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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Prev Res (Phila). Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
PMCID: PMC2789193
NIHMSID: NIHMS145202

The Cox inhibitor, sulindac sulfide inhibits EP4 expression and suppresses the growth of glioblastoma cells

Abstract

EP4 expression in human glioblastoma cells correlates with growth on soft agar. The cyclooxygenase (COX) inhibitor, sulindac sulfide, first altered specificity protein-1 (Sp-1) and, early growth response gene-1 (Egr1) expression, then increased the expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) and activating transcription factor-3 (ATF3), and then decreased EP4 expression. EP4 suppression was dependent on blocking the Sp-1 binding sites in the human EP4 promoter. Mutation in the Sp-1 sites in EP4 altered the promoter activity and abolished sulindac sulfide effects. The inhibitory effect of sulindac sulfide on EP4 expression was reversed by PD98059, an MEK-1/Erk inhibitor. Sp-1 phosphorylation was dependent on sulindac sulfide-induced Erk activation. ChIP assay confirmed that Sp-1 phosphorylation decreases Sp-1 binding to DNA and leads to the suppression of EP4. Inhibition of cell growth on soft agar assay was found to be a highly complex process and appears to require not only the inhibition of COX activity but also increased expression of NAG-1 and ATF3 and suppression of EP4 expression. Our data suggest that the suppression of EP4 expression by sulindac sulfide represents a new mechanism for understanding the tumor suppressor activity.

Keywords: Sp-1 phosphorylation, Cox-1/2, NAG-1/Gdf15, Egr-1

Introduction

Glioblastomas are the most common primary malignant brain tumors. Despite advancements in cancer therapies including surgery, radiation therapy, and chemotherapy, the median survival time is 12 to 15 months for patients newly diagnosed with glioblastoma (1). Shono and his colleagues reported that high cyclooxygenase-2 (COX-2) expression is associated with clinically more aggressive gliomas and is a strong predictor of poor survival (2). Thus, the activation of EP2 and EP4 is a potential modulator of glioblastoma progression. The overexpression of COX-2 is observed in several human cancers and increased COX-2 expression is associated with a poor prognosis in colon (3), breast (4), ovarian (5), and pancreatic cancers (6). The overproduction of PGE2 promotes tumor growth by binding to receptors designated EP1, EP2, EP3, and EP4. The EP receptor signaling pathways control cell proliferation, invasion, apoptosis, and angiogenesis. Deletion of the EP2 and EP4 receptors in mice causes a reduction of tumor growth in colorectal and breast cancers (7). EP2 and/or EP4 expression is up-regulated compared with normal tissues in colorectal (7) and breast (8) cancers and EP4 mRNA expression is up-regulated in human astrocytoma cells (9). Raza et al. reported that the EP4 expression in surgically resected glioblastoma tissues is also up-regulated compared to the expression in tissues from anaplastic astrocytomas (10).

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert effects by inhibiting COX activity, thereby reducing the levels of prostaglandins. Some NSAIDs have chemopreventive effects against the development of human cancers, including glioblastomas (1113). COX inhibitors may be useful, but the recently discovered toxic side effects of the selective COX-2 inhibitors appear to eliminate their clinical use. Sulindac, the pro-drug for sulindac sulfide, a non-selective COX inhibitor, is well documented as an effective drug in preventing the development of intestinal polyps in experimental animals and tumors in humans, but little information is available of prevention of glioblastoma by sulindac. The chemopreventive effect of sulindac sulfide and other NSAIDs, in general, seems to be complex and not exclusively dependent on inhibition of COX activity. Drugs like sulindac sulfide, as well as other chemopreventive chemicals, increase the expression of the tumor suppressor genes NAG-1 (1416) and ATF3 (17) and is mediated by the expression of the transcription factor Egr-1, which alters the expression of a number of genes. (18). Egr-1 sites frequently overlap with Sp-1 binding sites and interplay between Egr-1 and Sp-1 exists. Sulindac sulfide, like tolfenamic acid, independent of new protein synthesis, directly activates the epithelial-specific transcription factor, ESE-1 and facilitates the translocation from the nucleus, thereby increasing the expression of Egr-1 (19). Thus, sulindac sulfide may have two targets in the colorectal cancer cell, Cox inhibition and ESE-1 translocation; however, the relative contribution of non-COX targets like ESE-1 in cancer prevention is not known.

EP2 and EP4 receptors are critical proteins mediating prostaglandin responses. Drugs that inhibit or suppress EP2/EP4 expression could profoundly influence cancer development. The regulation of EP2 and EP4 has not been extensively investigated. PPARγ ligands inhibit the expression of EP2, while PPARγ ligands increase the expression of EP4 in human lung tumor cells (20, 21). The PPARγ ligand TGZ also increases Egr-1 expression by a mechanism independent of PPARγ activation and subsequently increases NAG-1 expression. Recently we characterized a functional Egr-1/Sp-1 site in the human EP4 promoter and demonstrated that TGZ first increased the expression of the EP4 receptor then suppressed EP4 expression (22). The TGZ-mediated increase in expression was dependent on Egr-1, while the reduction in expression was dependent on phosphorylation of Sp-1 protein. Because sulindac sulfide alters Egr-1 expression, we suspected that sulindac sulfide may decrease the expression of EP4 and this decrease in EP4 expression could be important in mediating the anti-tumor activity of sulindac sulfide. The response to sulindac sulfide is complex and may be dependent on both Cox inhibition and changes in the expression of the Egr-1 and altered EP4 expression. Because the expression of EP4 is up-regulated in glioblastoma (10), glioblastoma cells maybe a useful model system to investigate the complex mechanism for sulindac sulfide-induced chemopreventive activity.

In this report, we used glioblastoma cells expressing COX to investigate this problem. Sulindac sulfide reduced EP4 expression in human glioblastomas via changes in the Egr-1/Sp1 pathway and inhibited the growth of glioblastoma cells on soft agar. Growth inhibition as related to inhibition of COX activity, alteration in the expression of NAG-1 and ATF3, and suppression of EP4 expression was investigated.

Material and Methods

Cell Lines and Reagents

Human glioblastoma T98G, U118, and U87 cells and low grade glioma Hs683 cells were purchased from American Type Culture Collection (Manassas, VA). All cells were grown in Eagle’s minimal essential medium (EMEM) with 1mM MEM Sodium Pyruvate Solution (Gibco, Grand Island, NY), 2mM L-Glutamine (Gibco), 10 µg/ml gentamicin (Gibco), and 10% fetal bovine serum (FBS). Indomethacin, sulindac, sc-560 (COX-1 inhibitor), sc-58125 (COX-2 inhibitor), anti-EP4 antibody, anti-COX-1 antibody, and anti-COX-2 antibody were purchased from Cayman Chemical Co. Inc. (Ann Arbor, MI). Sulindac sulfide and acetaminophen were purchased from Sigma-Aldrich (St. Louis, MO). The MEK-1/Erk inhibitor PD98059 was purchased from EMD Biosciences (San Diego, CA) and the anti-phospho-Erk MAPK antibody (Thr202/Tyr204), anti-Egr-1, and anti-phospho-threonine antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Sp-1 (sc-59), anti-Sp-3 (sc-644), anti-Sp-4 (sc-645), anti-Erk 1 (sc-93), anti-Erk 2 (sc-154), anti-ATF-3 (sc-188), and anti-actin (sc-1615) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-NAG-1 antibody was reported previously in our laboratory (23).

Western Blot Analysis

After reaching 70 to 80% confluence, T98G cells were starved overnight with serum-free medium, then treated at the indicated concentrations and times with different drugs in the absence of serum. Total cell lysates were isolated in RIPA buffer [1X PBS, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF, 1 µM Okadaic acid, 10 mM β-glycerolphosphate, and Complete Mini protease inhibitor cocktail tablets from Roche (Indianapolis, IN)]. Quantitation of protein was performed by BCA assay (Pierce, Rockfold, IL), and 30 µg of total proteins were separated by SDS-PAGE 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA) and transferred onto a nitrocellulose membrane (Invitrogen). The blots were blocked for 1 h with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) (Sigma-Aldrich) and probed overnight at 4 °C with 5% skim milk in TBS-T with each primary antibody. After washing with TBS-T, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature with 5% skim milk in TBS-T and washed several times in TBS-T. Proteins were detected by the enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ).

Immunoprecipitation

One mg of nuclear extracts was prepared with the Nuclear Extract kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. The nuclear extract was precleaned with 60 µl of Protein A/G PLUS-Agarose (sc2003, Santa Cruz) by incubating for 1 h at 4 °C with rotator. After pelleting agarose beads, the supernatant was transferred to a new tube and immunoprecipitated with 5 µg of anti-Sp-1 antibody (sc-59 AC, Santa Cruz) for 3 h at 4 °C. For a negative control, normal rabbit IgG (sc-2027, Santa Cruz) was added to the sample instead of anti-Sp-1 antibody. After removal of the supernatant and washing the beads four times with RIPA buffer, the beads were resuspended in 30 µl of lysis buffer [1X LDS (Invitrogen), 1X sample reducing agent (Invitrogen), 1% SDS and 10 mM DTT] and boiled for 5 min at 100 °C. The samples were separated by SDS-PAGE 4–12% Bis-Tris gel and transferred onto a nitrocellulose membrane. The immunoprecipitated proteins were analyzed by immunoblotting using anti-phosphothreonine (p-Thr) antibody.

Real-time RT-PCR

Real-time RT-PCR assays using an ABI Prism 7700 (Applied Biosystems, Foster City, CA) were performed as previously described by our laboratory (24). Real time RT-PCR fluorescence detection was performed in 96-well plates with Quantitect SYBR Green buffer (Qiagen). The sequences of PCR primers (Invitrogen) for human EP4 and actin were designed according to the published data (9) and described previously (22). The experiments were performed in duplicate three times with individual time-matched vehicle-treated controls for each gene tested. Amplified product size was routinely checked by gel electrophoresis on a 1% agarose gel in the presence of 0.1 µg/ml ethidium bromide then visualized under UV light to confirm that only one product was formed.

Constructions of Plasmid

The human EP4 promoter luciferase constructs have been described previously (22). The Sp-1-dependent reporter plasmid (pGAGC6), the Egr-1-dependent reporter plasmid (pEBS14), the Sp-1 expression plasmid, and Egr-1 expression plasmid have been reported previously (16, 22, 23, 25). The mThr453/mThr739 Sp-1 expression plasmid, which has two mutations of residues Thr453 and Thr739 was produced using the PCR primers described previously (26). The EP4 cDNA being sub-cloned into pcDNA3.1was purchased from UMR cDNA Resource Center (Rolla, MO).

Luciferase Reporter Assay

T98G cells were seeded in 6-well plates at 2 × 105 cells/ well in EMEM supplemented with 10% fatal bovine serum and grown to 50–60% confluence. The plasmid mixtures containing 2 µg of EP4 promoter luciferase construct and 0.05 µg of pRL-null (Promega, Madison WI) were transfected using FuGENE 6 Transfection Reagent (Roche) according to the manufacturer’s protocol. The co-transfection experiment was carried out using plasmid mixtures containing 1 µg of pEP4-3 luciferase construct, 1 µg of expression plasmid (Sp-1, Egr-1, or mutant Sp-1) and 0.05 µg of pRL-null. The pcDNA3.1 empty vector (Invitrogen) was used as a negative control for the expression plasmid. After 24 h transfection, the cells were treated with indicated concentrations of drugs or control (0.1% Me2SO) for an additional 24h. Finally, the cells were harvested in 1× luciferase lysis buffer (Promega) and luciferase activity was measured and normalized with the values of pRL-null luciferase activity using a dual luciferase assay kit (Promega).

Colony Formation in Soft Agar Assay

T98G cells were suspended at 2 × 103 cells in 1 ml of 0.35% agar solution containing MEM, 10% FBS, 1mM MEM Sodium Pyruvate Solution, 2 mM L-Glutamine and the final concentration of sulindac sulfide, then layered on top of a 0.7% agar layer in 6-well plates. For EP4 knock-down or overexpression, T98G cells were transfected with 100 nM of EP4 siRNA (M-005714-00, Dharmacon) or 5 µg of EP4 cDNA and then the effect of EP4 knock-down or overexpression was confirmed by Western blot analysis. After 24 h transfection, the cells were trypsinized and re-suspended in 0.35% agar solution. Plates were incubated for 2 weeks at 37 °C in a 5% CO2 humidified atmosphere. Cell colonies were visualized following an overnight stain with 0.5 ml of p-iodonitrotetrazolium violet (Sigma-Aldrich) and examined microscopically. These were determined as mean colony number examined in 10 randomly chosen microscope fields.

Chromatin Immunoprecipitation (ChIP) Assay

A ChIP assay was performed using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s protocol. The details of the methods and conditions in the ChIP assay have been described previously (22). The immunoprecipitation was carried out using antibodies against Sp-1 (sc-59X, Santa Cruz), Egr-1 (sc-110X) or normal rabbit IgG overnight at 4 °C. The 136 bp products were resolved on a 2% agarose gel and visualized under UV light. PCR primers for a non-binding region of the promoter were used for controls.

Prostaglandin E2 Immunoassay

T98G cells were placed in a 10cm dish at 2 × 106 cells in complete media and grown to full confluence. The cells were pretreated with different concentrations of sulindac sulfide as indicated for 5 min before the addition of arachidonic acid (100 µM). After 1 h, cell culture supernatants were corrected, and the concentrations of PGE2 were measured using a PGE2 EIA kit (Cayman) according to the manufacturer’s protocol. Briefly, cell culture supernatants were incubated with PGE2 acetyl cholinesterase tracer and PGE2 antibody in 96-well plates for 18 h at 4 °C. After rinsed, the plates were incubated with Ellman’s reagent for 1 h and then read at a 405nm wavelength by a micro plate reader.

Statistical Analysis

Statistical differences between experimental groups were evaluated by the two-tailed unpaired Student t test.

Results

Sulindac Sulfide Suppresses the EP4 Expression in T98G Cells

T98G cells were treated with indomethacin (25 µM), sulindac sulfide (30 µM), sulindac (30 µM), sc-560 (COX-1 specific inhibitor, 25 µM), sc-58125 (COX-2 specific inhibitor, 25 µM), acetaminophen (100 µM), or vehicle for 48 h. As shown in Fig. 1A and 1B, only sulindac sulfide at these concentrations significantly suppressed EP4 expression at protein and mRNA levels. The inhibition of EP4 expression by sulindac sulfide was concentration-dependent (Fig. 1C). As shown on Fig. 2, the suppression of EP4 expression by sulindac sulfide was also examined in other glioblastoma cell lines (U87 and U118) and low grade glioma cell line (HS683) that have different patterns of COX-1 and COX-2 expression. T98G cells express COX-1, but not COX-2 (Fig. 2A). Treatment with 30 µM sulindac sulfide suppressed the expression of EP4 mRNA in several glioma cell lines (Fig. 2B). Thus, inhibition of EP4 expression by sulindac sulfide appears to be a general response in glioblastoma cells.

Figure 1
EP4 expression in T98G cells
Figure 2
COX and EP4 expression in several glioma cells

Sulindac Sulfide Suppresses EP4 Promoter Activity

To determine whether EP4 suppression by sulindac sulfide occurred at the transcriptional level and to examine the cis-acting elements in the human EP4 promoter, luciferase assays were done with human EP4 promoter constructs in T98G cells. As shown in Fig. 3A, a significant decrease in luciferase activities by sulindac sulfide treatment was observed with pEP4-1 to -3 constructs. However, sulindac sulfide treatment did not decrease luciferase activity after transfection of the pEP4-4 construct. This result suggests that the response element to sulindac sulfide could be located in the region between −197 and −160 of the EP4 promoter. In this region, the promoter contains two Sp-1 binding sites (Sp-1A and Sp-1B) and one AP-2α binding site overlapping with the Sp-1B site (26). We generated point mutations in the Sp-1 (ccgccc → cTTccc) and AP-2α (ccccccg → ccccTTg) binding sites of the pEP4-3 construct. Mutations in the AP-2α site did not alter the luciferase activities after sulindac sulfide treatment compared to the wild pEP4-3 construct. In contrast, point mutations in both Sp-1A and Sp-1B binding sites together caused a dramatic reduction in the sulindac sulfide inhibition of luciferase activity compared to the wild pEP4-3 construct, indicating that the two Sp-1 sites are important for transcription of the human EP4 gene (Fig. 3B).

Figure 3
EP4 promoter activity is induced by Sulindac Sulfide in T98G cells

Effects of Sp-1 or Egr-1 Binding on the Human EP4 Promoter

Proteins in the Sp family are known to bind to the GC-rich box. In addition, Egr-1 binding sites frequently overlap with the Sp-1 binding site. To determine which Sp protein, and if Egr-1 binds to the Sp-1/Egr-1 binding sites in the human EP4 promoter, we carried out a ChIP assay experiment using primers that amplify the response element containing two Sp-1 binding sites. As shown in Fig. 4A, only Sp-1 and Egr-1 were bound to the response element. Immunoprecipitation using anti-IgG, anti-Sp-3 or anti-Sp-4 failed to produce PCR products. The pEP4-3 luciferase construct and Egr-1 or Sp-1 expression plasmid were cotransfected into T98G cells. Interestingly, transfection of the Egr-1 expression plasmid increased EP4 luciferase activity, and this effect was further enhanced by sulindac sulfide treatment. In contrast, Sp-1 overexpression increased EP4 luciferase activity, but sulindac sulfide treatment markedly decreased the activity as compared to control (Fig. 4B). To further confirm the effect of Egr-1 and Sp-1 binding at the transcriptional level, the Egr-1 reporter plasmid that contains 4 conventional Egr-1 binding sites (pEBS14-luc) or the Sp-1 reporter plasmid that contains 6 conventional Sp-1 binding sites (pGAGC6-luc) was transfected into T98G cells and subsequently treated with several drugs as indicated. As shown in Fig. 4C and 4D, only sulindac sulfide significantly increased luciferase activity after transient transfection of pEBS14-luc or the Egr-1 binding sites while sulindac sulfide decreased luciferase activity after transfection with pGAGC6-luc or Sp-1 binding sites. These data support the hypothesis that Egr-1 and Sp-1 proteins have opposite effects on human EP4 promoter after sulindac sulfide treatment.

Figure 4
Effects of Sp-1 or Egr-1 binding on the human EP4 promoter

The Location of Putative Egr-1/Sp-1 Binding Site in the EP4 Promoter

pEP4-3 or pEP4-4 plasmids were transfected with Egr-1 expression plasmid into T98G cells, then treated with sulindac sulfide. The pcDNA3.1 plasmid was used as a control of Egr-1 expression plasmid. As shown in Fig. 5A, after transfection of pEP4-4, sulindac sulfide failed to increase EP4 luciferase activity relative to control. However, after transfection of pEP-4-3, Egr-1 expression further increased luciferase activity, suggesting that a putative Egr-1 binding site is located in the region between −197 and −160. This result coincided with the result of the ChIP assay experiment (Fig. 4A). We next transfected several mutant Sp-1 or AP-2α luciferase constructs with Egr-1 expression plasmid into T98G cells and subsequently measured EP4 luciferase activity after sulindac sulfide treatment. As shown in Fig. 5B, the luciferase activity of mutant Sp-1A and Sp-1A, B constructs failed to increase after Egr-1 expression. This result suggests that the putative Egr-1 binding site overlaps with the Sp-1A binding site (Fig. 5C).

Figure 5
The location of the putative Egr-1 binding site in the EP4 promoter

Time-dependent changes in EP4 Expression after Sulindac Sulfide Treatment

Egr-1 and Sp-1 proteins are induced after sulindac sulfide treatment at different times. As shown in Fig. 6A, Egr-1 expression was induced about 3.5-fold after 4 h treatment of sulindac sulfide and then declined. In contrast, Sp-1 protein expression increased at 8 and 16 h after treatment and then also declined. Sp-3 and Sp-4 expression did not change. EP4 protein first increased at 4 and 8 h after sulindac sulfide treatment and then declined to below initial levels at 24 and 48 h (Fig. 6B). We propose that the increase in Egr-1 expression mediates the increase in EP4 protein expression, while Sp-1 may be responsible for the decrease in expression. This hypothesis is in agreement with results obtained with Egr-1 or Sp-1 expression on EP4 promoter luciferase assays.

Figure 6
Time-dependent changes in Sp family, Egr-1, and EP4 expression after Sulindac Sulfide treatment of T98G cells

MEK-1/Erk Inhibition and Expression Changes of EP4 by Egr-1 and Sp-1

Phosphorylation of threonine-residues in Sp-1 by activated Erk is involved in the Troglitazone-induced EP4 suppression (22). Erk activation could be involved in sulindac sulfide-induced changes in EP4 expression mediated by Egr-1 and Sp-1. The phosphorylation of Erk protein after sulindac sulfide treatment was first confirmed by Western analysis. As shown in Fig. 7A, sulindac sulfide treatment markedly and rapidly induced Erk phosphorylation. We pretreated T98G cells with the MEK-1/Erk inhibitor, PD98059, for 1 h before the addition of sulindac sulfide. The MEK-1/Erk inhibitor reduced the phosphorylation of Erk1/2 and Egr-1 or Sp-1 expression induced by sulindac sulfide (Fig. 7B). Next, the cells were pretreated with PD98059 for 1 h followed by 8 h or 48 h sulindac sulfide treatment. As shown in Fig. 7C, the increase of EP4 protein observed at 8 h treatment with sulindac sulfide was significantly inhibited by PD98059. Furthermore, the suppression of EP4 protein observed at 48 h treatment with sulindac sulfide was reduced by co-treatment with the Erk inhibitor. Thus, an increase in EP4 protein expression is followed by a decrease in expression after sulindac sulfide treatment. Both the increase and decrease in expression are mediated by Egr-1 and Sp-1, respectively, and are also dependent on the Erk pathway.

Figure 7
Egr-1, Sp-1, and EP4 expression are dependent on the MEK-1/Erk pathway

Sp-1 Phosphorylation and EP4 Suppression

Sp-1 is phosphorylated at two specific threonine residues (Thr453, Thr739) and at other sites on the EP4 promoter by Erk (27). Phosphorylated threonine-residues in Sp-1 after sulindac sulfide treatment were measured by immunoprecipitation of Sp-1 followed by Western blot analysis using the phospho-threonine-specific antibody. The phosphorylated threonine residues in Sp-1 were detected after treatment with sulindac sulfide and were reduced by 1 h pretreatment with 30 µM PD98059 (Fig. 8A). Moreover, we cotransfected pEP4-3 and the mThr453/mThr739 Sp-1 expression plasmid, which has two mutations in residues Thr453 and Thr739 or the wild Sp-1 expression plasmid into T98G cells, and then treated the cells with sulindac sulfide. EP4 suppression was observed with Sp-1 expression while less inhibition was observed with transient transfection of the mutated Sp-1 expression plasmid (Fig. 8B). Taken together, these data indicate that threonine phosphorylation of Sp-1 induced by activated Erk is required for EP4 suppression by sulindac sulfide.

Figure 8
Phosphorylation of Sp-1 is required for EP4 suppression by sulindac sulfide

The DNA Binding Changes of Egr-1 and Sp-1 Influence the Expression of EP4

As shown in Fig. 6A, Egr-1 and Sp-1 proteins were induced at different time points by sulindac sulfide. We suspected that DNA binding of Egr-1 and Sp-1 to the human EP4 promoter may be dependent on phosphorylation.. We used the ChIP assay to measure DNA binding. To confirm that binding of Egr-1 and Sp-1 to DNA required MEK-1/Erk phosphorylation, we pretreated the cells with PD98059 for 1 h and subsequently added sulindac sulfide for 4 h (the peak for Egr-1 expression) or 16 h,(the peak for Sp-1 expression). The increase in Egr-1 binding and the decrease in Sp-1 binding were attenuated by PD98059 (Fig. 9). Thus, the DNA binding of Egr-1 and Sp-1 to the EP4 promoter requires MEK-1/Erk phosphorylation and correlates with an increase in EP4 expression followed by a decrease in EP4 expression. Thus Sp-1 is essential for the expression of EP4 in T98G cells, and the decrease of Sp-1 binding as a result of phosphorylation by Erk after sulindac sulfide treatment leads to the suppression of EP4 expression.

Figure 9
DNA binding changes of Egr-1 and Sp-1

Soft Agar Growth, Inhibition of PGE2 and Changes in Protein Expression

Soft agar assay was used to estimate the anti-tumorigenic activity of sulindac sulfide. Higher concentrations of drugs are necessary to inhibit the growth of T98G cells on soft agar because the cells require complete medium with 10% FBS for colony formation. In addition to the suppression of EP4 expression, the expression of the tumor suppression proteins NAG-1 and ATF3 can be increased by sulindac sulfide via the MEK-1/Erk/Egr-1 pathway in human colorectal cancer HCT-116 cells (16, 28). Changes in NAG-1 and ATF3 expression after drug treatment were measured to confirm the observations in HCT-116 cells would be observed also in T98G cells. The expressions of both proteins were markedly induced after 48 h treatment with 30 µM sulindac sulfide in T98G cells in serum-free conditions (Fig. 10A). T98G cells were then treated with sulindac sulfide at several concentrations under soft agar assay conditions to determine the dose response relationships for the inhibition of cell growth on soft agar. In addition, we determined the relationship between sulindac sulfide concentration and the inhibition of COX activity, the induction of NAG-1/ATF3 and suppression of EP4. Indomethacin was also used as negative control because at the concentration used, it does not alter Egr-1 and hence NAG-1 or ATF3 expression. As shown in Fig. 10B, tumor growth of the cells on soft agar was inhibited in a concentration-dependent manner with ~25% suppression at 60 µM and ~80% suppression at 120 µM concentration. Sulindac sulfide also inhibited prostaglandin formation, but was considerably less effective for inhibiting COX activity in the presence of serum. At the highest concentration tested, 120 µM, only 40% inhibition of prostaglandin formation was detected. Sulindac sulfide increased the expression of NAG-1 and ATF3, with an approximately 2-fold increase at 60 µM and a 6-fold increase at 120 µM concentrations. In contrast, sulindac sulfide (Fig. 10C) first increased expression then suppressed the expression of EP4 as observed previously. A 50% inhibition was observed at 120 µM. Incubation with indomethacin at dose up to120 µM did not alter colony formation (data not shown).

Figure 10
The association between cell growth, inhibition of PGE2, and changes in protein expressions in sulindac sulfide treatment

Soft agar growth and EP4 Expression in T98G Cells

T98G cells were transiently transfected with EP4 cDNA expression vector or siRNA to correspondingly increase or decrease EP4 expression. As shown in Fig. 10D, EP4 over-expression increased growth while suppression of EP4 by EP4 siRNA decreased growth. Thus, alteration in EP4 expression alters the growth of T98G cells on soft agar. Treatment with sulindac sulfide also inhibited soft agar growth and antagonized the increase in growth observed after transfection of EP4 receptor into the cell. Furthermore, transfection with siRNA for EP4 and treatment with sulindac sulfide inhibited colony formation to a greater extent than either sulindac sulfide or siRNA for EP4 alone. Taken together, these findings indicate that the expression level of EP4 has significant effect on soft agar growth. Thus, sulindac sulfide decreases cell growth, in part, by suppressing the expression of EP4 in T98G cells.

Discussion

In this report, we show for the first time that changes in the expression of EP4 influence the growth of glioblastoma cells and that sulindac sulfide suppresses EP4 expression by altering the expression of Egr-1/Sp-1 transcription factors. Further, we present evidence for a complex mechanism for the inhibition of glioblastoma cell growth by the COX inhibitor and chemopreventive drug sulindac sulfide. Treatment of glioblastoma cells, T98G, with sulindac sulfide inhibits the growth of these cells on soft agar and this is dependent on the concentration of the drug. In addition to COX inhibition, sulindac sulfide treatment alters the Egr-1/Sp1 pathways, resulting in increased expression of NAG-1 and ATF3 and reduced expression of the EP4 receptor. Treatment with another COX inhibitor, indomethacin at concentrations that do not alter the Egr-1/Sp1 pathway, did not alter soft agar growth. Previously, we have reported that Egr-1 protein expression is induced by sulindac sulfide and some other COX inhibitors. The transcription factor Egr-1 binds to the Egr-1 binding site presented in the NAG-1 (18) and ATF3 (28) promoters and results in the increased expression of these two proteins. Data from cells in culture (23) and studies of NAG-1 transgenic mice expressing human NAG-1 (29) support the hypothesis that NAG-1 acts to suppress the development of intestinal tumors. Because sulindac sulfide and other NSAIDs alter the Egr-1/Sp-1 pathways and because of our recent studies showing the suppression of EP4 expression by troglitazone, we suspected that sulindac sulfide and other COX inhibitors would also reduce the expression of EP4 in glioblastoma cells. Of the COX inhibitors examined, sulindac sulfide was the most effective in inhibiting EP4 expression, a finding in agreement with results showing that it is the most potent COX inhibitor for increasing the expression of Egr-1 and its downstream targets NAG-1 and ATF3 (29, 30).

Sulindac sulfide suppresses EP4 expression by increasing the phosphorylation of transcription factor Sp-1. The human EP4 promoter region contains two Sp-1 sites (31) that overlap with an Egr-1 site (22) and mutations of these sites in luciferase promoter studies confirmed that Sp-1/Egr-1 sites are important in regulating EP4 expression and responding to sulindac sulfide treatment. In addition, the ChIP assay and expression studies with Egr-1 and Sp-1 proteins confirmed that the Egr-1 sites are involved in the increased expression of EP4, while Sp-1 sites are important for sulindac sulfide suppression of EP4 expression. We also investigated the involvement of Sp-3 in EP4 suppression by sulindac sulfide, because Sp-3 is known to bind to GC-rich sites and inhibit Sp-1-induced transcription. Sp-3 protein expression was not changed by sulindac sulfide treatment. Additionally, the ChIP assay revealed that Sp-3 did not bind to the TGZ response element and that transfection of Sp-3 siRNA had no effect on EP4 suppression (data not shown). These data indicate that Sp-3 is not involved in sulindac sulfide-induced EP4 suppression.

Erk activation plays a role in the induction and suppression of EP4. Incubation with an Erk kinase inhibitor prevents the changes in NAG-1, ATF3, and EP4 expression by sulindac sulfide. Sp-1 protein is phosphorylated by several kinases including Erk. Five phosphorylation sites (Ser59, Ser131, Thr453, Thr579, and Thr739) have been identified in Sp-1 (27, 32, 33) and threonine phosphorylation at Thr453 and Thr739 occurs in response to Erk activation (27). Phosphorylation of Sp-1 decreases DNA binding activity and transcriptional activation of target genes. In this study, we detected phosphorylated threonine residues in Sp-1 activated by sulindac sulfide-induced Erk. The ChIP assay experiment revealed that sulindac sulfide decreases DNA binding activity of responsible Sp-1 binding sites in the human EP4 promoter (34). Taken together, these data suggest that phosphorylation of Sp-1 is critical and results in a decrease in Sp-1 DNA binding, and hence suppression of transcriptional activation of target genes like EP4 is observed.

Sp-1 is a member of a family of zinc finger transcription factors and binds to the GC-rich sequences, and Sp-1 sites in promoters can overlap with Egr-1 sites. Sp-1 plays important roles in a wide range of cellular processes including cell cycle regulation, hormonal activation, apoptosis, and angiogenesis (35). Egr-1, a transcription factor, is also involved in cell growth and differentiation. Egr-1 has been proposed as a tumor suppressor and regulates the expression of the NAG-1/MIC/GDF15 protein, which is reported to suppress intestinal tumor growth (29). Egr-1 also regulates the expression of ATF3, a transcription factor involved in controlling cell growth, apoptosis, and tumor invasion (28, 36). Sulindac sulfide increases the expression of Sp-1 and Egr-1 and alters the phosphorylation of these proteins via Erk (16, 37). As a consequence of these actions, NAG-1 and ATF3 are induced, followed by suppression of EP4 expression.

With the glioblastoma cells, the expression of EP4 appears to correlate with the grade of the tumor (22). The growth of T98G cells on soft agar, a measure of tumorigenicity, is either increased or decreased by a corresponding change in the expression levels of EP4 in these cells. Sulindac sulfide suppresses growth on soft agar, in part, by reducing EP4 expression. Expression of EP4 attenuates the response to sulindac, while inhibition of EP4 expression by siRNA enhances the sulindac sulfide inhibition of cell growth. Examination of the dose response relationship between growth on soft agar and inhibition of prostaglandin synthesis, induction of NAG-1/ATF3, and suppression of EP4 expression reveals complex relationships. One unexpected observation was that the inhibition (~40%) of prostaglandin formation was observed only at the highest drug concentration and indomethacin, at up to 120µM, did not inhibit cell growth. An increase in NAG-1 and ATF3 expression was observed at lower concentrations, while inhibition of EP4 expression was observed at the highest drug concentration, resulting in a 75% inhibition of growth. Thus, the inhibition of tumor growth by sulindac sulfide is very complex and involves a number of signal transduction pathways. After treatment with sulindac sulfide, an increase in the expression of NAG-1, ATF3, and EP4 was observed. Although NAG-1 and ATF3 expressions remained high, the level of EP4 then declined. The net effect of the changes in protein expression is the suppression of cell growth. Stimulation of Sp-1/Egr-1 pathways and the subsequent changes in the expression of NAG-1, ATF3, and EP-4 appear be very important in growth suppression in these cells.

Thus, we propose one explanation for the effect on chemoprevention activity of sulindac sulfide, in contrast to other COX inhibitors such as indomethacin, is that the drug has two targets, COX inhibition and the ESE-1/EGR-1 pathway. Sulindac sulfide inhibits COX activity and hence reduces the levels of prostaglandins, attenuating the down signaling events that result in tumor growth. In addition, sulindac sulfide activates the ESE-1/EGR-1 pathway that increases the expression of the anti-tumorigenic proteins NAG-1 and ATF3, and down regulates the expression of the EP4 receptor. Because EP4 is required for PGE2 activity, changes in the level of expression should have dramatic effect on prostaglandin biological activity mediated by EP4. The suppression of EP4 expression by sulindac sulfide is a novel mechanism to help explain the anti-tumorigenic property and suggests a new target for the development of drugs for the prevention or treatment of cancer. Because the side effects of specific COX-2 inhibitors appear to preclude their use as cancer prevention drugs, new approaches are necessary. Suppression of EP4 and the induction of NAG-1/ATF3 expression are likely candidates.

Acknowledgements

The authors thank Drs. Paul A. Wade, Karen L. Adelman (NIEHS), Misty R. Bailey (The University of Tennessee, Knoxville, TN) for their critical reading. This research was supported, in part, by the NIH, NIEHS Intramural Research Program.

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