) as analyzed by DAPI staining. The apoptotic cells were examined by fluorescence microscopy. Treatment of 15-deoxy-PGJ2 for 24 h caused apoptosis characterized by marked chromatin condensations, small membrane-bound bodies (apoptotic bodies), cytoplasmic condensations, and cellular shrinkage. The cell indicated by the arrow is an example of a morphological characteristic of apoptosis (magnification, 100x or 250x). Apoptotic cells were estimated by direct counting of fragmented nuclei after DAPI staining. The value are means ± S.D. of three experiments, with triplicate of each experiment.

The plot of DNA content analyzed by flow cytometry also showed that 15-deoxy-PGJ₂ increased the proportion of the cells in the sub-G₁ phase (apoptotic cells) in a dose-dependent manner (Fig. 4, A and B). The percentage of the cells present in the sub-G₁ phase was 2.2% (control), 6.7% (2 µM), 15.2% (4 µM), 24.7% (8 µM), 40.4% (16 µM), and 69.8% (16 µM) in SK-N-MC cells. The percentage of the cells present in the sub-G₁ phase was 3.2% (control) 5.7% (2 µM), 20.1% (4 µM), 26.5% (8 µM), 43.4% (16 µM), and 54.6% (16 µM) in SK-N-SH cells.

Expression of Cell Cycle and Apoptosis Regulatory Proteins. Expression of cell cycle regulatory proteins was also altered. 15-Deoxy-PGJ₂ increased the expression of G₂/M phase-regulating protein cyclin B1, whereas the expression of other proteins (cdk2, cdk4, and cyclin A and D) regulating G₁ phase was decreased with the similar pattern in the both cells (Fig. 6, A, a and b). Cdc25c protein, which modulates G₂/M phase by control the cyclin B1/cdc2 complex that can enter the cells into G₂/M phase, was decreased. Expression of proapoptotic proteins, Bax, and active form of caspases 3 and 9 was increased in a dose-dependent manner in the both cells treated by 15-deoxy-PGJ₂ for 24 h, whereas the expression of antiapoptotic protein Bcl-2 was decreased in both cells by the treatment of 15-deoxy-PGJ₂ (Fig. 6B).

View larger version (50K): [in this window] [in a new window]   Fig. 6. Expression of cell cycle (A) and apoptosis (B) related molecules in SK-N-SH cells (a) and SK-N-MC cells (b) treated with 15-deoxy-PGJ2 for 24 h. Equal amounts of whole cell lysates 50 µg were subjected to electrophoresis and analyzed by Western blot for cell cycle regulatory molecules (cdk4, cyclin D1, cdk2, cyclin A, cyclin E, cdc25C, and cyclin B1) and apoptosis regulatory molecules (caspase 3, caspase 9, Bax, and Bcl-2). Cell lysates were prepared, and levels of proteins were determined by using specific primary antibody followed by peroxidase-conjugated secondary antibody and visualization by enhanced chemiluminescence detection system. The protein bands were visualized with enhanced chemiluminescence, and relative density was analyzed by densitometry. Similar patterns of protein expression were obtained from three experiments (a). Values are mean ± S.D. of three experiments, with triplicate of each experiment (b).

Activation of ERK Signal Pathway. 15-Deoxy-PGJ₂ dose dependently increased the phosphorylated form of ERK2 (activation of ERK2) but not JNK1 and p38 MAP kinase determined by Western blotting with specific antibodies. Phosphorylated ERK2 expression was persistently increased by 15-deoxy-PGJ₂ dose dependently. No consistent change in the expression of total ERK2, JNK1, and p38 MAP kinase was detected in the SK-N-SH cells (Fig. 7A). Similar pattern and extent of activation of ERK2 were found in the SK-N-MC cells (Fig. 7B).

View larger version (50K): [in this window] [in a new window]   Fig. 7. Effect of 15-deoxy-PGJ2 on expression of MAP kinases in SK-N-SH cells (A) and in SK-N-MC cells (B). Cells were treated for 24 h with various concentrations of 15-deoxy-PGJ2. Equal amounts of whole cell lysate (50 µg) were subjected to electrophoresis and analysis by Western blot for total MAP kinases and phosphorylated MAP kinases. Density of immunoblotting bands of phosphorylated ERK MAP kinase was measured as described under Materials and Methods. The value are means ± S.D. of three experiments, with triplicate of each experiment.

Effect of PD98058 on the ERK2 Activation, Expression of PPAR-, Active Caspase-3, Cyclin B1, and Cell Growth Inhibition Induced by 15-Deoxy-PGJ₂. To determine whether the activation of ERK signal pathway is responsible for leading to the inhibition of cell growth or the induction of apoptosis, we pretreated the specific inhibitor of ERK signal pathway PD98058 30 min before treatment of 15-deoxy-PGJ₂ into the cells and then we assessed the ERK signal pathway activation, expression of PPAR-, and one of the most expressed apoptotic proteins (active caspase-3) and cell cycle-related protein (cyclin B1) induced by 15-deoxy-PGJ₂. As seen in Fig. 8, A and B, PD98058 concomitantly inhibited 15-deoxy-PGJ₂-induced ERK2 activation and expression of PPAR- dose dependently (12.5-50 µM) in both cells. In addition, the enhanced expression of cyclin B1-active caspase-3 was inhibited by PD98058 in a dose-dependent manner in both cells (Fig. 9, A and B). We also assessed the cell growth to investigate whether inhibition of ERK pathway activation prevents 15-deoxy-PGJ₂-induced cell growth inhibition. Correlated well with the inhibitory effect on the ERK2 activation and the expression of PPAR-, PD98058 prevented 15-deoxy-PGJ₂-induced inhibition of cell growth (Fig. 10A) and induction of apoptosis (Fig. 10B).

View larger version (26K): [in this window] [in a new window]   Fig. 8. Effect of PD98059 on the expression of phosphorylated ERK MAP kinase (A) and PPAR- (B) in SK-N-SH cells (a) and SK-N-MC cells (b). Cells were incubated with 15-deoxy-PGJ2 (12 µM) for 24 h, in the presence or absence of PD98059. Equal amounts of whole cell lysates (50 µg) were subjected to Western blot analysis with anti-ERK antibody for phosphorylated ERK MAP kinase (A) or anti-PPAR- (B). The values of the density of each band was obtained from means ± S.D. of three experiments, with triplicate of each experiment.

View larger version (26K): [in this window] [in a new window]   Fig. 9. Effect of inhibitor PD98059 on the 15-deoxy-PGJ2-induced expression of cyclin B1 (A) and caspase-3 (B) in SK-N-SH cells (a) and SK-N-MC cells (b). Cells were incubated with 15-deoxy-PGJ2 (12 µM) for 24 h, in the presence or absence of the inhibitor PD98059. Equal amounts of whole cell lysates (50 µg) were subjected to Western blot analysis with anti-cyclin B1 or active caspase-3 antibodies. The values of the density of each band were obtained from means ± S.D. of three experiments, with triplicate of each experiment.

View larger version (61K): [in this window] [in a new window]   Fig. 10. Effect of PD98059 on the 15-deoxy-PGJ2-induced inhibition of cell growth in SK-N-SH cells and in SK-N-MC cells. Cells were incubated with 15-deoxy-PGJ2 (12 µM) for 24 h, in the presence or absence of the inhibitor PD98059. Inhibitory effect of PD98059 on the 15-deoxy-PGJ2-induced inhibition of cell growth (A). Cell viability was determined by MTT assay as described under Materials and Methods. Values are mean ± S.D. of three experiments, with triplicate of each experiment. Morphological changes were observed as described under Materials and Methods under microscope (magnification, 200x; B).

Inhibition of NF-B Activation. It has been demonstrated that PPAR- negatively regulates nuclear transcription factor NF-B by means of protein-protein interaction. In addition, NF-B is known to be an inhibitory transcription factor of apoptosis. To investigate the hypothesis whether 15-deoxy-PGJ₂-induced translocation of PPAR- into nucleus can inactivate NF-B, and thereby prevent antiapoptotic ability of NF-B causing the cells go into apoptosis, we assessed NF-B activity in the cells treated for different times with 12 µM 15-deoxy-PGJ₂. NF-B was highly activated in these cells; however, the activation of NF-B was gradually decreased by the culture in the presence of 15-deoxy-PGJ₂ in the both cells (Fig. 11, A and B). The NF-B DNA complex in the treated cells was competed out with an unlabeled oligonucleotide but not irrelevant oligonucleotide (SP-1) (Fig. 11C) and was supershifted by anti-p65 antibody, both of which indicates the specificity of the NF-B band (Fig. 11D).

View larger version (42K): [in this window] [in a new window]   Fig. 11. Effect of 15-deoxy-PGJ2 on the DNA binding activity of NF-B in SK-N-SH cells and in SK-N-MC. Nuclear extracts were prepared from neuroblastoma cells, which were incubated with 12 µM 15-deoxy-PGJ2 for different time periods. Gel mobility shift assay was done as described under Materials and Methods. Similar pattern of DNA binding activity was seen from three different sets of experiments. NF-B DNA binding activity in SK-N-SH cells (A) and in SK-N-MC cells (B). Lane 1, 0 h; lane 2, 1 h; lane 3, 3 h; lane 4, 6 h; lane 5, 12 h; and lane 6, 24 h. Competition assay in the presence of unlabeled oligonucleotide or in the presence of labeled irrelevant oligonucleotides (C). Lane 1, cells treated 15-deoxy-PGJ2 (12 µM); lane 2, cells treated 15-deoxy-PGJ2 (12 µM) + unlabeled oligonucleotides of NF-B (200x excess); lane 3, cells treated 15-deoxy-PGJ2 (12 µM) + unlabeled oligonucleotides of NF-B (400x excess); and lane 4, competition assay with labeled irrelevant oligonucleotides of SP-1 (200x). Supershift assay of NF-B DNA binding in the presence of anti-p65 antibody. Lane 1, cells treated 15-deoxy-PGJ2 (12 µM); and lane 2, supershift band (*) by anti-p65 antibody (2 µg).

Effect of GW9662 on 15-Deoxy-PGJ₂-Induced Cell Growth Inhibition, Expression of PPAR-, and ERK2 Activation. To further investigate the dependence of PPAR- in the 15-deoxy-PGJ₂-induced cell growth inhibition and ERK2 activation, we cotreated the cells with PPAR- antagonist GW9662 and 15-deoxy-PGJ₂. 15-Deoxy-PGJ₂ (12 µM)-induced cell growth inhibition was blocked in a dose-dependent manner in both cells (Fig. 12A). The blocking effect of PPAR- antagonist GW9662 on the 15-deoxy-PGJ₂ (12 µM)-induced ERK2 activation (Fig. 12B) and PPAR- expression (Fig. 12C) was also dose-dependent in the SK-N-SH cells. Similar inhibitory effect of GW9662 was found in the SH-N-MC cells (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 12. Effect of GW9662 on the 15-deoxy-PGJ2-induced inhibition of cell growth (A) in SK-N-SH cells (a) and in SK-N-MC cells (b), ERK activation (B), and PPAR- expression (C) in SK-N-SH cells. Cells were incubated with 15-deoxy-PGJ2 (12 µM) in the presence or absence of GW9662 (12.5, 25, and 50 µM) for 72 h (cell growth inhibition assay) or for 24 h (ERK activation and PPAR- expression). Cell viability was determined by MTT assay, and ERK activation and PPAR- expression were determined by Western blotting as described under Materials and Methods. Values are mean ± S.D. of two experiments, with triplicate of each experiment. Lane A1, control; lane A2, cells treated with15-deoxy-PGJ2 (12 µM); lane A3, cells treated with GW9662 (50 µM); lane A4, cells treated with 15-deoxy-PGJ2 (12 µM) + GW9662 (12.5 µM); lane A5, cells treated with 15-deoxy-PGJ2 (12 µM) + GW9662 (25 µM); and lane 6, cells treated with 15-deoxy-PGJ2 (12 µM) + GW9662 (50 µM). Lanes B and C1, cells untreated; lanes B and C2, cell treated with 15-deoxy-PGJ2 (12 µM); and lanes B and C3, cells treated with 15-deoxy-PGJ2 (12 µM) + GW9662 (12.5 µM). Lanes B and C4, cells treated with 15-deoxy-PGJ2 (12 µM) + GW9662 (25 µM); and lanes B and C5, cells treated with 15-deoxy-PGJ2 (12 µM) + GW9662 (50 µM).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Inhibition of cancer cell growth via induction of differentiation and apoptosis is an attractive approach to human cancer therapy. PPAR- agonists, including 15-deoxy-PGJ₂ have been shown to inhibit cell growth and potentially induce apoptosis in several carcinoma cell lines such as breast, colon, prostate, pancreatic, and bladder cancer cells (Lee et al., 1995; Kubota et al., 1998; Mueller et al., 1998; Kitamura et al., 1999; Tsubouchi et al., 2000; Eibl et al., 2001; Inoue et al., 2001; Haydon et al., 2002; Yamakawa-Karakida et al., 2002). We attempted to extend this approach to human neuroblastoma cells. In this study, we first determined the expression of PPAR- in these cells because it is still controversial about the relevance of PPAR- in the biological function of PPAR- agonists in the inhibitory effect on cancer cell growth inhibition. We found that 15-deoxy-PGJ₂ increased expression of PPAR- in dose- and time-dependent manners and the treatment of 15-deoxy-PGJ₂ also translocated PPAR- from cytoplasm into nucleus in the neuroblastoma cells SK-N-MC and SK-N-SH. To further demonstrate the functional role of the PPAR- expression in these cells, and to investigate whether biological activity of 15-deoxy-PGJ₂ could be related to PPAR- activation, we determined the activation of PPAR- using a reporter gene assay system and found that 15-deoxy-PGJ₂ caused an significant increase in activation of PPAR- in these cells. We also determined DNA binding activity of PPAR-. The DNA binding activity of nuclear protein prepared from treated cells was increased in a dose-dependent manner. Furthermore, we examined the activity of NF-B because NF-B has been known to counteract with PPAR- by means of protein-protein interaction and found that treatment of 15-deoxy-PGJ₂ decreased NF-B activity exposure time dependently. These data showing the increase of PPAR- expression, and activation of PPAR--dependent transcription and DNA binding activity by treatment with PPAR- agonist 15-deoxy-PGJ₂, suggest that PPAR- is functionally expressed in these cells, and 15-deoxy-PGJ₂ may be a true endogenous ligand for PPAR- in these cells. We next investigated whether similar dose range of 15-deoxy-PGJ₂ to activate PPAR- could inhibit growth of these cells. Correlated well with the activation and expression of PPAR-, 15-deoxy-PGJ₂ induced inhibition cell growth in dose- (same dose required to activate PPAR-) and time-dependent manners. Similar inhibitory effect of 15-deoxy-PGJ₂ on the cancer cell growth and relevance of PPAR- expression in some cancer cells has been demonstrated in several malignancies described above. Although the dose causing cell growth inhibition and activation of PPAR- in these cells (0-16 µM) is similar to the dose used in other studies to inhibit cell growth of other cells, the precise or essential pharmacological dose reaching intracellular in vivo levels remains to be determined for use of this agent clinically. However, these data suggest that 15-deoxy-PGJ₂ may be a potential therapeutic agent in these malignancies.

Mechanisms by which 15-deoxy-PGJ₂ induced inhibition of cell growth are unknown. The present study therefore also aimed at investigation of the mechanisms of cell growth inhibition. We first investigated which point in the cell cycle is affected by15-deoxy-PGJ₂. Treatment with 12 µM 15-deoxy-PGJ₂ resulted in a significant cell cycle arrest in the G₂/M phase in the both cells. This observation is similar to that observed in the response to 15-deoxy-PGJ₂ treatment in JEG3 choriocarcinoma cells (Keelan et al., 1999). This G₂/M phase growth arrest was accompanied by a reduction of cells in the G₁ phase. The fluorescence-activated cell sorting analysis also showed the increase of proportion of cells arrested in the sub-G₁ phase of cell cycle by the treatment of 15-deoxy-PGJ₂, confirming that cells have undergone apoptosis. To further investigate the molecular mechanism underlying G₂/M phase arrest, we determined the levels of the regulatory proteins required for G₂/M restriction in the cell cycle. 15-Deoxy-PGJ₂ caused a dose-dependent increased level of cyclin B1 protein that has been known to be increased as cells enter into G₂/M phase (Vincent et al., 1997). Transition from G₂ into the M phase is controlled through the coordinated action by the protein cdc2 associated with cyclin B1 (Fletcher et al., 2002). However, the protein level of cdc2 and its phosphorylation were not changed by the treatment of 15-deoxy-PGJ₂ (data not shown). Because cdc25c protein inhibits the activity of cdc2/cyclin B1 complex, the level of cdc25c protein level was also determined. In agreement with the expectation, this protein level was decreased dose dependently. These alternations of the level of the proteins regulating G₂/M phase were accompanied with a reduction in the level of proteins (cyclin A and E, and cyclin-dependent kinases 2 and 4) regulating G₁ phase. This evidence, therefore, suggests that G₂/M phase arrest by the treatment of 15-deoxy-PGJ₂ seems to be involved in up-regulation of cyclin B1 accompanied by down-regulation of cdc25c protein, and this effect may at least be a possible mechanism of cell growth inhibition by 15-deoxy-PGJ₂.

We were also interested in investigating whether 15-deoxy-PGJ₂ treatment resulted in the induction of apoptosis. 15-Deoxy-PGJ₂ treatment for 24 h in the both cells resulted in a dose-dependent increase of apoptosis. It was also found that consistent with the increase of the induction of apoptosis, the expression of apoptotic proteins active caspase 3 and caspase 9, and Bax was dose dependently increased but that antiapoptotic protein Bcl-2 and transcription factor NF-B were decreased. Similar effects of PPAR- agonists, including 15-deoxy-PGJ₂ to induce apoptosis were found in the many carcinoma cell lines with several different mechanisms. In this study, active caspase-3 expression was markedly increased among the proteins in both cells, but caspase-9 and Bax expression was different between two cells. Cell-specific and stimuli-specific involvement of cell death signal (especially caspase signal) have been demonstrated in neuroblastoma (Shankar et al., 2001; Andoh et al., 2002; Racke et al., 2002); therefore, study to demonstrate differential or specific activation of caspase signaling in the 15-deoxy-PGJ₂-induced apoptosis of these cells remains to be conducted. These data suggest that 15-deoxy-PGJ₂ induced apoptosis of these cells and the alternation of the expression of apoptosis regulatory protein (active caspase-3), resulting in a shift the cells favoring apoptosis.

The mitogen-activated protein kinases (MAPKs) have been implicated in the control of cell growth and apoptosis. Among them, the role of ERK in cell death is somewhat controversial, but it may be responsible for the signal-inducing apoptosis (Goillot et al., 1997; Mohr et al., 1998). Recent data showed that ERK activation-mediated anticancer drug Zn²⁺, peroxinitrate, and ceramide induced apoptosis in neuroblastoma SH-SY5Y cells, dopaminergic PC 12 cells, and astrocytes, respectively (Blazquez et al., 2000; Seo et al., 2001; Kim et al., 2002). We therefore next examined the expression of proteins in the MAPK pathway to assess which signal pathway relays the cell growth inhibition and/or induction of apoptosis signals. The expression of the proteins of MAPK family (p38, ERK, and JNK proteins) was not changed by the treatment of 15-deoxy-PGJ₂ in these cells. However, the expression of phosphorylated form of ERK2 was increased in a dose-dependent manner in both cells, suggesting that ERK2 pathway is activated by 15-deoxy-PGJ₂. In addition, pretreatment with MEK1/2 inhibitor PD98059 in the cells resulted in reduced activation of ERK2. This inhibition correlated well with the reduction of 15-deoxy-PGJ₂-induced cell growth inhibition dose dependently. Moreover, PD98059 pretreatment also inhibited 15-deoxy-PGJ₂-induced increase in the expression of cyclin B1 and caspase-3 as well as PPAR- in a dose-dependent manner. These cause-effect results suggest that ERK2 signal may be the most involved in 15-deoxy-PGJ₂-induced inhibitory effect on the neuroblastoma cell growth and induction of apoptosis. The blocking effect of PPAR- antagonist GW9662 on the15-deoxy-PGJ₂-induced cell growth inhibition, ERK2 activation, and PPAR- expression further demonstrate the relevance of PPAR- in the 15-deoxy-PGJ₂-induced inhibitory effect of cell growth.

In summary, we showed for the first time that PPAR- is expressed in the human neuroblastoma cells SK-N-MC and SK-N-SH, and PPAR- agonist 15-deoxy-PGJ₂ increased its expression and transcriptional activation. 15-Deoxy-PGJ₂ also induced cell growth inhibition through increase in the induction of apoptosis in a dose-dependent manner. The inhibitory effect of 15-deoxy-PGJ₂ seems to be dependent on PPAR- through ERK signal pathway. These data suggest the possibility that specific PPAR- agonist can be a candidate as a preventive or a therapeutic agent for neuroblastoma.

	Footnotes

 
DOI: 10.1124/jpet.103.053876.

This work was supported by the Korean Research Foundation Grant KRF-2001-005-E2200

ABBREVIATIONS: PPAR, peroxisome proliferator activated receptor; 15-deoxy-PGJ₂, 15-deoxy-^12,14-prostaglandin J₂; MAP, mitogen-activated protein; JNK, c-Jun NH₂-terminal kinase; ERK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; DAPI, 4,6-diamidino-2-phenylindole; NF-B, nuclear factor-B; MAPK, mitogen-activated protein kinase; PD98059, 2'-amino-3'-methoxyflavone.

Address correspondence to: Dr. Jin Tae Hong, College of Pharmacy, Chungbuk National University, 48, Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Korea. E-mail: jinthong{at}cbucc.chungbuk.ac.kr

	References

Top Abstract Materials and Methods Results Discussion References

 

Andoh T, Chock PB, and Chiueh CC (2002) The roles of thioredoxin in protection against oxidative stress-induced apoptosis in SH-SY5Y cells. J Biol Chem 277: 9655-9660.[Abstract/Free Full Text]

Bentires-Alj M, Dejardin E, Viatour P, Lint CV, Froesch B, Reed JC, Merville MP, and Bours V (2001) Inhibition of the NF-B transcription factor increases Bax expression in cancer cell lines. Oncogene 20: 2805-2813.[CrossRef][Medline]

Berger J and Moller DE (2002) The mechanisms of action of PPARs. Annu Rev Med 53: 409-435.[CrossRef][Medline]

Blazquez C, Galve-Roperh I, and Guzman M (2000) De novo-synthesized ceramide signals apoptosis in astrocytes via extracellular signal-regulated kinase. FASEB J 14: 2315-2322.[Abstract/Free Full Text]

Boelsterli UA and Bedoucha M (2002) Toxicological consequences of altered peroxisome proliferator-activated receptor gamma (PPARgamma) expression in the liver: insights from models of obesity and type 2 diabetes. Biochem Pharmacol 63: 1-10.[CrossRef][Medline]

Dai Y, Yu C, Singh V, Tang L, Wang Z, McInistry R, Dent P, and Grant S (2001) Pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase/MAPK cascade interact synergistically with UCN-01 to induce mitochondrial dysfunction and apoptosis in human leukemia cells. Cancer Res 61: 5106-5115.[Abstract/Free Full Text]

Eibl G, Wente MN, Reber HA, and Hinesm OJ (2001) Peroxisome proliferator-activated receptor induces pancreatic cancer cell apoptosis. Biochem Biophys Res Commun 287: 522-529.[CrossRef][Medline]

Fletcher L, Cheng Y, and Muschel RJ (2002) Abolishment of the Tyr-15 inhibitory phosphorylation site on cdc2 reduces the radiation-induced G(2) delay, revealing a potential checkpoint in early mitosis. Cancer Res 62: 241-250.[Abstract/Free Full Text]

Goillot E, Raingeaud J, Ranger A, Tepper RI, Davis RJ, Harlow E, and Sanchez I (1997) Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway. Proc Natl Acad Sci USA 94: 3302-3307.[Abstract/Free Full Text]

Gregory JM and Julie CH (2000) PPAR- agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol Sci 21: 469-474.[CrossRef][Medline]

Han S, Wada RK, and Sidell N (2001b) Differentiation of human neuroblastoma by phenylacetate is mediated by peroxisome proliferator-activated receptor gamma. Cancer Res 61: 3998-4002.[Abstract/Free Full Text]

Han SW, Greene ME, Pitts J, Wada RK, and Novel NS (2001a) Expression and function of peroxisome proliferator-activated receptor gamma (PPAR) in human neuroblastoma cells. Clin Cancer Res 7: 98-104.[Abstract/Free Full Text]

Haydon RC, Zhou L, Feng T, Breyer B, Cheng H, Jiang W, Ishikawa A, Heabody T, Montag A, Simon MA, et al. (2002) Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma. Clin Cancer Res 8: 1288-1294.[Abstract/Free Full Text]

Hwang BY, Lee JH, Nam JB, Kim HS, Hong YS, and Lee JJ (2002) Two new furanoditerpenes from Saururus chinenesis and their effects on the activation of peroxisome proliferator-activated receptor gamma. J Nat Prod 65: 616-617.[CrossRef][Medline]

Jung KM, Park KS, Oh JH, Jung SY, Yang KH, Song YS, Son DJ, Park YH, Yun YP, Lee MK, et al. (2003) Activation of p38 mitogen-activated protein kinase and activator protein-1 during the promotion of neurite extension of PC-12 cells by 15-deoxy-delta12,14-prostaglandin J2. Mol Pharmacol 63: 607-616.[Abstract/Free Full Text]

Keelan JA, Sato TA, Marvin KW, Lander J, Gilmour RS, and Mitchell MD (1999) 15-Deoxy-delta(12,14)-prostaglandin J(2), a ligand for peroxisome proliferator-activated receptor-gamma, induces apoptosis in JEG3 choriocarcinoma cells. Biochem Biophys Res Commun 262: 579-585.[CrossRef][Medline]

Kim SS, Chae HS, Bach JH, Lee MW, Kim KY, Lee WB, Jung YM, Bonventre JV, and Suh YH (2002) P53 mediates ceramide-induced apoptosis in SK-N-SH cells. Oncogene 21: 2020-2028.[CrossRef][Medline]

Kitamura S, Miyazaki Y, Shinomura Y, Kondo S, Kanayama S, and Matsuzawa Y (1999) Peroxisome proliferator-activated receptor gamma induces growth arrest and differentiation markers of human colon cancer cells. Jpn J Cancer Res 90: 75-80.[CrossRef][Medline]

Koniaras K, Cuddihy AR, Christopoulos H, Hogg A, and O'Connell MJ (2001) Inhibition of Chk1-dependent G2 DNA damage checkpoint radiosensitizes p53 mutant human cells. Oncogene 20: 7453-7463.[CrossRef][Medline]

Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, Miyoshi I, and Koeffler HP (1998) Ligand for peroxisome proliferator-activated receptor (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 58: 3344-3352.[Abstract/Free Full Text]

Lee JH, Kim HS, Jeong SY, and Kim IK (1995) Induction of p53 and apoptosis by 12-PGJ2 in human hepatocarcinoma SK-HEP-1 cells. FEBS Lett 368: 348-352.[CrossRef][Medline]

Mackeigan JP, Collins TS, and Ting YPJ (2000) MEK inhibition enhances paclitaxel-induced tumor apoptosis. J Biol Chem 275: 38953-38956.[Abstract/Free Full Text]

Mauricio JR, Samuel LK, Shannon TB, and Mitchell AL (1998) Prostaglandins promote and block adipogenesis through opposing effects on peroxisome proliferator-activated receptor . J Biol Chem 273: 1855-1858.[Abstract/Free Full Text]

Mohr S, McCormick TS, and Lapetina EG (1998) Macrophages resistant to endogenously generated nitric oxide-mediated apoptosis are hypersensitive to exogenously added nitric oxide donors: dichotomous apoptotic response independent of caspase 3 and reversal by the mitogen-activated protein kinase kinase (MEK) inhibitor PD 098059. Proc Natl Acad Sci USA 95: 5045-5050.[Abstract/Free Full Text]

Mueller E, Sarraf P, Tontonoz P, Evans RM, Martin KJ, Zhang M, Fletcher C, Singer S, and Spiegelman BM (1998) Terminal differentiation of human breast cancer through PPAR gamma. Mol Cell 3: 465-701.

Oh-hashi K, Maruyama W, Yi H, Takahashi T, Naoi M, and Isobe K (1999) Mitogen-activated protein kinase pathway mediates peroxynitrite-induced apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells. Biochem Biophys Res Commun 263: 504-509.[CrossRef][Medline]

Racke MM, Mosior M, Kovacevic S, Chang CH, Glasebrook AL, Roehm NW, and Na S (2002) Activation of caspase-3 alone is insufficient for apoptotic morphological changes in human neuroblastoma cells. J Neurochem 80: 1039-1048.[CrossRef][Medline]

Schwartz PS and Waxman DJ (2001) Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells. Mol Pharmacol 60: 1268-1279.[Abstract/Free Full Text]

Seo SR, Chong SA, Lee SI, Sung JY, Ahn YS, Chung KC, and Seo JT (2001) Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells. J Neurochem 78: 600-610.[CrossRef][Medline]

Shankar SL, Mani S, O'Guin KN, Kandimalla ER, Agrawal S, and Shafit-Zagardo B (2001) Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways. J Neurochem 79: 426-436.[CrossRef][Medline]

Tsubouchi Y, Sano H, Kawahito Y, Mukai S, Yamada R, Kohno M, Ichiro K, Hla T, and Kondo M (2000) Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated receptor- agonists through induction of apoptosis. Biochem Biophys Res Commun 270: 400-405.[CrossRef][Medline]

Vincent I, Jicha G, Rosado M, and Dickson DW (1997) Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer's disease brain. J Neurosci 17: 3588-3598.[Abstract/Free Full Text]

Xin X, Yang S, Kowalski J, and Gerritsen ME (1999) Peroxisome proliferator-activated receptor ligands are potent inhibitors of angiogenesis in vitro and in vivo. Biol Chem 274: 9116-9121.[Abstract/Free Full Text]

Yamakawa-Karakida N, Sugita K, Inukai T, Goi K, Nakamura M, Uno K, Sato H, Kagami K, Barker N, and Nakazawa S (2002) Ligand activation of peroxisome proliferator-activated receptor gamma induces apoptosis of leukemia cells by down-regulating the c-myc gene expression via blockade of the Tcf-4 activity. Cell Death Differ 9: 513-526.[CrossRef][Medline]

Yu W, Liao QY, Hantah FM, Sanders BG, and Kline K (2001) Activation of extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR--tocopheryl succinate-induced apoptosis of human breast cancer cells. Cancer Res 61: 6569-6576.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Schwab, V. Reynders, S. Loitsch, Y. M. Shastri, D. Steinhilber, O. Schroder, and J. Stein PPAR{gamma} is involved in mesalazine-mediated induction of apoptosis and inhibition of cell growth in colon cancer cells Carcinogenesis, July 1, 2008; 29(7): 1407 - 1414. [Abstract] [Full Text] [PDF]

				S. Paruchuri, Y. Jiang, C. Feng, S. A. Francis, J. Plutzky, and J. A. Boyce Leukotriene E4 Activates Peroxisome Proliferator-activated Receptor {gamma} and Induces Prostaglandin D2 Generation by Human Mast Cells J. Biol. Chem., June 13, 2008; 283(24): 16477 - 16487. [Abstract] [Full Text] [PDF]

				A. Aiello, G. Pandini, F. Frasca, E. Conte, A. Murabito, A. Sacco, M. Genua, R. Vigneri, and A. Belfiore Peroxisomal Proliferator-Activated Receptor-{gamma} Agonists Induce Partial Reversion of Epithelial-Mesenchymal Transition in Anaplastic Thyroid Cancer Cells Endocrinology, September 1, 2006; 147(9): 4463 - 4475. [Abstract] [Full Text] [PDF]

				J. J. Schlezinger, J. K. Emberley, and D. H. Sherr Activation of Multiple Mitogen-Activated Protein Kinases in Pro/Pre-B Cells by GW7845, a Peroxisome Proliferator-Activated Receptor {gamma} Agonist, and Their Contribution to GW7845-Induced Apoptosis Toxicol. Sci., August 1, 2006; 92(2): 433 - 444. [Abstract] [Full Text] [PDF]

				S. Nakata, T. Yoshida, T. Shiraishi, M. Horinaka, J. Kouhara, M. Wakada, and T. Sakai 15-Deoxy-{Delta}12,14-prostaglandin J2 induces death receptor 5 expression through mRNA stabilization independently of PPAR{gamma} and potentiates TRAIL-induced apoptosis. Mol. Cancer Ther., July 1, 2006; 5(7): 1827 - 1835. [Abstract] [Full Text] [PDF]

				M. A. Peraza, A. D. Burdick, H. E. Marin, F. J. Gonzalez, and J. M. Peters The Toxicology of Ligands for Peroxisome Proliferator-Activated Receptors (PPAR) Toxicol. Sci., April 1, 2006; 90(2): 269 - 295. [Abstract] [Full Text] [PDF]

				S. Han, H. N. Rivera, and J. Roman Peroxisome Proliferator-Activated Receptor-{gamma} Ligands Inhibit {alpha}5 Integrin Gene Transcription in Non-Small Cell Lung Carcinoma Cells Am. J. Respir. Cell Mol. Biol., April 1, 2005; 32(4): 350 - 359. [Abstract] [Full Text] [PDF]

				N. Strakova, J. Ehrmann, P. Dzubak, J. Bouchal, and Z. Kolar The Synthetic Ligand of Peroxisome Proliferator-Activated Receptor-{gamma} Ciglitazone Affects Human Glioblastoma Cell Lines J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1239 - 1247. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.053876v1 307/2/505    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, E. J. Articles by Hong, J. T. Search for Related Content PubMed PubMed Citation Articles by Kim, E. J. Articles by Hong, J. T. Pubmed/NCBI databases Compound via MeSH Substance via MeSH