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

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.126193v1 323/2/746    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 Chen, Y.-L. Articles by Harn, H.-J. Search for Related Content PubMed PubMed Citation Articles by Chen, Y.-L. Articles by Harn, H.-J.

CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Activation of Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 via Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Revealed a Isochaihulactone-Triggered Apoptotic Pathway in Human Lung Cancer A549 Cells

Graduate Institute of Biotechnology (Y.-L.C.) and Department of Applied Animal Science (C.-C.L.), National Ilan University, Ilan, Taiwan, Republic of China; Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, Republic of China (P.-C.L.); Institute of Medical Sciences, Buddhist Tzu-Chi University, Hualien, Taiwan, Republic of China (S.-P.C.); School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, Republic of China (N.-M.T.); Division of Thoracic Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China (Y.-L.C.); School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, Republic of China (W.-L.C.); Center for Neuropsychiatry (S.-Z.L.) and Department of Pathology (H.-J.H.), China Medical University and Hospital, Taichung, Taiwan, Republic of China; and Department of Pathology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, Republic of China (H.-J.H.)

Received May 29, 2007; accepted August 21, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The novel lignan isochaihulactone inhibits cell proliferation and is an effective inducer of apoptosis in a variety of carcinoma cell lines. To determine the mechanisms underlying these effects, we examined isochaihulactone-induced changes in gene expression using oligodeoxynucleotide-based microarray screening of a human lung carcinoma cell line, A549. Isochaihulactone-inducible genes included the early growth response gene-1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene (NAG-1). Isochaihulactone increased EGR-1 and then NAG-1 mRNA and protein expression. Pure isochaihulactone induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Isochaihulactone-induced increases in EGR-1 and NAG-1 expression were reduced by the mitogen-activated protein kinase kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059), and this effect was not blocked by the phosphatidylinositol 3-kinase/protein kinase B pathway inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). Inhibition of isochaihulactone-induced NAG-1 expression by EGR-1 small interfering RNA blocked isochaihulactone-induced apoptosis in A549 cells, suggesting that induction of EGR-1 expression decreases survival of A549 cells. RNA interference using double-stranded RNA specific for NAG-1 also inhibited isochaihulactone-induced apoptosis, and cells transfected to increased NAG-1 expression had a greater apoptotic response to isochaihulactone and reduced colony formation efficiency. In addition, treatment of nude mice with isochaihulactone increased the in vivo NAG-1 expression as examined by immunohistochemistry from tumor biopsy. Isochaihulactone treatment increased the luciferase activity of NAG-1 in A549 cells transfected with the NAG-1 promoter construct. This induction increased expression of NAG-1 that was p53-independent and Sp1-dependent. Our findings suggest that NAG-1 expression is up-regulated by isochaihulactone through an ERK-dependent pathway involving the activation of EGR-1.

To identify genes that are involved in isochaihulactone-induced growth arrest and apoptosis, we used oligodeoxynucleotide-based microarray screening. We found that several isochaihulactone-induced genes are early response genes such as early growth response gene-1 (EGR-1) (also known as NGFI-A, Krox24, TIS8, and Zif/268). EGR-1 is a member of the zinc finger family of transcription factors and plays a role in cell growth and differentiation (Krishnaraju et al., 1995; Thiel and Cibelli, 2002). EGR-1 has been reported to be regulated by numerous growth-regulated genes such as c-myc and transforming growth factor- (TGF-), and to inhibit growth (Albo et al., 1994; Elkon et al., 2004). Induction of EGR-1 expression by antitumorigenic compounds is known to involve members of the family of mitogen-activated protein kinases (MAPKs) or phosphatidylinositol-3-kinase (PI3K)-dependent pathways. For example, induction of EGR-1 expression by the peroxisome proliferator-activated receptor- (PPAR) ligand troglitazone occurs by the ERK phosphorylation pathway rather than by the PPAR pathway (Baek et al., 2003, 2004b, 2005). In contrast, PPAR ligands such as 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methane compounds induced EGR-1 through a PI3K-dependent pathway, which in turn activated serum-response elements in the EGR-1 promoter (Baek et al., 2003).

We also found that several isochaihulactone-induced genes are induced by nonsteroidal anti-inflammatory drugs (NSAIDs). NSAID-activated gene-1 (NAG-1) (also known as MIC-1, GDF-15, placental TGF-, and PLAB) was highly induced in our study. NAG-1 is a transforming growth factor--like secreted protein. It was initially characterized as a p53-regulated gene (Baek et al., 2002; Bottone et al., 2002; Wilson et al., 2003). Overexpression of NAG-1 in breast cancer cells both in vitro and in vivo results in growth arrest and apoptosis; similar results were also observed for colon cancer cells (Baek et al., 2004a, 2005; Eling et al., 2006) and for treatment of prostate cancer cells with purified NAG-1, which induced apoptosis (Liu et al., 2003). These findings suggest that NAG-1 is linked to apoptosis and that reduced expression of NAG-1 may enhance tumorigenesis.

It is known that the PPAR-dependent activation of NAG-1 by troglitazone is due to induction of EGR-1, which in turn activates NAG-1 (Rokos and Ledwith, 1997; Baek et al., 2003, 2004b; Yamaguchi et al., 2006). Like troglitazone, the PPAR-active 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes also induce EGR-1, which in turn interacts with proximal (GC-rich) EGR-1 motifs in the NAG-1 promoter (Chintharlapalli et al., 2005, 2006). This reaction represents a pathway for induction of EGR-1 and NAG-1, and these responses contribute to the induction of growth inhibition and apoptosis by antitumoral compounds in cancer cells.

During this investigation, we found that isochaihulactone induced growth inhibition and apoptosis by activating EGR-1 and NAG-1 through an ERK-dependent pathway and did not involve activation of PI3K signaling. NAG-1 induction after isochaihulactone treatment was followed by EGR-1 activation. Also, NAG-1 expression in the xenograft animal model was correlated with inhibition of tumor development. These results distinguish the mechanism of action of isochaihulactone from that of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl) methanes and provide a model for understanding the downstream effectors for isochaihulactone-induced apoptosis in A549 cells. Thus, NAG-1 seems to exert its antitumoral activity in the tumor suppression pathway.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Fraction Purification of Isochaihulactone and Structure Determination. B. scorzonerifolium roots were supplied by Chung-Yuan Co. (Taipei, Taiwan, Republic of China), and the plant was identified by Professor Lin of the National Defense Medicinal Center, where a voucher specimen was deposited (NDMCP 900801). Isochaihulactone (4-benzo[1,3]dioxol-5-ylmethyl-3-(3,4,5-trimethoxyl-benzylidene)-dihydro-furan-2-one) was prepared as described previously (Chen et al., 2006).

Chemicals and Reagents. RPMI 1640 medium, Eagle's minimum essential medium, fetal bovine serum (FBS), penicillin, streptomycin, trypsin/EDTA, and a NuPAGE Bis-Tris Electrophoresis System (precast polyacrylamide minigel) were purchased from Invitrogen (Carlsbad, CA). An RNA isolation kit was purchased from QIAGEN (Valencia, CA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), the PKC inhibitor GF109203X, paclitaxel, and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemical (St. Louis, MO). The ERK1/2 kinase inhibitor PD98059 and the JNK inhibitor SP600125 were purchased from R&D Systems (Minneapolis, MN). The p38 inhibitor SB203580 and the PI3K/AKT inhibitor LY294002 were purchased from Calbiochem (San Diego, CA). EGR-1 rabbit polyclonal antibody (1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). NAG-1/PTGF- (1:1000) and phosphor-GSK-3 (1:1000) rabbit polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Phosphor-AKT (1:1000), phosphor-ERK1/2 (1:2000), ERK1/2(1:1000), phosphor-p38 (1:1000), p38 (1:2000), phosphor-JNK1/2(1:2000), and JNK1/2(1:1000) monoclonal antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Mycoplasma removal reagent was from Dainippon Pharmaceutical Co. (Osaka, Japan). The annexin V-FLOUS Staining Kit was from Roche Molecular Biochemicals (Mannheim, Germany). Polyvinylidene difluoride membranes, a bovine serum albumin protein assay kit, and Western blot chemiluminescence reagent were purchased from Amersham Biosciences (Arlington Heights, IL).

Cell Lines and Culture. A549 human lung adenocarcinoma cells, H460 human lung large cell carcinoma cells, H1299, p53-null and p16-deficient human non-small cell carcinoma cells, and HT29 human colon adenocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA). The HepG2 human hepatoma cell lines were kindly provided by M. J. Chou and C. S. Yang (Hosono et al., 1991). A549, H460, H1299, and HT29 cancer cells were maintained with RPMI 1640 medium containing 10% FBS and 100 ng/ml each of penicillin and streptomycin at 37°C in a humidified atmosphere with 5% CO₂. HepG2 cancer cells were maintained in Eagle's minimum essential medium with 10% FBS and 100 ng/ml each of penicillin and streptomycin at 37°C in a humidified atmosphere with 5% CO₂. All cultures were free of Mycoplasma.

RNA Extraction and Expression Profiling. For microarray experiments, human lung carcinoma A549 cells were treated with 20 µM isochaihulactone for 3 and 48 h. RNA was extracted with TRIzol (Invitrogen) and then purified using RNeasy (QIAGEN), according to the manufacturer's specifications. RNA quality was assessed using either agarose gel electrophoresis or an Agilent Bioanalyzer. A total of 100 ng of total RNA was then amplified using the Affymetrix small sample protocol (GeneChip Eukaryotic Small Sample Target Labeling Technical Note), and 15 µg of cRNA was then hybridized on each U133A GeneChip and scanned according to the manufacturer's instructions (Affymetrix, Santa Clara, CA). Image files were processed using MAS 5.0 to produce CHP files. Images were masked to remove any streaks/smears present, and no scaling of data was performed during analysis. Data were then imported into GeneSpring (version 7.2; Silicon Genetics, Redwood City, CA) and per chip normalization was performed, using the 50.0th percentile of all measurements in that sample. Differential expressions of cDNA were confirmed by RT-PCR analysis.

RT-PCR Analysis. Total RNA from A549 cells was isolated as described above. cDNA was synthesized by reverse transcription of 2 µg of total RNA using oligo(dT)_12–18 and SuperScript II RNA reverse transcriptase (Invitrogen). The cDNA was then used as the template to amplify the corresponding DNA fragments by PCR using two sets of synthetic oligonucleotide primers. DNA amplification was performed by PCR with the Thermocycler 2400 (PerkinElmer Life and Analytical Sciences, Boston, MA) using the following parameters: 35 cycles of denaturing at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min. Primers used for the PCR amplifications are listed in Table 1. The PCR products were separated on 2% agarose gels, stained with ethidium bromide, and visualized using the FluorChem imaging system (Alpha InnoTech, San Leandro, CA), and levels of glyceraldehyde-3-phosphate dehydrogenase were used as the control.

Western Blot Analysis. Approximately 5 x 10⁶ cells were cultured in 100-mm² dishes and then incubated in various concentration of isochaihulactone for the indicated time. The cells were lysed on ice with 200 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM MgCl₂, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 50 µg/ml leupeptin) and centrifuged at 13,000g for 20 min at 4°C. The protein concentrations in the supernatants were quantified using a bovine serum albumin protein assay kit. Electrophoresis was performed on a NuPAGE Bis-Tris Electrophoresis System using 50 µg of reduced protein extract per lane. Resolved proteins were then transferred to polyvinylidene difluoride membranes. Filters were blocked with 5% nonfat milk overnight and probed with an appropriate dilution of primary antibodies for 1 h at room temperature. Membranes were washed three times with 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 1 h at room temperature. All proteins were detected using Western Lightning Chemiluminescence Reagent Plus and quantified using a densitometer.

Growth Inhibition Assay. The viability of the cells after treatment with various chemicals was evaluated using an MTT assay preformed in triplicate. Briefly, the cancer cells (5 x 10³) were incubated in 96-well plates containing 200 µl of serum-containing medium. Cells were permitted to adhere for 12 to 18 h and then were washed with phosphate-buffered saline (PBS). Solutions were always prepared fresh by dissolving 0.2% DMSO or drugs in culture medium and added to A549 cells. For inhibitor treatment experiments, cells were treated with 20 µM isochaihulactone and preincubated for 1 h with 25 and 50 µM ERK1/2 kinase inhibitor PD98059, 10 and 20 µM PI3K inhibitor LY294002, or 10 and 20 µM PKC inhibitor GF109203X. After 48 h of exposure, the drug-containing medium was removed, washed with PBS, and replaced by fresh medium. The cells in each well were then incubated in culture medium with 500 µg/ml MTT for 4 h. After the medium were removed, 200 µl of DMSO and 25 µl of glycine buffer (0.1 M glycine and 0.1 M NaCl, pH 10.5) were added to each well. Absorbance at 570 nm of the maximum was detected by a PowerWave X Microplate ELISA Reader (Bio-Tek Instruments, Winooski, VT). The absorbance for DMSO-treated cells was considered as 100%. The results were determined by three independent experiments.

Detection of Apoptosis. Apoptosis was analyzed according to the method described by van Engeland et al. (1998) to detect the integrity of cellular membrane and the externalization of phosphatidylserine. In brief, approximately 10⁶ cells were grown in 35-mm diameter plates. The cells were treated with various herbal extracts and chemicals according to the experimental design and then were labeled with 10 µg/ml annexin V-FLOUS and 20 µg/ml PI before harvesting. After labeling, the cells were washed with binding buffer and harvested by scraping. Cells were resuspended in binding buffer at a concentration of 2 x 10⁵ cells/ml before analysis by flow cytometry (FACScan). The data were analyzed with WinMDI V2.8 software. The percentage of cells undergoing apoptosis was determined by three independent experiments.

Transfection with siRNA. EGR-1 siRNA targeting the sequence AGA GGC AUA CCA AGA UCC A and NAG-1 siRNA targeting the sequence GAC UCC AGA UUC CGA GAG U were synthesized (Ambion, Austin, TX). Cells at 50 to 60% confluence were transfected using GeneJammer Transfection Reagent according to the manufacturer's protocol (Stratagene, La Jolla, CA) with EGR-1 or NAG-1 siRNA (Ambion) (in various concentrations of 10–50 nM) for 48 h. Medium was removed, and the cells were treated with isochaihulactone or vehicle for up to 48 h. After incubation, RNA was isolated for RT-PCR, protein was isolated for Western blot analysis, and cells were collected for analysis of apoptosis.

In Vitro Transfection. The full-length NAG-1 cDNA containing the entire coding region was isolated by RT-PCR using two primers from the PTGFB sequence (GenBank accession no. AF008303): sense strand, 5'-ACCTGCACAGCCATGCCCGGGCA-3'; and antisense strand, 5'-CAGTGGAAGGACCAGGACTGCTC-3'. To create the pNAG-1-expressing A549 stable cell line, A549 was transfected with 2 µg of pCDNA3.1-NAG-1 or pCDNA3.1-neoplasmids using GeneJammer reagent. After 48 h, the cells were subjected to selection for stable integrants by exposure to 500 µg/ml G418 (Invitrogen) in complete medium containing 10% FBS for 3 weeks to select the transfected cells. The cells were then assessed for overexpression of NAG-1 by Western blot analysis.

Colony Formation Assay. NAG-1-transfected cells were plated onto 10-cm culture dishes with 500 cells/dish in 5 ml of RPMI 1640 in the presence or absence of isochaihulactone for 24 h. The cells were then cultured in fresh medium for 10 to 15 days. When the colonies became visible, they were stained with 2% methyl blue solution to assess the survival rate. Single colonies were selected and grown separately. The results were determined by three independent experiments.

Construction of Plasmids. Human NAG-1 promoter was isolated from volunteer blood using a DNA extraction kit (QIAGEN) according to the manufacturer's protocol. The –499/+70 (pNAG499/+70) and –137/+70 (pNAG137/+70) NAG-1 promoter regions were generated using the following two primers: pNAG499/+70: sense, 5'-CGACGCGTCACCTCTCCAGTGAGAGTCTC-3', and antisense, 5'-GGAAGATCTTGAGAGCCATTCACCGTCC-3'; pNAG137/+70: sense, 5'-CGACGCGTTAAATACACCCCCAGACCCCG-3', and antisense, 5'-GGAAGATCTTGAGAGCCATTCACCGTCC-3'. The amplified product was digested with MluI and BglII restriction enzymes and ligated into pGL3-basic luciferase vector (Promega, Madison, WI) digested with the same enzymes. The Sp1 binding site was mutated using the Quick Change site mutagenesis kit (Stratagene). For the point mutation of Sp1-BC sites on the –137/+70 promoter region, the following primers were used: pNAG137mut: sense, 5'-GGAGTTCGGGACTGAGCATTCGGAGACGGA-3', and antisense, 5'-TCCGTCTCCGAATGCTCAGTCCCGAACTCC-3'. Site-specific mutations were confirmed by DNA sequencing.

Transfection of the Luciferase Reporter System. A549 cells were plated in six-well plates at 2 x 10⁵ cells/well in RPMI 1640 medium supplemented with 10% fetal bovine serum. After growth overnight, plasmid mixtures containing 2 µg of NAG-1 promoter linked to luciferase and 0.2 µg of pRT-null (Promega) were transfected by GeneJammer reagent according to the manufacturer's protocol. After 48 h of transfection, the cells were harvested in 1x luciferase lysis buffer, and luciferase activity was determined and normalized to the pRL-TK luciferase activity with a dual luciferase assay kit (Promega). For isochaihulactone treatment, the cells were treated with the drug in the absence of serum for 24 h and then assayed for luciferase activity. Cells were treated with 29 µM sulindac sulfide for 24 h as a positive control. The results were determined by three independent experiments.

Antitumor Activity in Vivo. Xenograft mice were used as a model system to study the cytotoxicity effect of isochaihulactone in vivo; implantation of cancer cells was performed similarly to previous reports. Female congenital athymic BALB/c nude (nu/nu) mice were purchased from the National Sciences Council (Taipei, Taiwan), and all procedures were performed in compliance with the standard operating procedures of the Laboratory Animal Center of Tzu Chi University (Hualien, Taiwan). All experiments were carried out using 6- to 8-week-old mice weighing 18 to 22 g. The mice were implanted with 1 x 10⁷ cells s.c. into their backs. When the tumors reached 80 to 120 mm³ in volume, animals were divided randomly into control and test groups consisting of six mice per group (day 0). Daily s.c. administration of isochaihulactone, dissolved in a vehicle of 20% Tween 80 in normal saline (v/v), was performed from days 0 to 4, far from the inoculated tumor sites (>1.5 cm). The control group was treated with vehicle only. The mice were weighed three times a week up to days 21 to 28 to monitor effects and at the same time the tumor volume was determined by measurement of the length (L) and width (W) of the tumor. The tumor volume at day n (TVn) was calculated as TV (cubic millimeters) = (L x W²)/². The relative tumor volume at day n (RTVn) versus that at day 0 was expressed according to the following formula: RTVn = TVn/TV0. Tumor regression [T/C (percent)] in treated versus control mice was calculated using T/C (percent) = (mean RTV of treated group)/(mean RTV of control group) x 100. Xenograft tumors as well as other vital organs of treated and control mice were harvested and fixed in 4% formalin, embedded in paraffin, and cut in 4-mm sections for histologic study.

Immunohistochemical Staining. All tumor tissues (s.c. A549 tumors with or without isochaihulactone treatment) were fixed in 4% formalin at 4°C for 16 h and then embedded in paraffin. Paraffin sections (5 µm) were deparaffinized in xylene and rehydrated through a graded series of ethanol solutions. The sections were incubated with blocking solution (5% milk powder and 1% bovine serum albumin in phosphate-buffered saline) for 60 min at room temperature, followed by a 4°C overnight incubation with anti-NAG-1/PTGF- rabbit polyclonal antibody in blocking solution. Subsequently, the immune complexes were visualized using horseradish peroxidase-conjugated anti-goat IgG secondary antibodies (1:1000 dilution; Santa Cruz Biotechnology Inc.) and the LSAB2 system (Dako North America Inc., Carpinteria, CA), respectively, and then incubated for 10 min with 0.5 mg/ml diaminobenzidine and 0.03% (v/v) H₂O₂ in PBS. Finally, sections were counterstained with hematoxylin, mounted, observed under a light microscope at magnifications of 400x, and photographed.

Statistical Analysis. The data are shown as mean ± S.D.. The statistical difference was analyzed using the Student's t test for normal distributed values and the nonparametric Mann-Whitney U test for values of non-normal distribution. Values of P < 0.05 were considered significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Microarrays. To obtain insight into the mechanism by which isochaihulactone induces growth inhibition and apoptosis, we used oligodeoxynucleotide-based microarray screening to identify isochaihulactone-induced changes in gene expression. Human non-small cell lung carcinoma A549 cells were treated with 20 µM isochaihulactone. After 3- and 48-h incubations, RNA was extracted and used in microarray screening. Primers were designed for different genes. The sequence of primers is shown in Table 1. Among the genes, EGR-1, NAG-1, NRG-1, FGF-2, FEM-1, and Cullin 5 expression were up-regulated time dependently. This result was also confirmed by RT-PCR (Fig. 1). EGR-1 and NAG-1, two genes implicated in growth inhibition and apoptosis (Baek et al., 2004, 2005; Chintharlapalli et al., 2005; Lim et al., 2007), were highly induced by drug treatment. We investigated these effects further.

View larger version (69K): [in this window] [in a new window]   Fig. 1. Isochaihulactone induces EGR-1, NAG-1, NRG-1, FGF-2, FEM-1, and Cullin 5 transcription in A549 cells. Human non-small lung carcinoma A549 cells were treated with 20 µM isochaihulactone for various durations from 0 to 48 h, and total RNAs were isolated. RT-PCR analysis was performed using the respective primers.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Isochaihulactone induces NAG-1 in human cancer cells. A, induction of EGR-1 and NAG-1 protein expression in A549 cells. Cells were treated with 20 µM isochaihulactone at the indicated times. B, A549, H460, H1299, HT29, and HepG2 cells were treated with 20 µM isochaihulactone or vehicle (0.2% DMSO) for 24 h, and cell lysates were isolated. Western blot analysis was performed using NAG-1 antibody. STD indicates cell lysates from CD437-treated H460 cells. C, the level of NAG-1 protein was quantitated with a PhosphorImage analyzer, and the relative amount of NAG-1 protein was plotted. D, expression of NAG-1 after 20 µM isochaihulactone treatment of A549 (p53+/+) and H1299 (p53–/–) cells for the indicated times.

The induction of NAG-1 was not limited to lung carcinoma A549 cells. Isochaihulactone increased the level of NAG-1 protein expression in other lung carcinoma cell lines including H460 and H1299 cells, in human colorectal carcinoma HT29 cells, and in human hepatocellular carcinoma HepG2 cells (Fig. 2, B and C). Because H1299 is a p53-null cell and NAG-1 can be induced by isochaihulactone in this cell line, induction by isochaihulactone must be p53-independent (Fig. 2D).

Isochaihulactone-Induced ERK1/2 Activation Followed by Growth Inhibition in A549 Cells. A role for MAPKs in the regulation of NAG-1 and NAG-1 as a novel downstream target of the PI3K/AKT/GSK-3 (PI3K/AKT) pathway has been reported in several studies (Yamaguchi et al., 2004). To determine whether MAPKs, PKC, and PI3K/AKT play a role in isochaihulactone-induced growth inhibition in A549 cells, cells were treated with isochaihulactone in the presence and absence of the MEK1/2 inhibitor PD98059, the PKC inhibitor GF109203X, or the PI3K/AKT inhibitor LY294002 (10–50 µM). Only PD98059 (Fig. 3A) abrogated isochaihulactone-induced growth inhibition in a concentration-dependent manner. We also found that isochaihulactone had no effect on the activation of PKC or AKT/GSK-3 (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Inhibition of NAG-1 expression and growth inhibition by ERK1/2 inhibitor. A, MTT assay of A549 cells with culture or serum-containing medium pretreated with the MEK1/2 inhibitor PD98059 (25 and 50 µM), the PKC inhibitor GF109203X (10 and 20 µM), or the PI3K/AKT inhibitor LY294002 (10 and 20 µM) for 1 h and then treated with 20 µM isochaihulactone for 24 h. Lane 1 shows A549 cells treated with serum-containing media and no test compound as a negative control. The data represent the means ± S.D. of three different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus vehicle. B, Western blot analysis of PKC, phosphor-PKC (pPKC), AKT, phosphor-AKT (pAKT), GSK-3, and phosphor-GSK-3 (pGSK-3) in A549 cells after treatment with 20 µM isochaihulactone for the indicated times. C, A549 cells were treated with 20 µM isochaihulactone for the indicated times. Total ERK1/2, phosphor-ERK1/2 (pERK1/2), total p38, phosphor-p38 (pp38), total JNK, and phosphor-JNK (pJNK) were detected by Western blot. D, Western blot of A549 cells pretreated with the MEK1/2 inhibitor PD98059 (25 and 50 µM), the p38 inhibitor SB203580 (10 and 20 µM), or the JNK1/2 inhibitor SP600125 (10 and 20 µM) for 1 h and then treated with 20 µM isochaihulactone for 24 h.

ERK1/2 Signaling Pathway Was Involved in Isochaihulactone-Induced EGR-1 and NAG-1 Expressions. To investigate a possible role for ERK1/2 in the regulation of EGR-1 and NAG-1, A549 cells were treated with isochaihulactone in the presence and absence of the MEK1/2 inhibitor PD98059 (25 and 50 µM), the p38 inhibitor SB203580 (10 and 20 µM), or the JNK1/2 inhibitor SP600125 (10 and 20 µM). Using Western blot analysis, we found that inhibition of ERK1/2 expression with PD98059 reduced both EGR-1 and NAG-1 protein levels in a dose-dependent manner after treatment with isochaihulactone in A549 cells (Fig. 3D). In contrast, inhibition of JNK1/2 had little effect on the induction of NAG-1, whereas inhibition of p38 had no effect on either of the two genes. These observations suggest that activation of the ERK1/2 signaling pathway was involved in isochaihulactone-induced EGR-1 and NAG-1 expression.

Induction of NAG-1 Requires EGR-1 Activation and EGR-1 Is Involved in Isochaihulactone-Induced Apoptosis. We determined whether EGR-1 is critical in the induction of NAG-1. The suppression of EGR-1 by EGR-1 siRNA (siEGR-1) or NAG-1 by NAG-1 siRNA (siNAG-1) in a concentration-dependent manner was confirmed by RT-PCR and Western blot analysis (Fig. 4, A and B). To further characterize the role of EGR-1 in isochaihulactone-induced growth inhibition, flow cytometry analysis was performed to identify the percentage of apoptotic cells after treatment of EGR-1 with siRNA. Between 20 and 40% of apoptosis was inhibited by 20 and 50 nM EGR-1 siRNA after exposure of cells to 20 µM isochaihulactone (Fig. 4C). NAG-1 siRNA decreased apoptosis 12 to 35% (Fig. 4D). To evaluate the significance of EGR-1 to the induction of NAG-1, cells were transiently transfected with siEGR-1 and subsequently treated with isochaihulactone. EGR-1 siRNA significantly blocked NAG-1 mRNA and protein expression induced by isochaihulactone, whereas NAG-1 siRNA had no obviously inhibitory effect on EGR-1 mRNA and protein expression (Fig. 4, E and F).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effect of EGR-1 and NAG-1 on the induction of apoptosis by isochaihulactone in A549 cells. A549 cells were transfected with various concentrations of EGR-1 or NAG-1 siRNA (10–50 nM) using GeneJammer transfection reagent. After 48 h (A) RT-PCR and (B) Western blot analysis were performed for EGR-1 and NAG-1. C and D, apoptosis was measured by flow cytometry PI/annexin V staining in A549 cells transfected with either EGR-1 (C) or NAG-1 (D) siRNA (20 and 50 nM) for 48 h and then treated with 20 µM isochaihulactone for 24 h. N indicates cells transfected with 20 µM Silencer Negative Control siRNA (catalog no. 4636; Ambion). P indicates cells treated with 5 nM paclitaxel for 24 h. The data represent the means ± S.D. of three different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus vehicle. E and F, suppression of isochaihulactone-induced NAG-1 expression in A549 cells by EGR-1 siRNA. A549 cells were transfected with various concentrations of either EGR-1 siRNA or NAG-1 siRNA (10–50 nM) using GeneJammmer transfection reagent. After 48 h, RT-PCR (E) and Western blot (F) analysis was performed for NAG-1.

NAG-1 Enhances Isochaihulactone-Induced Apoptosis. NSAIDs induce apoptosis in colorectal cancer cells (Wilson et al., 2003; Baek et al., 2004a; Rosetti et al., 2006). In our study, A549 cells were stably transfected with an expression vector containing the full-length NAG-1 coding region in the sense orientation. A pooled population of cells obtained after selection with G418 was then used. Western blot analysis indicated that these cells express NAG-1 protein at a 2.0-fold greater rate than do vector-transfected cells (Fig. 5A). These stably transfected cells were incubated with isochaihulactone for 48 h, and apoptosis was again determined by flow cytometry. A higher percentage of pCDNA3.1-NAG-1 transfected cells than vector-transfected cells underwent apoptosis (Fig. 5B). A colony formation assay also confirmed the fact that overexpression of NAG-1 enhanced isochaihulactone toxicity. In the presence of 20 µM isochaihulactone, NAG-1 overexpression resulted in a significant reduction (70%) of the clonogenic capacity of A549 cells (Fig. 5C).

View larger version (10K): [in this window] [in a new window]   Fig. 5. Effects of NAG-1 expression on isochaihulactone-induced apoptosis. A and B, antitumorigenic activity of NAG-1 in vitro. A, expression of NAG-1 protein in transfected A549 cells. Full-length NAG-1 cDNA was cloned into pCDNA3.1, yielding the plasmid pCDNA3.1-NAG-1. A549 cells were transfected with either pCDNA3.1 (Vector Laboratories, Burlingame, CA) or pCDNA3.1-NAG-1 (NAG-1), and stably transfected clones were selected by exposure to G418 (500 µg/ml) for 3 weeks. The stably transfected clones were treated with either vehicle or 20 µM isochaihulactone for 24 h. B, apoptosis was measured by flow cytometry PI/annexin V staining in stably transfected A549 cells treated with vehicle or 20 µM isochaihulactone for 24 h. C, colony formation assay using NAG-1 overexpression cells. A549 cells stably transfected with pCDNA3.1 (Vector) or pCDNA3.1-NAG-1 (NAG-1) were incubated in the presence of 20 µM isochaihulactone for 24 h and then grown in culture medium for 2 weeks. The data represent the means ± S.D. of three different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus vehicle.

View larger version (15K): [in this window] [in a new window]   Fig. 6. The Sp1/EGR-1 binding site has an important role in isochaihulactone-induced NAG-1 expression. A, comparison of wild-type and mutant sequences between –137 and +70 in the NAG-1 promoter region. Mutated base pairs are indicated in each mutated sequence. B, wild-type (pNAG137/+70 and pNAG499/+70) or Sp1/EGR-1 mutant vector (pNAG137mut) and pRL-TK cotransfected into A549 cells. These indicated promoter regions were fused to a luciferase report gene. Each construct (2 µg) was cotransfected with 0.2 µg of pRL-TK vector into A549 cells using GeneJammmer transfection reagent followed by treatment with 20 µM isochaihulactone for 24 h. Cells were lysed, and luciferase activity was measured. Transfection efficiency for luciferase activity was normalized to Renilla luciferase (pRL-TK vector). The x-axis shows relative luciferase units (RLU) (firefly luciferase/Renilla luciferase). C, NGA-1 promoter activity is attenuated by PD98059. A549 cells were transfected with pNAG137/+70 reporter plasmid and pRL-TK plasmid. Forty-eight hours after transfection, cells were pretreated with PD98059 for 1 h and then treated with isochaihulactone for another 24 h. pNAG137/+70 promoter construct activity was normalized to Renilla luciferase (pRL-TK vector). The data represent the means ± S.D. of three different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus vehicle.

View larger version (29K): [in this window] [in a new window]   Fig. 7. In situ expression of NAG-1 and tumor growth. A and B, antitumorigenic activity of NAG-1 in vivo. A549 tumor-bearing mice were administered vehicle control () and 50 mg/kg isochaihulactone () s.c. on days 0 to 4 for 5 days. The figures shows the relative tumor volume (A) and body weight (B) of control and treated groups. C and D, immunohistochemical staining was analyzed in A549 tumor tissues (at day 35 after initiation of drug treatment). Representative photographs of sections of the control group (C) and isochaihulactone-treated group (D) A549 tumors, immunohistochemically stained with NAG-1/PTGF- rabbit polyclonal antibody, and NAG-1/PTGF--positive cells were stained brown (black arrowheads) (400x). Scale bars, 100 µm. Data represent means ± S.D. of tumor volume and body weight at each time point.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, oligodeoxynucleotide-based microarray analysis was used to investigate isochaihulactone-induced genes encoding proteins involving cell growth and apoptosis. Analysis with the Affymetrix Human Genome-U133 Plus 2.0 GeneChip identified more than 100 genes that were up-regulated or down-regulated by isochaihulactone treatment. We then tested 6 of these genes in validation experiments using quantitative RT-PCR. Some of the genes up-regulated by isochaihulactone represent immediate-early response genes and genes induced by NSAIDs (Table 2). We focused on NAG-1. NAG-1 is a target gene for NSAIDs, and a unique member of the transforming growth factor- superfamily. Increases in NAG-1 expression result in the induction of apoptosis in several cancer cell lines (Wilson et al., 2003; Baek et al., 2005). Also, NAG-1 expression is induced not only by NSAIDs but also by several antitumorigenic compounds. These include dietary compounds, peroxisome proliferator-activated receptor- ligands, and phytochemicals (Baek et al., 2001b; Yamaguchi et al., 2006; Joshua et al., 2007) as well as resveratrol, genistein, diallyldisulfide, 5F203, and retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (Newman et al., 2003; Lee et al., 2005; Eling et al., 2006).

To determine whether NSAIDs induce expression of the NAG-1 protein, A549 tumor cells were treated with isochaihulactone or sulindac sulfide, a cyclooxygenase inhibitor, and Western blot analysis was performed. As shown in Fig. 2A, isochaihulactone time-dependently induced NAG-1 protein expression. By using colony formation assays and transfection viability assays, we showed that isochaihulactone induced growth arrest and apoptosis in A549 cells through the up-regulation of the antitumorigenic protein NAG-1 (Fig. 5C). Furthermore, we had confirmed the antitumoral effect of isochaihulactone in our previously study (Chen et al., 2006), and here we extended the inhibition of tumor growth based on NAG-1 protein expression in a nude mice xenograft animal model (Fig. 7, C and Fig. D). These data indicate that the expression of NAG-1 is an essential factor in the antitumoral activity of isochaihulactone. In addition, by using an siRNA technique, our experiments also support the idea that NAG-1 expression has an apoptosis-promoting effect by silencing NAG-1 expression (Fig. 4D). Finally, because the up-regulation of NAG-1 by isochaihulactone is followed by up-regulation of EGR-1, the silencing of EGR-1 expression by siRNAs could also inhibit NAG-1 RNA and protein expression (Fig. 4E). In this examination, we found that NAG-1 siRNA would not affect levels of EGR-1 mRNA and protein expression (Fig. 4F). In summary, we found that isochaihulactone increased EGR-1 expression, suggesting that the antitumor effect of isochaihulactone is mediated via this tumor suppressor protein. NAG-1 appears to be a key downstream target of EGR-1.

To determine which MAPK family is involved in the major signaling pathway for isochaihulactone-mediated NAG-1 up-regulation and growth inhibition, MAPK inhibitors were applied to study the growth inhibition induced by isochaihulactone. In our study, PD98059 significantly decreased the growth inhibition induced by isochaihulactone (Fig. 3A), but neither the p38 inhibitor SB203580 or the JNK inhibitor SP600125 could reverse isochaihulactone-induced growth inhibition (data not shown). Furthermore, only the MEK1/2 inhibitor PD98059 reduced isochaihulactone-induced EGR-1 and NAG-1 protein expression (Fig. 3D). These data support the idea that isochaihulactone-induced ERK1/2 activity is critical in regulating EGR-1 and NAG-1 expression.

In a report by Baek et al. (2004), it was shown that a PI3K/AKT/GSK3 pathway regulates NAG-1 expression in human colorectal cancer cells as assessed by the inhibition of PI3K, AKT, and GSK-3. PI3K inhibition by LY294002 showed an increase in NAG-1 protein and mRNA expression, and 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (an AKT inhibitor) also induced NAG-1 expression (Yamaguchi et al., 2004; Shim and Eling, 2005; Martinez et al., 2006; Pang et al., 2007). In an attempt to identify the signaling pathway through which PI3K/AKT/GSK3 is involved in receptor signal transduction through tyrosine kinase receptors for isochaihulactone, the effect of LY294002, a PI3K inhibitor, was examined. We found that LY294002 did not reverse tumor growth inhibition caused by isochaihulactone (Fig. 3A). Compared with the effects of other phytochemical agents, this result suggests that the ability of our drug to cause tumor apoptosis might not go through this pathway.

By using a gel retardation method, many researchers have shown that EGR-1 protein binds to the NAG-1 promoter and trans-activates NAG-1 expression by competing against Sp1 and NAB1/2 transcription factors (Baek et al., 2001). The NAG-1 promoter (–1739 to + 70) containing two p53 motifs and the Sp1 motifs were also present. Several dietary compounds induced NAG-1 expression by increasing p53 expression (Fu et al., 2002; Lim et al., 2007). However, mutations commonly occur at the p53 tumor suppressor locus in many forms of cancer, including colorectal cancer. The increase in NAG-1 expression due to NSAIDs is independent of p53, suggesting that NSAIDs can still increase NAG-1 expression in tumors with p53 mutations. To determine whether p53 is the target motif of isochaihulactone, we used the p53-null cell line H1299. We found that even H1299 cells show NAG-1 expression that is induced by isochaihulactone (Fig. 2D). This result indicates that the expressions of NAG-1 and of the p53 motif are independent.

Because we already excluded the idea that p53 has a required role in isochaihulactone effects on the NAG promoter, we explored the possible regulatory role of two Sp1s on the NAG-1 promoter. With our NAG-1 promoter, which contained two Sp1 motifs with an engineered luciferase reporter, isochaihulactone induced a 5.0 to 7.5-fold increase in luciferase activity compared with vehicle (Fig. 6B). To further test whether these two Sp1 motifs were isochaihulactone functional motifs, mutation of the two Sp1 DNA sequences was done. Isochaihulactone did not increase reporter gene expression (Fig. 6B). Together, these result indicate that isochaihulactone is associated with the Sp1 site rather than with p53. Figure 6C illustrates a model for ERK1/2 regulation of the NAG-1 promoter after isochaihulactone treatment. Treatment with the MAPK inhibitor PD98059 resulted in the reduction of NAG-1 promoter activity in the presence of isochaihulactone, thus supporting the hypothesis that isochaihulactone stimulates ERK1/2 activation and then induces ERG-1, which is required for expression of NAG-1.

In summary, we studied mechanisms underlying the antitumor activity of isochaihulactone because of our previous studies showing growth inhibition and antitumor activity in specific tumor cell lines in vitro and inhibition of a variety of xenografts including A549 lung cancer cell in vivo (Cheng et al., 2005; Chen et al., 2006). cDNA microarray analysis showed that when A549 cells are exposed to isochaihulactone, NAG-1 is the most highly induced gene. ERK1/2 MAPK signaling pathways have been implicated in the regulation of Egr-1 and NAG-1 because isochaihulactone induces activation of phosphor-ERK in A549 cells. These results suggest a gene target for isochaihulactone, which may be useful for future clinical applications.

	Footnotes

 
This work was supported by Grants 96-2320-B-197-001-MY2 and 96-2320-B-303-001-MY3 from National Science Council of the Republic of China. Y.-L.C. and P.-C.L. contributed equally as first authors, and S.-Z.L. and H.-J.H. share equal corresponding authorship.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.126193.

ABBREVIATIONS: EGR-1, early growth response gene-1; TGF-, transforming growth factor-; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3-kinase; PPAR, peroxisome proliferator-activated receptor-; ERK, extracellular signal-regulated kinase; NSAID, nonsteroidal anti-inflammatory drug; NAG-1, nonsteroidal anti-inflammatory drug-activated gene; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide; PKC, protein kinase C; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; PD98059, 2-amino-3-methoxyflavone; JNK, c-Jun NH₂-terminal kinase; SP600125, anthra[1,9-cd] pyrazol-6 (2H)-one; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; AKT, protein kinase B; PTGF, placental transforming growth factor; GSK, glycogen synthase kinase; RT, reverse transcription; PCR, polymerase chain reaction; si, small interfering; PBS, phosphate-buffered saline; NRG-1, neuregulin-1; FGF-1, fibroblast growth factor-1; MEK, mitogen-activated protein kinase kinase; 5F203, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole.

Address correspondence to: Dr. Horng-Jyh Harn, Neuro-Medical Scientific Center, Department of Pathology, Tzu-Chi General Hospital, 707, Section 3, Chung-Yang Rd., 970 Hualien, Taiwan, R.O.C. E-mail address: duke_harn{at}yahoo.com.tw

	References

Top Abstract Materials and Methods Results Discussion References

 

Albo D, Arnoletti JP, and Castiglioni A (1994) Thrombospondin (TSP) and transforming growth factor 1 (TGF-) promote human A549 lung carcinoma cell plasminogen activator inhibitor type 1 (PAI-1) production and stimulate tumor cell attachment in vitro. Biochem Biophys Res Commun 203: 857–865.[CrossRef][Medline]

Baek SJ, Horowitz JM, and Eling TE (2001a) Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. J Biol Chem 276: 33384–33392.[Abstract/Free Full Text]

Baek SJ, Kim JS, Jackson FR, Eling TE, McEntee MF, and Lee SH (2004a) Epicatechin gallate-induced expression of NAG-1 is associated with growth inhibition and apoptosis in colon cancer cells. Carcinogenesis 25: 2425–2432.[Abstract/Free Full Text]

Baek SJ, Kim JS, Moore SM, Lee SH, Martinez J, and Eling TE (2005) Cyclooxygenase inhibitors induce the expression of the tumor suppressor gene EGR-1, which results in the up-regulation of NAG-1, an antitumorigenic protein. Mol Pharmacol 67: 356–364.[Abstract/Free Full Text]

Baek SJ, Kim JS, Nixon JB, DiAugustine RP, and Eling TE (2004b) Expression of NAG-1, a transforming growth factor- superfamily member, by troglitazone requires the early growth response gene EGR-1. J Biol Chem 279: 6883–6892.[Abstract/Free Full Text]

Baek SJ, Kim KS, Nixon JB, Wilson LC, and Eling TE (2001b) Cyclooxygenase inhibitors regulate the expression of a TGF- superfamily member that has proapoptotic and antitumorigenic activities. Mol Pharmacol 59: 901–908.[Abstract/Free Full Text]

Baek SJ, Wilson LC, and Eling TE (2002) Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53. Carcinogenesis 5: 425–434.

Baek SJ, Wilson LC, Hsi LC, and Eling TE (2003) Troglitazone, a peroxisome proliferator-activated receptor g (PPAR) ligand, selectively induces the early growth response-1 gene independently of PPAR: a novel mechanism for its anti-tumorigenic activity. J Biol Chem 278: 5845–5853.[Abstract/Free Full Text]

Bottone FG Jr, Baek SJ, Nixon JB, and Eling TE (2002) Diallyl disulfide (DADS) induces the antitumorigenic NSAID-activated gene (NAG-1) by a p53-dependent mechanism in human colorectal HCT 116 cells. J Nutr 132: 773-778.[Abstract/Free Full Text]

Chen YL, Lin SZ, Chang JY, Cheng YL, Tsai NM, Chang WL, and Harn HJ (2006) In vitro and in vivo studies of a novel potential anticancer agent of isochaihulactone on human lung cancer A. Biochem Pharmacol 72: 308–319.[CrossRef][Medline]

Chen YL, Lin SZ, Cheng YL, Chang WL, and Harn HJ (2005) Requirement for ERK activation in acetone extract identified from Bupleurum scorzonerifolium induced A549 tumor cell apoptosis and keratin 8 phosphorylation. Life Sci 76: 2409–2420.[CrossRef][Medline]

Cheng YL, Lee CC, Lin SZ, Chang WL, Chen YL, Tsai NM, Liu YC, Tzao C, Yu DS, and Harn HJ (2005) Anti-proliferative activity of Bupleurum scorzonerifolium in A549 human lung cancer cells in vitro and in vivo. Cancer Lett 222: 183–193.[CrossRef][Medline]

Chintharlapalli S, Papineni S, and Safe S (2006) 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through PPAR-dependent and PPAR-independent pathways. Mol Cancer Ther 5: 1362–1370.[Abstract/Free Full Text]

Chintharlapalli S, Papineni S, Baek SJ, Liu S, and Safe S (2005) 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes are peroxisome proliferator-activated receptor- agonists but decrease HCT-116 colon cancer cell survival through receptor-independent activation of early growth response-1 and nonsteroidal anti-inflammatory drug-activated gene-1. Mol Pharmacol 66: 1783–1792.

Eling TE, Baek SJ, Shim M, and Lee CH (2006) NSAID activated gene (NAG-1), a modulator of tumorigenesis. J Biochem Mol Biol 39: 649–655.[Medline]

Elkon R, Zeller KI, Linhart C, Dang CV, Shamir R, and Shilon Y (2004) In silico identification of transcriptional regulators associated with c-Myc. Nucleic Acids Res 32: 4955–4961.[Abstract/Free Full Text]

Fu M, Zhang J, Lin Y, Zhu X, Ehrenqruber MU, and Chen YE (2002) Early growth response factor-1 is a critical transcriptional mediator of peroxisome proliferator-activated receptor- 1 gene expression in human aortic smooth muscle cells. J Biol Chem 277: 26808–26814.[Abstract/Free Full Text]

Hosono S, Lee CS, Chou MJ, Yang CS, and Shih CH (1991) Molecular analysis of the p53 alleles in primary hepatocellular carcinomas and cell lines. Oncogene 6: 237–243.[Medline]

Joshua KS, Leung WC, Ho WK, and Chiu P (2007) Herbal diterpenoids induce growth arrest and apoptosis in colon cancer cells with increased expression of the nonsteroidal anti-inflammatory drug-activated gene. Eur J Pharmacol 559: 1–13.[CrossRef][Medline]

Krishnaraju K, Nguyen HQ, Liebermann DA, and Hoffman B (1995) The zinc finger transcription factor EGR-1 potentiates macrophage differentiation of hematopoietic cells. Mol Cell Biol 15: 5499–5507.[Abstract]

Lee SH, Kim JS, Yamagucki K, Eling TE, and Baek SJ (2005) Indole-3-carbinol and 3,3'-diindolylmethane induce expression of NAG-1 in a p53-independent manner. Biochem Biophys Res Commun 328: 63–69.[CrossRef][Medline]

Lim JH, Park JW, Min DS, Chang JS, Lee YH, Park YB, Choi KS, and Kwon TK (2007) NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells. Apoptosis 12: 411–421.[CrossRef][Medline]

Liu T, Bauskin AR, Zaunders J, Brown DA, Pankurst S, Russell PJ, and Breit SN (2003) Macrophage inhibitory cytokine 1 reduces cell adhesion and induces apoptosis in prostate cancer cells. Cancer Res 63: 5034–5040.[Abstract/Free Full Text]

Martinez JM, Sali T, Akazaki R, Anna C, Hollingshead M, Hose C, Monks A, Walker NJ, Baek SJ, and Eling TE (2006) Drug-induced expression of nonsteroidal anti-inflammatory drug-activated gene/macrophage inhibitory cytokine-1/prostate-derived factor, a putative tumor suppressor, inhibits tumor growth. J Pharmacol Exp Ther 318: 899–906.[Abstract/Free Full Text]

Newman D, Sakaue M, Koo JS, Kim KS, Baek SJ, Eling T, and Jetten AM (2003) Differential regulation of nonsteroidal anti-inflammatory drug-activated gene in normal human tracheobronchial epithelial and lung carcinoma cells by retinoids. Mol Pharmacol 63: 557–764.[Abstract/Free Full Text]

Pang RP, Zhou JG, Zeng ZR, Li XY, Chen W, Chen MH, and Hu PJ (2007) Celecoxib induces apoptosis in COX-2 deficient human gastric cancer cells through Akt/GSK3/NAG-1 pathway. Cancer Lett 251: 268–277.[CrossRef][Medline]

Rokos CL and Ledwith BJ (1997) Peroxisome proliferators activate extracellular signal-regulated kinases in immortalized mouse liver cells. J Biol Chem 272: 13452–13457.[Abstract/Free Full Text]

Rosetti M, Tesei A, Ulini P, Fabbri F, Vannini I, Brigliadori G, Amadori D, Bolla M, and Zoli W (2006) Molecular characterization of cytotoxic and resistance mechanisms induced by NCX 4040, a novel NO-NSAID, in pancreatic cancer cell lines. Apoptosis 11: 1321–1330.[CrossRef][Medline]

Shim M and Eling TE (2005) Protein kinase C-dependent regulation of NAG-1/placental bone morphogenic protein/MIC-1 expression in LNCaP prostate carcinoma cells. J Biol Chem 280: 18636–18642.[Abstract/Free Full Text]

Thiel G and Cibelli G (2002) Regulation of life and death by zinc finger transcription factor Egr-1. J Cell Physiol 193: 287–292.[CrossRef][Medline]

Tomioka K, Ishiguro T, Iitaka Y, and Koga K (1986) Stereoselective reactions. XII. Synthesis of antitumor-active steganacin analogs, picrosteganol and epipicrosteganol, by selective isomerization. Chem Pharm Bull (Tokyo) 34: 1501–1504.[Medline]

van Engeland M, Ramaekers FC, Schutte B, and Reutelingsperger CP (1998) Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31: 1–9.[CrossRef][Medline]

Wilson LC, Baek SJ, Call A, and Eling TE (2003) Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is induced by genistein through the expression of p53 in colorectal cancer cells. Int J Cancer 105: 747–753.[CrossRef][Medline]

Yamaguchi K, Lee SH, Eling TE, and Baek SJ (2006) A novel peroxisome proliferator-activated receptor ligand, MCC-555, induces apoptosis via posttranscriptional regulation of NAG-1 in colorectal cancer cells. Mol Cancer Ther 5: 1352–1361.[Abstract/Free Full Text]

Yamaguchi K, Lee SH, Eling TE, and Baek SJ (2004) Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3 pathway. J Biol Chem 279: 49617–49623.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Cekanova, S.-H. Lee, R. L. Donnell, M. Sukhthankar, T. E. Eling, S. M. Fischer, and S. J. Baek Nonsteroidal Anti-inflammatory Drug-Activated Gene-1 Expression Inhibits Urethane-Induced Pulmonary Tumorigenesis in Transgenic Mice Cancer Prevention Research, May 1, 2009; 2(5): 450 - 458. [Abstract] [Full Text] [PDF]

				W. Zhang, Y. Chen, H. Wei, C. Zheng, R. Sun, J. Zhang, and Z. Tian Antiapoptotic Activity of Autocrine Interleukin-22 and Therapeutic Effects of Interleukin-22-Small Interfering RNA on Human Lung Cancer Xenografts Clin. Cancer Res., October 15, 2008; 14(20): 6432 - 6439. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.126193v1 323/2/746    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 Chen, Y.-L. Articles by Harn, H.-J. Search for Related Content PubMed PubMed Citation Articles by Chen, Y.-L. Articles by Harn, H.-J.