Cyclooxygenase Inhibitors Induce the Expression of the Tumor Suppressor Gene EGR-1, Which Results in the Up-Regulation of NAG-1, an Antitumo
Laboratory of Molecular Carcinogenesis (S.J.B., J.-S.K., S.M.M., T.E.E.) and Laboratory of Computational Biology and Risk Analysis (J.M.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee (S.J.B., S.-H.L.)
Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to have chemopreventive activity, but the mechanisms involved are not clearly understood. Although NSAIDs inhibit cyclooxygenase activity, they also increase the expression of a divergent member of the transforming growth factor- superfamily, termed NSAID-activated gene 1 (NAG-1), a protein with an antitumorigenic and proapoptotic activity that could in part be linked to the chemoprevention activity of NSAIDs. NAG-1 is induced by some NSAIDs, but the mechanisms responsible are not clear. In this report, we have identified a cis-acting element responsive to NSAIDs located within the eC73 to eC51 region of the NAG-1 promoter. This region contains overlapping EGR-1 and Sp1 binding sites, and mutations in this region suggest that the transcription factors have an important role in NSAID-induced NAG-1 expression. EGR-1 was found to play a critical role in the induction of NAG-1 by sulindac sulfide and other NSAIDs. NSAIDs increase EGR-1 protein expression that occurs before the induction of NAG-1 expression, supporting the hypothesis that EGR-1 is necessary for NSAID-induced NAG-1 expression. Thus, NSAIDs induce the expression of EGR-1, a tumor suppressor gene, providing a novel mechanism to explain, in part, the antitumorigenic properties of some NSAIDs. NAG-1 seems to be an important downstream target protein of this transcription factor, EGR-1, and may mediate the chemopreventive activity of some NSAIDs.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of inflammatory disease, and their anti-inflammatory effects are believed to result from inhibition of prostaglandin H synthase (also known as cyclooxygenase, COX). Two isoforms of prostaglandin H synthase, COX-1 and COX-2, are known; COX-1 is constitutively expressed in many tissues, whereas mitogens, tumor promoters, and growth factors up-regulate the expression of COX-2. COX-2 protein is also up-regulated in human colorectal tumors and regulates tumor growth in animal models (Taketo, 1998; Reddy and Rao, 2002; Ricchi et al., 2003). NSAIDs reduce the number and size of polyps in animal models, and epidemiological studies reveal a 40 to 50% reduction in mortality from colorectal cancer (Thun and Heath, 1995; Thun, 1996). Chemopreventive effects of certain NSAIDs seem to be mediated through both COX-dependent and -independent pathways. However, the target molecules that mediate chemopreventive effects are not elucidated. Identifying possible targets is important to understanding the mechanism of the chemopreventive actions. A number of molecular mechanisms responsible for the chemopreventive effects of NSAIDs have been proposed. One hypothesis is the obvious involvement of COX-2 inhibition, but it is clear that prostaglandin-independent mechanisms are also involved (Jones et al., 1999; McEntee et al., 1999; Baek et al., 2002b; Ferrandez et al., 2003; Rice et al., 2003). In either case, the mechanisms responsible for the chemopreventive activity are not clear. Changes in gene expression by COX inhibitors could provide an explanation for the activity. A number of studies with microarray and PCR-based subtractive hybridization have identified potential target genes (Baek et al., 2001b; Iizaka et al., 2002; Bottone et al., 2003). NAG-1 was identified in our laboratory and is a highly promising target gene because it is a proapoptotic and antitumorigenic protein, and its expression is highly induced by NSAIDs (Baek et al., 2001b). The human NAG-1 cDNA encodes a secreted protein with homology to members of the transforming growth factor- superfamily and has been identified previously as macrophage inhibitory cytokine-1 (Bootcov et al., 1997), placental transformation growth factor- (Li et al., 2000), prostate-derived factor (Paralkar et al., 1998), growth differentiation factor 15 (Bottner et al., 1999), and placental bone morphogenetic protein (Hromas et al., 1997). In mature intestinal epithelial cells, NAG-1 is expressed, but the expression is significantly reduced in human colorectal carcinoma samples and neoplastic intestinal polyps of Min mice (Kim et al., 2002). NAG-1 overexpression from a recombinant adenoviral vector results in an 80% reduction of MDA-MB-468 and MCF-7 breast cancer cell viability (Li et al., 2000), and treatment of prostate cancer cells with purified NAG-1 induces apoptosis (Liu et al., 2003). These data support the hypothesis that NAG-1 is linked to apoptosis and that its reduced expression may enhance tumorigenesis. NAG-1 expression is up-regulated independently of prostaglandin formation in human colorectal cancer cells by several NSAIDs (Baek et al., 2002b), In addition, antitumorigenic compounds such as resveratrol (Baek et al., 2002a), genistein (Wilson et al., 2003), diallyl disulfide (Bottone et al., 2002), retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (Newman et al., 2003), 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (Monks et al., 2003), and peroxisome proliferator-activated receptor- ligands induce NAG-1 expression (Baek et al., 2004). These findings suggest a critical role for NAG-1 in the chemopreventive activity of a number of unrelated chemopreventive chemicals.
The aim of the present study was to identify cis- and trans-acting elements that are responsible for NAG-1 induction by sulindac sulfide and other NSAIDs. During this investigation, we found NSAIDs to increase the expression of EGR-1, a transcription factor and tumor suppressor protein, suggesting that the chemopreventive activity of NSAIDs is mediated via this tumor suppressor protein. NAG-1 was characterized as a key downstream target of EGR-1.
Materials and Methods
Cell Lines and Reagents. Cell lines in this study were purchased from American Type Culture Collection (Manassas, VA). Human colorectal carcinoma cells, HCT-116, were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and gentamicin. All NSAIDs in this study were purchased from Sigma-Aldrich (St. Louis, MO), except for sulindac sulfide, SC-560 (both from Cayman Chemical, Ann Arbor, MI), and [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone] (DFU) (Merck, Whitehouse Station, NJ). All NSAIDs were dissolved in DMSO.
Western Blot Analysis. After reaching 60 to 80% confluence in 10-cm plates, the cells were treated at the indicated concentrations and times with each different NSAID in the absence of serum. The level of NAG-1 was evaluated using Western blot analysis with anti-human NAG-1 antibody reported previously (Baek et al., 2001b). Cell lysates were harvested, and 30 e of total proteins was separated by 15% SDS-polyacrylamide gel electrophoresis and transferred for 1 h onto nitrocellulose membrane (ECL Hybond; Amersham Biosciences Inc., Piscataway, NJ). The blots were blocked for 1 h with 5% skim milk in Tris-buffered saline/0.05% Tween 20 and probed with anti-NAG-1 antibody (1:1000 dilution, 5% skim milk in Tris-buffered saline/0.05% Tween 20 at 4°C overnight). After washing, the blots were treated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed several times. The signal was detected by the enhanced chemiluminescence system (Amersham) and autoradiography.
Transfection of Cells with the Luciferase Reporter System. HCT-116 cells were plated in six-well plates at 2 x 105 cells/well in McCoy's 5A media supplemented with 10% fetal bovine serum. After growth for 16 h, plasmid mixtures containing 1 e of promoter linked to luciferase and 0.05 e of pRL-null (Promega, Madison, WI) were transfected by LipofectAMINE (Invitrogen, Carlsbad, CA). For the cotransfection experiment, 0.5 e of reporter vector and 0.5 e of expression vector were transfected with 0.05 e of pRL-null vector 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-null luciferase activity with a Dual Luciferase Assay Kit (Promega).
Construction of Plasmids. Several NAG-1 promoter clones linked to luciferase have been reported previously (Baek et al., 2001a). The full-length EGR-1 cDNA was generated by PCR from human universal QUICK-Clone cDNA (BD Biosciences Clontech, Palo Alto, CA) using the following primers: 5'-GACACCAGCTCTCCAGCCTGCTCGTCCAGG-3' (top strand) and 5'-TTCCCTTTAGCAAATTTCAATTGTCCTGGG-3' (bottom strand). The amplified products were cloned into pCR2.1 TOPO vector (Stratagene, La Jolla, CA) and followed by cloning into pCDNA3.1/NEO expression vector (Invitrogen). The Sp1 expression vector was described previously (Baek et al., 2001a). The EGR-1 promoter (eC1260 to +35) linked to the luciferase gene (pEGR1260) was cloned by PCR from human Genomic DNA (Promega) with the following primers: 5'-CGGCTCGAGCGGGAGGAGGAGCGAGGAGGCGGCGG-3' (top strand, XhoI site was underlined) and 5'-CCCAAGCTTGGGCGGCGGCGGCTCCCCAAGTTCTGCGGC-3' (bottom strand, HindIII site was underlined). After PCR amplification, the fragment was digested with XhoI and HindIII and ligated into pGLBasic3 luciferase vector. The deletion clones of EGR-1 promoter were generated from pEGR1260 using ExoIII nuclease. All plasmids were sequenced for verification.
Real-Time PCR. HCT-116 cells were pretreated with vehicle or 5 e/ml, followed by the treatment of 30 e sulindac sulfide for 12 h. Cells were washed with phosphate-buffered saline, removed by scraping, and lysed using Nucleic Acid Purification Lysis solution (1x) (Applied Biosystems, Foster City, CA). Total RNA was isolated using ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems) according to the manufacturer's recommendations, quantified, diluted with diethyl pyrocarbonate water, and stored at eC70°C. Total RNA (50 ng) was denatured at 70°C for 10 min and then reverse-transcribed with oligo(dT) primers (1 e) and Moloney murine leukemia virus reverse transcriptase (1.25 U/e) in 10-e reactions at 37°C for 1 h. Real-time fluorescence detection was carried with ABI Prism 7900 Sequence Detection System (Applied Biosystems). 18S rRNA was amplified with a probe dye VIC-MGB using TaqMan Universal PCR Master Mix 1x. NAG-1 was amplified using the following primers: forward, 5'-TGCCCGCCAGCTACAATC-3'; reverse, 5'-TCTTTGGCTAACAAGTCATCATAGGT-3' (0.2 e each) (Research Genetics, Huntsville, AL) with SYBR Green PCR Master Mix (1x) and cDNA (10 e) in a final PCR reaction volume of 50 e. PE 7900 amplification parameters were the following: denaturation at 94°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Reverse transcription and PCR reagents were purchased from Applied Biosystems.
Results
NAG-1 Induction Requires de novo Synthesis. NAG-1 is induced by several NSAIDs at the transcriptional level (Baek et al., 2001b). Sulindac sulfide is very potent, and thus, this NSAID was used as a model for COX inhibitors. To confirm that NSAIDs induce NAG-1 at the protein level, HCT-116 cells were treated with two conventional NSAIDs (sulindac sulfide and indomethacin), a COX-1eCspecific inhibitor (SC-560), a COX-2eCspecific inhibitor (DFU), or a non-COX inhibitor (acetaminophen), and Western analysis was performed. As shown in Fig. 1A, sulindac sulfide highly induces NAG-1 protein expression, whereas indomethacin and SC-560 induce NAG-1 expression to a lesser extent. However, DFU and acetaminophen do not induce NAG-1 expression. This result is consistent with our previous report showing that NAG-1 mRNA expression is induced by sulindac sulfide and indomethacin but not by DFU and acetaminophen (Baek et al., 2001b). To examine whether NSAID-induced NAG-1 expression requires de novo synthesis, HCT-116 cells were pretreated with or without cycloheximide for 30 min, and then followed by incubation with 30 e sulindac sulfide. As shown in Fig. 1B, NAG-1 mRNA was induced by sulindac sulfide treatment by 15.9-fold, but sulindac sulfide could not increase the level of NAG-1 mRNA in the presence of cycloheximide, suggesting that sulindac sulfide-induced NAG-1 expression requires de novo protein synthesis. These data are compatible with the notion that the increase in NAG-1 biosynthesis conferred by sulindac sulfide and presumably other NSAIDs requires, at least in part, de novo synthesis at the transcriptional level.
Effects of NSAIDs on the NAG-1 Promoter Activity. Because NSAIDs induce the expression of NAG-1 mRNA and protein, we examined whether NAG-1 promoter activity is increased in the presence of several NSAIDs. A construct, pNAG1739/LUC, containing the eC1739 to +41 NAG-1 promoter region and luciferase reporter gene was transfected into HCT-116 cells. The cells were then treated with different concentrations of NSAIDs and subsequently assayed for luciferase activity. As shown in Table 1, sulindac sulfide treatment was the most potent inducer of luciferase activity, with an estimated ED50 of 16 e, followed by indomethacin, diclofenac, ibuprofen, piroxicam, naproxen, sodium salicylate, and aspirin. These data are very consistent with Northern and Western data reported previously (Baek et al., 2001b). In addition, celecoxib and acetaminophen did not increase the NAG-1 promoter activity (data not shown), which is consistent with previous data (Fig. 1A), showing that celecoxib and acetaminophen do not induce NAG-1 protein expression in HCT-116 cells.
A construct, pNAG1739/LUC, was transfected into HCT-116 cells and treated with the indicated NSAIDs at different concentrations. As an internal control, pRL-null vector was cotransfected and used to adjust transfection efficiencies. The data shown is the average from three independent experiments. The estimated ED50 was calculated from the 50% induction of their maximum luciferase activity.
Deletion Analysis of NAG-1 Promoter. To evaluate the importance of cis-acting elements regulating NSAID-inducible NAG-1 expression, the 3.5-kb NAG-1 promoter and other deletion clones were transfected into HCT-116 cells and treated with the model NSAID, sulindac sulfide. As an internal control, the plasmid pRL-null (Promega) was used for adjusting transfection efficiency. As shown in Fig. 2A, a large increase in luciferase activity induced by sulindac sulfide treatment was observed with all constructs. These data suggest that there is a positive cis-acting element responsible for sulindac sulfide within eC133-base pair NAG-1 promoter region. As a negative control, the promoterless vector, pGLBasic3, was also transfected into HCT-116 cells and showed no significant luciferase activity after treatment of the cells with sulindac sulfide. A p53 binding site present in the NAG-1 promoter at the +43 position responds to several dietary antitumorigenic compounds (Baek et al., 2002a; Wilson et al., 2003). To examine the importance of the p53 site in sulindac sulfide induced NAG-1 expression, we generated two constructs containing a p53 site at the +43 position in the pNAG133/+70 and pNAG41/+70. These constructs were transfected into HCT-116 cells and treated with sulindac sulfide, and the luciferase activity was measured. As shown in Fig. 2B, sulindac sulfide did not increase the activity of the pNAG41/+70 construct, which has the p53 site, but did increase the activity of the pNAG133/+70 construct. Thus the p53 site at +43 is not involved in the induction of NAG-1 expression by sulindac sulfide. These data are in agreement with a previous report that NSAID-induced NAG-1 expression is p53-independent (Baek et al., 2002b). The NSAID response element is thus located between eC133 and eC41 in the NAG-1 promoter.
Sulindac Sulfide Response Element Is Located in eC73 to eC51 of NAG-1 Promoter Region. Three Sp1 binding sites, Sp1A, Sp1B, and Sp1C, are present in the eC133 to eC41 region of the NAG-1 promoter (Baek et al., 2001a). To further define the responsible element for sulindac sulfide, internal deletion clones pNAG133SP1-I and pNAG133SP1-II were generated and transfected into HCT-116 cells. The pNAG133SP1-I clone has a deletion in the Sp1A site, whereas pNAG133SP1-II has a deletion in the Sp1B and Sp1C sites in the NAG-1 promoter region. It is interesting that Sp1B and Sp1C sites are overlapped with two EGR-1 sites. As shown in Fig. 3A, the deletion mutant pNAG133SP1-II showed no significant induction of luciferase activity after sulindac sulfide treatment, indicating that the sulindac sulfide response element is located in this region containing two Sp1 sites and two EGR-1 sites. To investigate whether other NSAIDs also required this site for NAG-1 induction, pNAG133/LUC-transfected cells were treated with indomethacin, diclofenac, sulindac sulfide, or acetaminophen. As shown in Fig. 3B, all of the compounds except for acetaminophen increase the luciferase activity. In contrast, NSAID treatment of HCT-116 cells transfected with the pNAG133SP1-II construct did not exhibit an increase in luciferase activity, suggesting that this site is critical for NAG-1 induction by NSAIDs, including sulindac sulfide. It should be noted that 30 e sulindac sulfide was used, whereas 100 e was used for the other NSAIDs.
Sp1, EGR-1, and Sulindac Sulfide-Induced NAG-1 Expression. Sp1 and EGR-1 sites have a pivotal role in the sulindac sulfide-induced NAG-1 expression. We next determined whether the expression of Sp1 and/or EGR-1 protein would alter sulindac sulfide-induced NAG-1 expression. An Sp1 or EGR-1 expression vector was cotransfected along with the pNAG133/LUC reporter vector into HCT-116 cells and the cells incubated with sulindac sulfide. Sp1 expression in the absence of sulindac sulfide increased the luciferase activity 1.5-fold, consistent with previous data (Baek et al., 2001a). However, sulindac sulfide treatment in the Sp1-transfected cells did not enhance the luciferase activity relative to the sulindac sulfide-treated vector-transfected cells. In fact, a marginal reduction in the luciferase activity in the Sp1-transfected cells, 6.9-versus 5.5-fold, respectively (Fig. 4A), was observed. These data suggest that Sp1 expression may interfere with sulindac sulfide-induced NAG-1 expression. The promoters of many genes contain a GC box, which may share its binding proteins with Sp1 and EGR-1 (Khachigian et al., 1995; Raychowdhury et al., 2002; Davis et al., 2003). The NAG-1 promoter activity was measured after EGR-1 expression. The cells expressing EGR-1 were treated with sulindac sulfide, and a 2-fold increase luciferase activity (6.9-versus 12.6-fold) was observed. Thus, EGR-1 expression seems to be critical for sulindac sulfide-induced NAG-1 expression in HCT-116 cells. In contrast, Sp1 may compete with EGR-1 in controlling NAG-1 expression by sulindac sulfide. Therefore, to elucidate the relationship between Sp1 and EGR-1 expression, both Sp1 and EGR-1 were transfected with the pNAG133/LUC reporter vector, and luciferase activity was measured with and without sulindac sulfide treatment. As shown in Fig. 4B, the more EGR-1 is expressed, the more NAG-1 promoter activity is induced by sulindac sulfide. The expression of SP-1 protein attenuated the increase in NAG-1 expression by sulindac sulfide. These results indicate that EGR-1 is a key mediator of sulindac sulfide-induced NAG-1 expression, whereas Sp1 may inhibit the sulindac sulfide-induced NAG-1 expression, probably by competing for the same binding sites in the NAG-1 promoter region.
Expression of EGR-1 Is Induced by Sulindac Sulfide. Because EGR-1 plays an important role in sulindac sulfide-induced NAG-1 expression in HCT-116 cells at the transcription level, we sought to determine whether EGR-1 protein is altered during sulindac sulfide treatment in HCT-116 cells. HCT-116 cells were treated with 30 e sulindac sulfide at the indicated times, and cell extracts were analyzed for EGR-1 by Western analysis. EGR-1 expression was increased with treatment with sulindac sulfide. An increase in protein was observed as early as 2 h and then decreased after 16 h of treatment (Fig. 5). In contrast, Sp1 protein expression was not changed during the sulindac sulfide treatment. NAG-1 expression was also increased by sulindac sulfide, but it was observed after the increase in EGR-1 expression. In contrast, incubation with the prodrug sulindac did not increase the expression of EGR-1, Sp1, or NAG-1 in HCT-116 cells. This result is consistent with previous reports that sulindac sulfide induces NAG-1 expression, whereas sulindac does not induce NAG-1 expression (Baek et al., 2001b). These data support the hypothesis that sulindac sulfide-induced NAG-1 expression requires de novo protein synthesis (Fig. 1B) and is mediated by EGR-1.
EGR-1 Promoter Is Activated by NSAIDs. To confirm that sulindac sulfide induces EGR-1 at the transcription level, the EGR-1 promoter was cloned into the luciferase reporter vector. A plasmid, pEGR1260/LUC (Baek et al., 2004), was transfected into HCT-116 cells, and the luciferase activity was measured in response to several NSAIDs. As shown in Fig. 6A, sulindac sulfide greatly enhanced EGR-1 promoter activity by 25-fold, whereas indomethacin, ibuprofen, and diclofenac were less effective in stimulating the EGR-1 promoter activity. Aspirin, piroxicam, and naproxen marginally enhanced the EGR-1 promoter activity at the indicated concentrations. Sulindac sulfone, sulindac, acetaminophen, and DFU did not enhance the luciferase activity (data not shown). Thus, NSAID-induced EGR-1 promoter activity and NSAID-induced NAG-1 promoter activity show a similar pattern of responses to different NSAIDs. To determine whether enhanced EGR-1 production by NSAIDs results in the transactivation of EGR-1 target genes, a construct containing four copies of EGR-1 binding sites pEBS14luc construct was transfected into HCT-116 cells, and the cells were treated with several NSAIDs. As shown in Fig. 6B, sulindac sulfide dramatically enhanced promoter activity, followed by diclofenac and indomethacin. These data indicate that NSAID induces the expression of a functionally active protein, EGR-1, that binds and transactivates EGR-1 target genes.
Discussion
Many epidemiological studies have reported a consistent 40 to 50% reduction in the risk of developing colorectal cancer associated with the use of NSAIDs (Thun and Heath, 1995; Thun, 1996; Thun et al., 2002). Although many reports suggest a clear relationship between NSAID usage and cancer chemoprevention, the exact molecular mechanism by which NSAIDs exert their antitumorigenic effect is not clear. A substantial body of data suggests that COX-2 overexpression promotes tumor progression, including resistance to apoptosis, increased invasiveness, and angiogenesis (Tsujii et al., 1998). Furthermore, COX-1 expression also plays an important role in tumorigenesis (Tiano et al., 2002). Thus, COX inhibition by NSAIDs probably plays an important role in antitumorigenesis. Other findings indicate that COX-independent mechanisms are also responsible for the chemopreventive activity of COX inhibitors. For instance, the R-enantiomer of flurbiprofen does not inhibit COX but has chemopreventive activity in the mouse model of intestinal polyposis (Wechter et al., 1997), prostate cancer (Wechter et al., 2000), and in vitro (Grosch et al., 2003). In addition, sulindac sulfone, which is not a COX inhibitor, inhibits azoxymethane-induced colon tumors in rats (Piazza et al., 1997). Furthermore, noneCCOX-expressing cells including human colorectal HCT-116 cells were shown to undergo NSAID-induced apoptosis (Baek et al., 2001b). Thus, both COX-dependent and COX-independent pathways seem to be involved in the chemopreventive activity of NSAIDs.
NAG-1 was identified as a target gene for NSAIDs, and a significant increase in the expression of this protein was observed in cultured cells treated with some NSAIDs and mice treated with chemopreventive doses of the prodrug sulindac (Baek et al., 2001b; Kim et al., 2002). The changes in NAG-1 expression were not dependent on the inhibition of COX but could provide a possible explanation for antitumorigenic activities independent of COX inhibition. NAG-1 is a unique member of the transforming growth factor- superfamily with incompletely characterized biological activity, but studies with xenograft mouse models confirm that NAG-1 has antitumorigenic activity. The increase in NAG-1 expression results in the induction of apoptosis in several cancer cell lines, including human colorectal HCT-116 cells (Li et al., 2000; Tan et al., 2000; Baek et al., 2001b). NAG-1 expression is induced not only by NSAIDs, but also by several antitumorigenic compounds, including dietary chemicals, peroxisome proliferator-activated receptor- ligands, and p53 activators. NAG-1 contains a p53 binding site in the 5' upstream region (Li et al., 2000; Baek et al., 2001a), and several dietary compounds induce NAG-1 expression by increasing p53 expression (Baek et al., 2002a; Wilson et al., 2003). In fact, NAG-1 was recently reported to be the most highly induced gene by p53 as measured with cDNA array technology (Robles et al., 2001). However, mutations commonly occur at the p53 tumor suppressor locus in many forms of cancer, including colorectal cancer. The increase in NAG-1 expression by NSAIDs is independent of p53, suggesting that NSAIDs could still increase NAG-1 expression in tumors with mutations in p53. In this report, we found that the regulation of NAG-1 by NSAIDs is mediated by the transcription factor EGR-1. Our findings indicate that an increase in EGR-1 expression occurs before NAG-1 expression and is required for the increased transcriptional activity of NAG-1 by NSAIDs (Fig. 5). An EGR-1 binding site was found in the NAG-1 promoter that overlaps with an Sp1 site. Thus, the transcriptional activity of NAG-1 depends on the balance of EGR-1 and Sp1 family members. Indeed, this site in the NAG-1 promoter was identified previously as a troglitazone-response element, and EGR-1 was shown to binds to this site as assessed by electrophoretic mobility shift assay (Baek et al., 2004). The expression of Sp1 is not altered in the presence of sulindac sulfide, whereas EGR-1 expression is increased. The expression of EGR-1 also increases NAG-1 transcription and will enhance sulindac sulfide-induced NAG-1 expression. Overlapping consensus sequences for EGR-1 and Sp1 have been described in the regulatory elements of numerous cytokine genes, including M-CSF (Srivastava et al., 1998) and IL-2 (Decker et al., 1998). Competition for DNA binding between the inducible product of EGR-1 and the constitutively produced Sp1 provides a well-defined means of transcriptional regulation. Troglitazone, a chemopreventive drug but not an NSAID, increases NAG-1 expression also via EGR-1 expression (Baek et al., 2004). In both cases, the expression of EGR-1 occurred before the increase in NAG-1 expression. The EGR-1 expression results in the transcriptional activation of the NAG-1 promoter and hence an increase in NAG-1 expression. However, the mechanisms responsible for the regulation of EGR-1 expression are different. Although sulindac sulfide dramatically increased the promoter activity of EGR-1, troglitazone does not. Troglitazone increased EGR-1 by altering the stability of EGR-1 RNA mediated by increased extracellular signal-regulated kinase 1/2 activity (Baek et al., 2003).
The EGR-1 transcription factor (also know as NGFI-A, Zif268, krox24, and TIS8) is a member of a transcription factor family that contains three zinc fingers and preferentially binds to the GC-rich DNA core sequence. EGR-1 is also a member of the immediate early gene family and encodes a nuclear phosphoprotein involved in the regulation of cell growth and differentiation in response to diverse stimuli. Although several downstream target genes of EGR-1, including growth factors, adhesion molecules, cytokines, cell-cycle components, and coagulation system, are identified, the expression of EGR-1 and its role in cancer are complex (Liu et al., 1998). A number of reports indicate that EGR-1 acts as a tumor suppressor gene. EGR-1 is down-regulated in several types of neoplasia as well as in an array of tumor cell lines (Huang et al., 1997). EGR-1 is induced very early in the apoptotic process, in which it mediates the activation of downstream regulators such as p53 (Nair et al., 1997), but EGR-1eCinduced apoptosis has also been reported in p53eC/eCcells, indicating the existence of both p53-dependent and -independent pathways. EGR-1 may also activate the phosphatase and tensin homolog tumor suppressor gene during UV irradiation (Virolle et al., 2001). The expression of EGR-1 suppresses the growth of transformed cells both in soft agar and in athymic nude mice (Huang et al., 1995). One of the important downstream targets of EGR-1 seems to be NAG-1, a protein that will suppress the growth of cells on soft agar and inhibit tumor growth in the xenographic nude mouse model. NAG-1 may mediate some of the tumor-suppressor activity of this tumor-suppressor gene, but this transcription factor regulates the expression a number of genes. These genes are linked to the regulation of other biological processes such as angiogenesis, vascular injury, and inflammatory stress (Fahmy et al., 2003). The discovery that some COX inhibitors at physiological concentrations increase the expression of EGR-1 opens a new area of investigation and may provide a better understanding of the chemopreventive, pharmacological, and toxicological activities of this class of drugs.
Acknowledgements
We thank Leigh Wilson for technical assistance. We also thank Drs. Tina Sali and Richard DiAugustine of the National Institute of Environmental Health Sciences for their comments and suggestions.
The project was supported by National Institutes of Health grant K22-ES011657 (to S.J.B.).
doi:10.1124/mol.104.005108.
References
Baek SJ, Horowitz JM, and Eling TE (2001a) Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3. J Biol Chem 276: 33384eC33392.
Baek SJ, Kim JS, Nixon JB, DiAugustine RP, and Eling TE (2004) Expression of NAG-1, a transforming growth factor- superfamily member, by troglitazone requires the early growth response gene EGR-1. J Biol Chem 279: 6883eC6892.
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: 901eC908.
Baek SJ, Wilson LC, and Eling TE (2002a) Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53. Carcinogenesis 23: 425eC434.
Baek SJ, Wilson LC, Hsi LC, and Eling TE (2003) Troglitazone, a peroxisome proliferator-activated receptor (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: 5845eC5853.
Baek SJ, Wilson LC, Lee CH, and Eling TE (2002b) Dual function of nonsteroidal anti-inflammatory drugs (NSAIDs): inhibition of cyclooxygenase and induction of NSAID-activated gene. J Pharmacol Exp Ther 301: 1126eC1131.
Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, Zhang HP, Donnellan M, Mahler S, Pryor K, et al. (1997) MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF- superfamily. Proc Natl Acad Sci USA 94: 11514eC11519.
Bottner M, Laaff M, Schechinger B, Rappold G, Unsicker K, and Suter-Crazzolara C (1999) Characterization of the rat, mouse and human genes of growth/differentiation factor-15/macrophage inhibiting cytokine-1 (GDF-15/MIC-1). Gene 237: 105eC111.
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: 773eC778.
Bottone FG Jr, Martinez JM, Collins JB, Afshari CA, and Eling TE (2003) Gene modulation by the cyclooxygenase inhibitor, sulindac sulfide, in human colorectal carcinoma cells: possible link to apoptosis. J Biol Chem 278: 25790eC25801.
Davis W Jr, Chen ZJ, Ile KE, and Tew KD (2003) Reciprocal regulation of expression of the human adenosine 5'-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors. Nucleic Acids Res 31: 1097eC1107.
Decker EL, Skerka C, and Zipfel PF (1998) The early growth response protein (EGR-1) regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated T cells. J Biol Chem 273: 26923eC26930.
Fahmy RG, Dass CR, Sun LQ, Chesterman CN, and Khachigian LM (2003) Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat Med 9: 1026eC1032.
Ferrandez A, Prescott S, and Burt RW (2003) COX-2 and colorectal cancer. Curr Pharm Des 9: 2229eC2251.
Grosch S, Tegeder I, Schilling K, Maier TJ, Niederberger E, and Geisslinger G (2003) Activation of c-Jun-N-terminal-kinase is crucial for the induction of a cell cycle arrest in human colon carcinoma cells caused by flurbiprofen enantiomers. FASEB J 17: 1316eC1318.
Hromas R, Hufford M, Sutton J, Xu D, Li Y, and Lu L (1997) PLAB, a novel placental bone morphogenetic protein. Biochim Biophys Acta 1354: 40eC44.
Huang RP, Fan Y, de Belle I, Niemeyer C, Gottardis MM, Mercola D, and Adamson ED (1997) Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation. Int J Cancer 72: 102eC109.
Huang RP, Liu C, Fan Y, Mercola D, and Adamson ED (1995) Egr-1 negatively regulates human tumor cell growth via the DNA-binding domain. Cancer Res 55: 5054eC5062.
Iizaka M, Furukawa Y, Tsunoda T, Akashi H, Ogawa M, and Nakamura Y (2002) Expression profile analysis of colon cancer cells in response to sulindac or aspirin. Biochem Biophys Res Commun 292: 498eC512.
Jones MK, Wang H, Peskar BM, Levin E, Itani RM, Sarfeh IJ, and Tarnawski AS (1999) Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing. Nat Med 5: 1418eC1423.
Khachigian LM, Williams AJ, and Collins T (1995) Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells. J Biol Chem 270: 27679eC27686.
Kim KS, Baek SJ, Flake GP, Loftin CD, Calvo BF, and Eling TE (2002) Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in human and mouse tissue. Gastroenterology 122: 1388eC1398.
Li PX, Wong J, Ayed A, Ngo D, Brade AM, Arrowsmith C, Austin RC, and Klamut HJ (2000) Placental TGF- is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression. J Biol Chem 275: 20127eC20135.
Liu C, Rangnekar VM, Adamson E, and Mercola D (1998) Suppression of growth and transformation and induction of apoptosis by EGR-1. Cancer Gene Ther 5: 3eC28.
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: 5034eC5040.
McEntee MF, Chiu CH, and Whelan J (1999) Relationship of beta-catenin and Bcl-2 expression to sulindac-induced regression of intestinal tumors in Min mice. Carcinogenesis 20: 635eC640.
Monks A, Harris E, Hose C, Connelly J, and Sausville EA (2003) Genotoxic profiling of MCF-7 breast cancer cell line elucidates gene expression modifications underlying toxicity of the anticancer drug 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole. Mol Pharmacol 63: 766eC772.
Nair P, Muthukkumar S, Sells SF, Han SS, Sukhatme VP, and Rangnekar VM (1997) Early growth response-1-dependent apoptosis is mediated by p53. J Biol Chem 272: 20131eC20138.
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: 557eC564.
Paralkar VM, Vail AL, Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H, Owen TA and Thompson DD (1998) Cloning and characterization of a novel member of the transforming growth factor-b/bone morphogenetic protein family. J Biol Chem 273: 13760eC13767.
Piazza GA, Alberts DS, Hixson LJ, Paranka NS, Li H, Finn T, Bogert C, Guillen JM, Brendel K, Gross PH, et al. (1997) Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels. Cancer Res 57: 2909eC2915.
Raychowdhury R, Schafer G, Fleming J, Rosewicz S, Wiedenmann B, Wang TC, and Hocker M (2002) Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1) and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter. Mol Endocrinol 16: 2802eC2818.
Reddy BS and Rao CV (2002) Novel approaches for colon cancer prevention by cyclooxygenase-2 inhibitors. J Environ Pathol Toxicol Oncol 21: 155eC164.
Ricchi P, Zarrilli R, Di Palma A, and Acquaviva AM (2003) Nonsteroidal anti-inflammatory drugs in colorectal cancer: from prevention to therapy. Br J Cancer 88: 803eC807.
Rice PL, Kelloff J, Sullivan H, Driggers LJ, Beard KS, Kuwada S, Piazza G, and Ahnen DJ (2003) Sulindac metabolites induce caspase- and proteasome-dependent degradation of -catenin protein in human colon cancer cells. Mol Cancer Ther 2: 885eC892.
Robles AI, Bemmels NA, Foraker AB, and Harris CC (2001) APAF-1 is a transcriptional target of p53 in DNA damage-induced apoptosis. Cancer Res 61: 6660eC6664.
Srivastava S, Weitzmann MN, Kimble RB, Rizzo M, Zahner M, Milbrandt J, Ross FP, and Pacifici R (1998) Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1. J Clin Investig 102: 1850eC1859.
Taketo MM (1998) Cyclooxygenase-2 inhibitors in tumorigenesis (part I). J Natl Cancer Inst 90: 1529eC1536.
Tan M, Wang Y, Guan K, and Sun Y (2000) PTGF-, a type beta transforming growth factor (TGF-) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF- signaling pathway. Proc Natl Acad Sci USA 97: 109eC114.
Thun MJ (1996) NSAID use and decreased risk of gastrointestinal cancers. Gastroenterol Clin North Am 25: 333eC348.
Thun MJ and Heath CW Jr (1995) Aspirin use and reduced risk of gastrointestinal tract cancers in the American Cancer Society prospective studies. Prev Med 24: 116eC118.
Thun MJ, Henley SJ, and Patrono C (2002) Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic and clinical issues. J Natl Cancer Inst 94: 252eC266.
Tiano HF, Loftin CD, Akunda J, Lee CA, Spalding J, Sessoms A, Dunson DB, Rogan EG, Morham SG, Smart RC, et al. (2002) Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis. Cancer Res 62: 3395eC3401.
Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, and DuBois RN (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells [published erratum appears in Cell 94(2):following 271, 1998]. Cell 93: 705eC716.
Virolle T, Adamson ED, Baron V, Birle D, Mercola D, Mustelin T, and de Belle I (2001) The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling. Nat Cell Biol 3: 1124eC1128.
Wechter WJ, Kantoci D, Murray ED Jr, Quiggle DD, Leipold DD, Gibson KM, and McCracken JD (1997) R-flurbiprofen chemoprevention and treatment of intestinal adenomas in the APC(Min)/+ mouse model: implications for prophylaxis and treatment of colon cancer. Cancer Res 57: 4316eC4324.
Wechter WJ, Leipold DD, Murray ED Jr, Quiggle D, McCracken JD, Barrios RS, and Greenberg NM (2000) E-7869 (R-flurbiprofen) inhibits progression of prostate cancer in the TRAMP mouse. Cancer Res 60: 2203eC2208.
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: 747eC753., 百拇医药(Seung Joon Baek, Jong-Sik)
Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee (S.J.B., S.-H.L.)
Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to have chemopreventive activity, but the mechanisms involved are not clearly understood. Although NSAIDs inhibit cyclooxygenase activity, they also increase the expression of a divergent member of the transforming growth factor- superfamily, termed NSAID-activated gene 1 (NAG-1), a protein with an antitumorigenic and proapoptotic activity that could in part be linked to the chemoprevention activity of NSAIDs. NAG-1 is induced by some NSAIDs, but the mechanisms responsible are not clear. In this report, we have identified a cis-acting element responsive to NSAIDs located within the eC73 to eC51 region of the NAG-1 promoter. This region contains overlapping EGR-1 and Sp1 binding sites, and mutations in this region suggest that the transcription factors have an important role in NSAID-induced NAG-1 expression. EGR-1 was found to play a critical role in the induction of NAG-1 by sulindac sulfide and other NSAIDs. NSAIDs increase EGR-1 protein expression that occurs before the induction of NAG-1 expression, supporting the hypothesis that EGR-1 is necessary for NSAID-induced NAG-1 expression. Thus, NSAIDs induce the expression of EGR-1, a tumor suppressor gene, providing a novel mechanism to explain, in part, the antitumorigenic properties of some NSAIDs. NAG-1 seems to be an important downstream target protein of this transcription factor, EGR-1, and may mediate the chemopreventive activity of some NSAIDs.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of inflammatory disease, and their anti-inflammatory effects are believed to result from inhibition of prostaglandin H synthase (also known as cyclooxygenase, COX). Two isoforms of prostaglandin H synthase, COX-1 and COX-2, are known; COX-1 is constitutively expressed in many tissues, whereas mitogens, tumor promoters, and growth factors up-regulate the expression of COX-2. COX-2 protein is also up-regulated in human colorectal tumors and regulates tumor growth in animal models (Taketo, 1998; Reddy and Rao, 2002; Ricchi et al., 2003). NSAIDs reduce the number and size of polyps in animal models, and epidemiological studies reveal a 40 to 50% reduction in mortality from colorectal cancer (Thun and Heath, 1995; Thun, 1996). Chemopreventive effects of certain NSAIDs seem to be mediated through both COX-dependent and -independent pathways. However, the target molecules that mediate chemopreventive effects are not elucidated. Identifying possible targets is important to understanding the mechanism of the chemopreventive actions. A number of molecular mechanisms responsible for the chemopreventive effects of NSAIDs have been proposed. One hypothesis is the obvious involvement of COX-2 inhibition, but it is clear that prostaglandin-independent mechanisms are also involved (Jones et al., 1999; McEntee et al., 1999; Baek et al., 2002b; Ferrandez et al., 2003; Rice et al., 2003). In either case, the mechanisms responsible for the chemopreventive activity are not clear. Changes in gene expression by COX inhibitors could provide an explanation for the activity. A number of studies with microarray and PCR-based subtractive hybridization have identified potential target genes (Baek et al., 2001b; Iizaka et al., 2002; Bottone et al., 2003). NAG-1 was identified in our laboratory and is a highly promising target gene because it is a proapoptotic and antitumorigenic protein, and its expression is highly induced by NSAIDs (Baek et al., 2001b). The human NAG-1 cDNA encodes a secreted protein with homology to members of the transforming growth factor- superfamily and has been identified previously as macrophage inhibitory cytokine-1 (Bootcov et al., 1997), placental transformation growth factor- (Li et al., 2000), prostate-derived factor (Paralkar et al., 1998), growth differentiation factor 15 (Bottner et al., 1999), and placental bone morphogenetic protein (Hromas et al., 1997). In mature intestinal epithelial cells, NAG-1 is expressed, but the expression is significantly reduced in human colorectal carcinoma samples and neoplastic intestinal polyps of Min mice (Kim et al., 2002). NAG-1 overexpression from a recombinant adenoviral vector results in an 80% reduction of MDA-MB-468 and MCF-7 breast cancer cell viability (Li et al., 2000), and treatment of prostate cancer cells with purified NAG-1 induces apoptosis (Liu et al., 2003). These data support the hypothesis that NAG-1 is linked to apoptosis and that its reduced expression may enhance tumorigenesis. NAG-1 expression is up-regulated independently of prostaglandin formation in human colorectal cancer cells by several NSAIDs (Baek et al., 2002b), In addition, antitumorigenic compounds such as resveratrol (Baek et al., 2002a), genistein (Wilson et al., 2003), diallyl disulfide (Bottone et al., 2002), retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (Newman et al., 2003), 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (Monks et al., 2003), and peroxisome proliferator-activated receptor- ligands induce NAG-1 expression (Baek et al., 2004). These findings suggest a critical role for NAG-1 in the chemopreventive activity of a number of unrelated chemopreventive chemicals.
The aim of the present study was to identify cis- and trans-acting elements that are responsible for NAG-1 induction by sulindac sulfide and other NSAIDs. During this investigation, we found NSAIDs to increase the expression of EGR-1, a transcription factor and tumor suppressor protein, suggesting that the chemopreventive activity of NSAIDs is mediated via this tumor suppressor protein. NAG-1 was characterized as a key downstream target of EGR-1.
Materials and Methods
Cell Lines and Reagents. Cell lines in this study were purchased from American Type Culture Collection (Manassas, VA). Human colorectal carcinoma cells, HCT-116, were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and gentamicin. All NSAIDs in this study were purchased from Sigma-Aldrich (St. Louis, MO), except for sulindac sulfide, SC-560 (both from Cayman Chemical, Ann Arbor, MI), and [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone] (DFU) (Merck, Whitehouse Station, NJ). All NSAIDs were dissolved in DMSO.
Western Blot Analysis. After reaching 60 to 80% confluence in 10-cm plates, the cells were treated at the indicated concentrations and times with each different NSAID in the absence of serum. The level of NAG-1 was evaluated using Western blot analysis with anti-human NAG-1 antibody reported previously (Baek et al., 2001b). Cell lysates were harvested, and 30 e of total proteins was separated by 15% SDS-polyacrylamide gel electrophoresis and transferred for 1 h onto nitrocellulose membrane (ECL Hybond; Amersham Biosciences Inc., Piscataway, NJ). The blots were blocked for 1 h with 5% skim milk in Tris-buffered saline/0.05% Tween 20 and probed with anti-NAG-1 antibody (1:1000 dilution, 5% skim milk in Tris-buffered saline/0.05% Tween 20 at 4°C overnight). After washing, the blots were treated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed several times. The signal was detected by the enhanced chemiluminescence system (Amersham) and autoradiography.
Transfection of Cells with the Luciferase Reporter System. HCT-116 cells were plated in six-well plates at 2 x 105 cells/well in McCoy's 5A media supplemented with 10% fetal bovine serum. After growth for 16 h, plasmid mixtures containing 1 e of promoter linked to luciferase and 0.05 e of pRL-null (Promega, Madison, WI) were transfected by LipofectAMINE (Invitrogen, Carlsbad, CA). For the cotransfection experiment, 0.5 e of reporter vector and 0.5 e of expression vector were transfected with 0.05 e of pRL-null vector 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-null luciferase activity with a Dual Luciferase Assay Kit (Promega).
Construction of Plasmids. Several NAG-1 promoter clones linked to luciferase have been reported previously (Baek et al., 2001a). The full-length EGR-1 cDNA was generated by PCR from human universal QUICK-Clone cDNA (BD Biosciences Clontech, Palo Alto, CA) using the following primers: 5'-GACACCAGCTCTCCAGCCTGCTCGTCCAGG-3' (top strand) and 5'-TTCCCTTTAGCAAATTTCAATTGTCCTGGG-3' (bottom strand). The amplified products were cloned into pCR2.1 TOPO vector (Stratagene, La Jolla, CA) and followed by cloning into pCDNA3.1/NEO expression vector (Invitrogen). The Sp1 expression vector was described previously (Baek et al., 2001a). The EGR-1 promoter (eC1260 to +35) linked to the luciferase gene (pEGR1260) was cloned by PCR from human Genomic DNA (Promega) with the following primers: 5'-CGGCTCGAGCGGGAGGAGGAGCGAGGAGGCGGCGG-3' (top strand, XhoI site was underlined) and 5'-CCCAAGCTTGGGCGGCGGCGGCTCCCCAAGTTCTGCGGC-3' (bottom strand, HindIII site was underlined). After PCR amplification, the fragment was digested with XhoI and HindIII and ligated into pGLBasic3 luciferase vector. The deletion clones of EGR-1 promoter were generated from pEGR1260 using ExoIII nuclease. All plasmids were sequenced for verification.
Real-Time PCR. HCT-116 cells were pretreated with vehicle or 5 e/ml, followed by the treatment of 30 e sulindac sulfide for 12 h. Cells were washed with phosphate-buffered saline, removed by scraping, and lysed using Nucleic Acid Purification Lysis solution (1x) (Applied Biosystems, Foster City, CA). Total RNA was isolated using ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems) according to the manufacturer's recommendations, quantified, diluted with diethyl pyrocarbonate water, and stored at eC70°C. Total RNA (50 ng) was denatured at 70°C for 10 min and then reverse-transcribed with oligo(dT) primers (1 e) and Moloney murine leukemia virus reverse transcriptase (1.25 U/e) in 10-e reactions at 37°C for 1 h. Real-time fluorescence detection was carried with ABI Prism 7900 Sequence Detection System (Applied Biosystems). 18S rRNA was amplified with a probe dye VIC-MGB using TaqMan Universal PCR Master Mix 1x. NAG-1 was amplified using the following primers: forward, 5'-TGCCCGCCAGCTACAATC-3'; reverse, 5'-TCTTTGGCTAACAAGTCATCATAGGT-3' (0.2 e each) (Research Genetics, Huntsville, AL) with SYBR Green PCR Master Mix (1x) and cDNA (10 e) in a final PCR reaction volume of 50 e. PE 7900 amplification parameters were the following: denaturation at 94°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Reverse transcription and PCR reagents were purchased from Applied Biosystems.
Results
NAG-1 Induction Requires de novo Synthesis. NAG-1 is induced by several NSAIDs at the transcriptional level (Baek et al., 2001b). Sulindac sulfide is very potent, and thus, this NSAID was used as a model for COX inhibitors. To confirm that NSAIDs induce NAG-1 at the protein level, HCT-116 cells were treated with two conventional NSAIDs (sulindac sulfide and indomethacin), a COX-1eCspecific inhibitor (SC-560), a COX-2eCspecific inhibitor (DFU), or a non-COX inhibitor (acetaminophen), and Western analysis was performed. As shown in Fig. 1A, sulindac sulfide highly induces NAG-1 protein expression, whereas indomethacin and SC-560 induce NAG-1 expression to a lesser extent. However, DFU and acetaminophen do not induce NAG-1 expression. This result is consistent with our previous report showing that NAG-1 mRNA expression is induced by sulindac sulfide and indomethacin but not by DFU and acetaminophen (Baek et al., 2001b). To examine whether NSAID-induced NAG-1 expression requires de novo synthesis, HCT-116 cells were pretreated with or without cycloheximide for 30 min, and then followed by incubation with 30 e sulindac sulfide. As shown in Fig. 1B, NAG-1 mRNA was induced by sulindac sulfide treatment by 15.9-fold, but sulindac sulfide could not increase the level of NAG-1 mRNA in the presence of cycloheximide, suggesting that sulindac sulfide-induced NAG-1 expression requires de novo protein synthesis. These data are compatible with the notion that the increase in NAG-1 biosynthesis conferred by sulindac sulfide and presumably other NSAIDs requires, at least in part, de novo synthesis at the transcriptional level.
Effects of NSAIDs on the NAG-1 Promoter Activity. Because NSAIDs induce the expression of NAG-1 mRNA and protein, we examined whether NAG-1 promoter activity is increased in the presence of several NSAIDs. A construct, pNAG1739/LUC, containing the eC1739 to +41 NAG-1 promoter region and luciferase reporter gene was transfected into HCT-116 cells. The cells were then treated with different concentrations of NSAIDs and subsequently assayed for luciferase activity. As shown in Table 1, sulindac sulfide treatment was the most potent inducer of luciferase activity, with an estimated ED50 of 16 e, followed by indomethacin, diclofenac, ibuprofen, piroxicam, naproxen, sodium salicylate, and aspirin. These data are very consistent with Northern and Western data reported previously (Baek et al., 2001b). In addition, celecoxib and acetaminophen did not increase the NAG-1 promoter activity (data not shown), which is consistent with previous data (Fig. 1A), showing that celecoxib and acetaminophen do not induce NAG-1 protein expression in HCT-116 cells.
A construct, pNAG1739/LUC, was transfected into HCT-116 cells and treated with the indicated NSAIDs at different concentrations. As an internal control, pRL-null vector was cotransfected and used to adjust transfection efficiencies. The data shown is the average from three independent experiments. The estimated ED50 was calculated from the 50% induction of their maximum luciferase activity.
Deletion Analysis of NAG-1 Promoter. To evaluate the importance of cis-acting elements regulating NSAID-inducible NAG-1 expression, the 3.5-kb NAG-1 promoter and other deletion clones were transfected into HCT-116 cells and treated with the model NSAID, sulindac sulfide. As an internal control, the plasmid pRL-null (Promega) was used for adjusting transfection efficiency. As shown in Fig. 2A, a large increase in luciferase activity induced by sulindac sulfide treatment was observed with all constructs. These data suggest that there is a positive cis-acting element responsible for sulindac sulfide within eC133-base pair NAG-1 promoter region. As a negative control, the promoterless vector, pGLBasic3, was also transfected into HCT-116 cells and showed no significant luciferase activity after treatment of the cells with sulindac sulfide. A p53 binding site present in the NAG-1 promoter at the +43 position responds to several dietary antitumorigenic compounds (Baek et al., 2002a; Wilson et al., 2003). To examine the importance of the p53 site in sulindac sulfide induced NAG-1 expression, we generated two constructs containing a p53 site at the +43 position in the pNAG133/+70 and pNAG41/+70. These constructs were transfected into HCT-116 cells and treated with sulindac sulfide, and the luciferase activity was measured. As shown in Fig. 2B, sulindac sulfide did not increase the activity of the pNAG41/+70 construct, which has the p53 site, but did increase the activity of the pNAG133/+70 construct. Thus the p53 site at +43 is not involved in the induction of NAG-1 expression by sulindac sulfide. These data are in agreement with a previous report that NSAID-induced NAG-1 expression is p53-independent (Baek et al., 2002b). The NSAID response element is thus located between eC133 and eC41 in the NAG-1 promoter.
Sulindac Sulfide Response Element Is Located in eC73 to eC51 of NAG-1 Promoter Region. Three Sp1 binding sites, Sp1A, Sp1B, and Sp1C, are present in the eC133 to eC41 region of the NAG-1 promoter (Baek et al., 2001a). To further define the responsible element for sulindac sulfide, internal deletion clones pNAG133SP1-I and pNAG133SP1-II were generated and transfected into HCT-116 cells. The pNAG133SP1-I clone has a deletion in the Sp1A site, whereas pNAG133SP1-II has a deletion in the Sp1B and Sp1C sites in the NAG-1 promoter region. It is interesting that Sp1B and Sp1C sites are overlapped with two EGR-1 sites. As shown in Fig. 3A, the deletion mutant pNAG133SP1-II showed no significant induction of luciferase activity after sulindac sulfide treatment, indicating that the sulindac sulfide response element is located in this region containing two Sp1 sites and two EGR-1 sites. To investigate whether other NSAIDs also required this site for NAG-1 induction, pNAG133/LUC-transfected cells were treated with indomethacin, diclofenac, sulindac sulfide, or acetaminophen. As shown in Fig. 3B, all of the compounds except for acetaminophen increase the luciferase activity. In contrast, NSAID treatment of HCT-116 cells transfected with the pNAG133SP1-II construct did not exhibit an increase in luciferase activity, suggesting that this site is critical for NAG-1 induction by NSAIDs, including sulindac sulfide. It should be noted that 30 e sulindac sulfide was used, whereas 100 e was used for the other NSAIDs.
Sp1, EGR-1, and Sulindac Sulfide-Induced NAG-1 Expression. Sp1 and EGR-1 sites have a pivotal role in the sulindac sulfide-induced NAG-1 expression. We next determined whether the expression of Sp1 and/or EGR-1 protein would alter sulindac sulfide-induced NAG-1 expression. An Sp1 or EGR-1 expression vector was cotransfected along with the pNAG133/LUC reporter vector into HCT-116 cells and the cells incubated with sulindac sulfide. Sp1 expression in the absence of sulindac sulfide increased the luciferase activity 1.5-fold, consistent with previous data (Baek et al., 2001a). However, sulindac sulfide treatment in the Sp1-transfected cells did not enhance the luciferase activity relative to the sulindac sulfide-treated vector-transfected cells. In fact, a marginal reduction in the luciferase activity in the Sp1-transfected cells, 6.9-versus 5.5-fold, respectively (Fig. 4A), was observed. These data suggest that Sp1 expression may interfere with sulindac sulfide-induced NAG-1 expression. The promoters of many genes contain a GC box, which may share its binding proteins with Sp1 and EGR-1 (Khachigian et al., 1995; Raychowdhury et al., 2002; Davis et al., 2003). The NAG-1 promoter activity was measured after EGR-1 expression. The cells expressing EGR-1 were treated with sulindac sulfide, and a 2-fold increase luciferase activity (6.9-versus 12.6-fold) was observed. Thus, EGR-1 expression seems to be critical for sulindac sulfide-induced NAG-1 expression in HCT-116 cells. In contrast, Sp1 may compete with EGR-1 in controlling NAG-1 expression by sulindac sulfide. Therefore, to elucidate the relationship between Sp1 and EGR-1 expression, both Sp1 and EGR-1 were transfected with the pNAG133/LUC reporter vector, and luciferase activity was measured with and without sulindac sulfide treatment. As shown in Fig. 4B, the more EGR-1 is expressed, the more NAG-1 promoter activity is induced by sulindac sulfide. The expression of SP-1 protein attenuated the increase in NAG-1 expression by sulindac sulfide. These results indicate that EGR-1 is a key mediator of sulindac sulfide-induced NAG-1 expression, whereas Sp1 may inhibit the sulindac sulfide-induced NAG-1 expression, probably by competing for the same binding sites in the NAG-1 promoter region.
Expression of EGR-1 Is Induced by Sulindac Sulfide. Because EGR-1 plays an important role in sulindac sulfide-induced NAG-1 expression in HCT-116 cells at the transcription level, we sought to determine whether EGR-1 protein is altered during sulindac sulfide treatment in HCT-116 cells. HCT-116 cells were treated with 30 e sulindac sulfide at the indicated times, and cell extracts were analyzed for EGR-1 by Western analysis. EGR-1 expression was increased with treatment with sulindac sulfide. An increase in protein was observed as early as 2 h and then decreased after 16 h of treatment (Fig. 5). In contrast, Sp1 protein expression was not changed during the sulindac sulfide treatment. NAG-1 expression was also increased by sulindac sulfide, but it was observed after the increase in EGR-1 expression. In contrast, incubation with the prodrug sulindac did not increase the expression of EGR-1, Sp1, or NAG-1 in HCT-116 cells. This result is consistent with previous reports that sulindac sulfide induces NAG-1 expression, whereas sulindac does not induce NAG-1 expression (Baek et al., 2001b). These data support the hypothesis that sulindac sulfide-induced NAG-1 expression requires de novo protein synthesis (Fig. 1B) and is mediated by EGR-1.
EGR-1 Promoter Is Activated by NSAIDs. To confirm that sulindac sulfide induces EGR-1 at the transcription level, the EGR-1 promoter was cloned into the luciferase reporter vector. A plasmid, pEGR1260/LUC (Baek et al., 2004), was transfected into HCT-116 cells, and the luciferase activity was measured in response to several NSAIDs. As shown in Fig. 6A, sulindac sulfide greatly enhanced EGR-1 promoter activity by 25-fold, whereas indomethacin, ibuprofen, and diclofenac were less effective in stimulating the EGR-1 promoter activity. Aspirin, piroxicam, and naproxen marginally enhanced the EGR-1 promoter activity at the indicated concentrations. Sulindac sulfone, sulindac, acetaminophen, and DFU did not enhance the luciferase activity (data not shown). Thus, NSAID-induced EGR-1 promoter activity and NSAID-induced NAG-1 promoter activity show a similar pattern of responses to different NSAIDs. To determine whether enhanced EGR-1 production by NSAIDs results in the transactivation of EGR-1 target genes, a construct containing four copies of EGR-1 binding sites pEBS14luc construct was transfected into HCT-116 cells, and the cells were treated with several NSAIDs. As shown in Fig. 6B, sulindac sulfide dramatically enhanced promoter activity, followed by diclofenac and indomethacin. These data indicate that NSAID induces the expression of a functionally active protein, EGR-1, that binds and transactivates EGR-1 target genes.
Discussion
Many epidemiological studies have reported a consistent 40 to 50% reduction in the risk of developing colorectal cancer associated with the use of NSAIDs (Thun and Heath, 1995; Thun, 1996; Thun et al., 2002). Although many reports suggest a clear relationship between NSAID usage and cancer chemoprevention, the exact molecular mechanism by which NSAIDs exert their antitumorigenic effect is not clear. A substantial body of data suggests that COX-2 overexpression promotes tumor progression, including resistance to apoptosis, increased invasiveness, and angiogenesis (Tsujii et al., 1998). Furthermore, COX-1 expression also plays an important role in tumorigenesis (Tiano et al., 2002). Thus, COX inhibition by NSAIDs probably plays an important role in antitumorigenesis. Other findings indicate that COX-independent mechanisms are also responsible for the chemopreventive activity of COX inhibitors. For instance, the R-enantiomer of flurbiprofen does not inhibit COX but has chemopreventive activity in the mouse model of intestinal polyposis (Wechter et al., 1997), prostate cancer (Wechter et al., 2000), and in vitro (Grosch et al., 2003). In addition, sulindac sulfone, which is not a COX inhibitor, inhibits azoxymethane-induced colon tumors in rats (Piazza et al., 1997). Furthermore, noneCCOX-expressing cells including human colorectal HCT-116 cells were shown to undergo NSAID-induced apoptosis (Baek et al., 2001b). Thus, both COX-dependent and COX-independent pathways seem to be involved in the chemopreventive activity of NSAIDs.
NAG-1 was identified as a target gene for NSAIDs, and a significant increase in the expression of this protein was observed in cultured cells treated with some NSAIDs and mice treated with chemopreventive doses of the prodrug sulindac (Baek et al., 2001b; Kim et al., 2002). The changes in NAG-1 expression were not dependent on the inhibition of COX but could provide a possible explanation for antitumorigenic activities independent of COX inhibition. NAG-1 is a unique member of the transforming growth factor- superfamily with incompletely characterized biological activity, but studies with xenograft mouse models confirm that NAG-1 has antitumorigenic activity. The increase in NAG-1 expression results in the induction of apoptosis in several cancer cell lines, including human colorectal HCT-116 cells (Li et al., 2000; Tan et al., 2000; Baek et al., 2001b). NAG-1 expression is induced not only by NSAIDs, but also by several antitumorigenic compounds, including dietary chemicals, peroxisome proliferator-activated receptor- ligands, and p53 activators. NAG-1 contains a p53 binding site in the 5' upstream region (Li et al., 2000; Baek et al., 2001a), and several dietary compounds induce NAG-1 expression by increasing p53 expression (Baek et al., 2002a; Wilson et al., 2003). In fact, NAG-1 was recently reported to be the most highly induced gene by p53 as measured with cDNA array technology (Robles et al., 2001). However, mutations commonly occur at the p53 tumor suppressor locus in many forms of cancer, including colorectal cancer. The increase in NAG-1 expression by NSAIDs is independent of p53, suggesting that NSAIDs could still increase NAG-1 expression in tumors with mutations in p53. In this report, we found that the regulation of NAG-1 by NSAIDs is mediated by the transcription factor EGR-1. Our findings indicate that an increase in EGR-1 expression occurs before NAG-1 expression and is required for the increased transcriptional activity of NAG-1 by NSAIDs (Fig. 5). An EGR-1 binding site was found in the NAG-1 promoter that overlaps with an Sp1 site. Thus, the transcriptional activity of NAG-1 depends on the balance of EGR-1 and Sp1 family members. Indeed, this site in the NAG-1 promoter was identified previously as a troglitazone-response element, and EGR-1 was shown to binds to this site as assessed by electrophoretic mobility shift assay (Baek et al., 2004). The expression of Sp1 is not altered in the presence of sulindac sulfide, whereas EGR-1 expression is increased. The expression of EGR-1 also increases NAG-1 transcription and will enhance sulindac sulfide-induced NAG-1 expression. Overlapping consensus sequences for EGR-1 and Sp1 have been described in the regulatory elements of numerous cytokine genes, including M-CSF (Srivastava et al., 1998) and IL-2 (Decker et al., 1998). Competition for DNA binding between the inducible product of EGR-1 and the constitutively produced Sp1 provides a well-defined means of transcriptional regulation. Troglitazone, a chemopreventive drug but not an NSAID, increases NAG-1 expression also via EGR-1 expression (Baek et al., 2004). In both cases, the expression of EGR-1 occurred before the increase in NAG-1 expression. The EGR-1 expression results in the transcriptional activation of the NAG-1 promoter and hence an increase in NAG-1 expression. However, the mechanisms responsible for the regulation of EGR-1 expression are different. Although sulindac sulfide dramatically increased the promoter activity of EGR-1, troglitazone does not. Troglitazone increased EGR-1 by altering the stability of EGR-1 RNA mediated by increased extracellular signal-regulated kinase 1/2 activity (Baek et al., 2003).
The EGR-1 transcription factor (also know as NGFI-A, Zif268, krox24, and TIS8) is a member of a transcription factor family that contains three zinc fingers and preferentially binds to the GC-rich DNA core sequence. EGR-1 is also a member of the immediate early gene family and encodes a nuclear phosphoprotein involved in the regulation of cell growth and differentiation in response to diverse stimuli. Although several downstream target genes of EGR-1, including growth factors, adhesion molecules, cytokines, cell-cycle components, and coagulation system, are identified, the expression of EGR-1 and its role in cancer are complex (Liu et al., 1998). A number of reports indicate that EGR-1 acts as a tumor suppressor gene. EGR-1 is down-regulated in several types of neoplasia as well as in an array of tumor cell lines (Huang et al., 1997). EGR-1 is induced very early in the apoptotic process, in which it mediates the activation of downstream regulators such as p53 (Nair et al., 1997), but EGR-1eCinduced apoptosis has also been reported in p53eC/eCcells, indicating the existence of both p53-dependent and -independent pathways. EGR-1 may also activate the phosphatase and tensin homolog tumor suppressor gene during UV irradiation (Virolle et al., 2001). The expression of EGR-1 suppresses the growth of transformed cells both in soft agar and in athymic nude mice (Huang et al., 1995). One of the important downstream targets of EGR-1 seems to be NAG-1, a protein that will suppress the growth of cells on soft agar and inhibit tumor growth in the xenographic nude mouse model. NAG-1 may mediate some of the tumor-suppressor activity of this tumor-suppressor gene, but this transcription factor regulates the expression a number of genes. These genes are linked to the regulation of other biological processes such as angiogenesis, vascular injury, and inflammatory stress (Fahmy et al., 2003). The discovery that some COX inhibitors at physiological concentrations increase the expression of EGR-1 opens a new area of investigation and may provide a better understanding of the chemopreventive, pharmacological, and toxicological activities of this class of drugs.
Acknowledgements
We thank Leigh Wilson for technical assistance. We also thank Drs. Tina Sali and Richard DiAugustine of the National Institute of Environmental Health Sciences for their comments and suggestions.
The project was supported by National Institutes of Health grant K22-ES011657 (to S.J.B.).
doi:10.1124/mol.104.005108.
References
Baek SJ, Horowitz JM, and Eling TE (2001a) Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3. J Biol Chem 276: 33384eC33392.
Baek SJ, Kim JS, Nixon JB, DiAugustine RP, and Eling TE (2004) Expression of NAG-1, a transforming growth factor- superfamily member, by troglitazone requires the early growth response gene EGR-1. J Biol Chem 279: 6883eC6892.
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: 901eC908.
Baek SJ, Wilson LC, and Eling TE (2002a) Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53. Carcinogenesis 23: 425eC434.
Baek SJ, Wilson LC, Hsi LC, and Eling TE (2003) Troglitazone, a peroxisome proliferator-activated receptor (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: 5845eC5853.
Baek SJ, Wilson LC, Lee CH, and Eling TE (2002b) Dual function of nonsteroidal anti-inflammatory drugs (NSAIDs): inhibition of cyclooxygenase and induction of NSAID-activated gene. J Pharmacol Exp Ther 301: 1126eC1131.
Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, Zhang HP, Donnellan M, Mahler S, Pryor K, et al. (1997) MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF- superfamily. Proc Natl Acad Sci USA 94: 11514eC11519.
Bottner M, Laaff M, Schechinger B, Rappold G, Unsicker K, and Suter-Crazzolara C (1999) Characterization of the rat, mouse and human genes of growth/differentiation factor-15/macrophage inhibiting cytokine-1 (GDF-15/MIC-1). Gene 237: 105eC111.
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: 773eC778.
Bottone FG Jr, Martinez JM, Collins JB, Afshari CA, and Eling TE (2003) Gene modulation by the cyclooxygenase inhibitor, sulindac sulfide, in human colorectal carcinoma cells: possible link to apoptosis. J Biol Chem 278: 25790eC25801.
Davis W Jr, Chen ZJ, Ile KE, and Tew KD (2003) Reciprocal regulation of expression of the human adenosine 5'-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors. Nucleic Acids Res 31: 1097eC1107.
Decker EL, Skerka C, and Zipfel PF (1998) The early growth response protein (EGR-1) regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated T cells. J Biol Chem 273: 26923eC26930.
Fahmy RG, Dass CR, Sun LQ, Chesterman CN, and Khachigian LM (2003) Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat Med 9: 1026eC1032.
Ferrandez A, Prescott S, and Burt RW (2003) COX-2 and colorectal cancer. Curr Pharm Des 9: 2229eC2251.
Grosch S, Tegeder I, Schilling K, Maier TJ, Niederberger E, and Geisslinger G (2003) Activation of c-Jun-N-terminal-kinase is crucial for the induction of a cell cycle arrest in human colon carcinoma cells caused by flurbiprofen enantiomers. FASEB J 17: 1316eC1318.
Hromas R, Hufford M, Sutton J, Xu D, Li Y, and Lu L (1997) PLAB, a novel placental bone morphogenetic protein. Biochim Biophys Acta 1354: 40eC44.
Huang RP, Fan Y, de Belle I, Niemeyer C, Gottardis MM, Mercola D, and Adamson ED (1997) Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation. Int J Cancer 72: 102eC109.
Huang RP, Liu C, Fan Y, Mercola D, and Adamson ED (1995) Egr-1 negatively regulates human tumor cell growth via the DNA-binding domain. Cancer Res 55: 5054eC5062.
Iizaka M, Furukawa Y, Tsunoda T, Akashi H, Ogawa M, and Nakamura Y (2002) Expression profile analysis of colon cancer cells in response to sulindac or aspirin. Biochem Biophys Res Commun 292: 498eC512.
Jones MK, Wang H, Peskar BM, Levin E, Itani RM, Sarfeh IJ, and Tarnawski AS (1999) Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing. Nat Med 5: 1418eC1423.
Khachigian LM, Williams AJ, and Collins T (1995) Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells. J Biol Chem 270: 27679eC27686.
Kim KS, Baek SJ, Flake GP, Loftin CD, Calvo BF, and Eling TE (2002) Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in human and mouse tissue. Gastroenterology 122: 1388eC1398.
Li PX, Wong J, Ayed A, Ngo D, Brade AM, Arrowsmith C, Austin RC, and Klamut HJ (2000) Placental TGF- is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression. J Biol Chem 275: 20127eC20135.
Liu C, Rangnekar VM, Adamson E, and Mercola D (1998) Suppression of growth and transformation and induction of apoptosis by EGR-1. Cancer Gene Ther 5: 3eC28.
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: 5034eC5040.
McEntee MF, Chiu CH, and Whelan J (1999) Relationship of beta-catenin and Bcl-2 expression to sulindac-induced regression of intestinal tumors in Min mice. Carcinogenesis 20: 635eC640.
Monks A, Harris E, Hose C, Connelly J, and Sausville EA (2003) Genotoxic profiling of MCF-7 breast cancer cell line elucidates gene expression modifications underlying toxicity of the anticancer drug 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole. Mol Pharmacol 63: 766eC772.
Nair P, Muthukkumar S, Sells SF, Han SS, Sukhatme VP, and Rangnekar VM (1997) Early growth response-1-dependent apoptosis is mediated by p53. J Biol Chem 272: 20131eC20138.
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: 557eC564.
Paralkar VM, Vail AL, Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H, Owen TA and Thompson DD (1998) Cloning and characterization of a novel member of the transforming growth factor-b/bone morphogenetic protein family. J Biol Chem 273: 13760eC13767.
Piazza GA, Alberts DS, Hixson LJ, Paranka NS, Li H, Finn T, Bogert C, Guillen JM, Brendel K, Gross PH, et al. (1997) Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels. Cancer Res 57: 2909eC2915.
Raychowdhury R, Schafer G, Fleming J, Rosewicz S, Wiedenmann B, Wang TC, and Hocker M (2002) Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1) and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter. Mol Endocrinol 16: 2802eC2818.
Reddy BS and Rao CV (2002) Novel approaches for colon cancer prevention by cyclooxygenase-2 inhibitors. J Environ Pathol Toxicol Oncol 21: 155eC164.
Ricchi P, Zarrilli R, Di Palma A, and Acquaviva AM (2003) Nonsteroidal anti-inflammatory drugs in colorectal cancer: from prevention to therapy. Br J Cancer 88: 803eC807.
Rice PL, Kelloff J, Sullivan H, Driggers LJ, Beard KS, Kuwada S, Piazza G, and Ahnen DJ (2003) Sulindac metabolites induce caspase- and proteasome-dependent degradation of -catenin protein in human colon cancer cells. Mol Cancer Ther 2: 885eC892.
Robles AI, Bemmels NA, Foraker AB, and Harris CC (2001) APAF-1 is a transcriptional target of p53 in DNA damage-induced apoptosis. Cancer Res 61: 6660eC6664.
Srivastava S, Weitzmann MN, Kimble RB, Rizzo M, Zahner M, Milbrandt J, Ross FP, and Pacifici R (1998) Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1. J Clin Investig 102: 1850eC1859.
Taketo MM (1998) Cyclooxygenase-2 inhibitors in tumorigenesis (part I). J Natl Cancer Inst 90: 1529eC1536.
Tan M, Wang Y, Guan K, and Sun Y (2000) PTGF-, a type beta transforming growth factor (TGF-) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF- signaling pathway. Proc Natl Acad Sci USA 97: 109eC114.
Thun MJ (1996) NSAID use and decreased risk of gastrointestinal cancers. Gastroenterol Clin North Am 25: 333eC348.
Thun MJ and Heath CW Jr (1995) Aspirin use and reduced risk of gastrointestinal tract cancers in the American Cancer Society prospective studies. Prev Med 24: 116eC118.
Thun MJ, Henley SJ, and Patrono C (2002) Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic and clinical issues. J Natl Cancer Inst 94: 252eC266.
Tiano HF, Loftin CD, Akunda J, Lee CA, Spalding J, Sessoms A, Dunson DB, Rogan EG, Morham SG, Smart RC, et al. (2002) Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis. Cancer Res 62: 3395eC3401.
Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, and DuBois RN (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells [published erratum appears in Cell 94(2):following 271, 1998]. Cell 93: 705eC716.
Virolle T, Adamson ED, Baron V, Birle D, Mercola D, Mustelin T, and de Belle I (2001) The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling. Nat Cell Biol 3: 1124eC1128.
Wechter WJ, Kantoci D, Murray ED Jr, Quiggle DD, Leipold DD, Gibson KM, and McCracken JD (1997) R-flurbiprofen chemoprevention and treatment of intestinal adenomas in the APC(Min)/+ mouse model: implications for prophylaxis and treatment of colon cancer. Cancer Res 57: 4316eC4324.
Wechter WJ, Leipold DD, Murray ED Jr, Quiggle D, McCracken JD, Barrios RS, and Greenberg NM (2000) E-7869 (R-flurbiprofen) inhibits progression of prostate cancer in the TRAMP mouse. Cancer Res 60: 2203eC2208.
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: 747eC753., 百拇医药(Seung Joon Baek, Jong-Sik)