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Experimental Therapeutics, Preclinical Pharmacology |
in Human Cervical Cancer Cells
Division of Research, Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322 [S. H., L. C. F., N. S.], and Department of Pharmacology, National Cardiovascular Center Research Institute, Osaka, 565-8565, Japan [H. I.]
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ABSTRACT |
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(PPAR), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the control of cyclooxygenase (COX) 2 expression in some tissue, although the exact mechanism(s) of this activity has not been elucidated. In this study we explored the possible mechanism(s) of control of COX-2 gene expression through PPAR signaling in human cervical cancer.
Experimental Design: Using primary human cervical tissues and the CaSki human cervical cancer cell line, we assayed for PPAR and COX-2 mRNA expression by reverse transcription-PCR. Nuclear protein binding activities to three response elements located in the COX-2 promoter [nuclear factor 
B (NFB), cyclic AMP response element, and activator protein (AP)-2] were measured by gel mobility shift assays. We used transient transfection assays with COX-2 promoter reporter gene constructs to determine the regulatory sites in this promoter, which mediates PPAR regulation of COX-2 activity.
Results: We showed, for the first time, that primary human cervical cancer tissues express PPAR. Using CaSki cells, we demonstrated that COX-2 and PPAR mRNA levels were inversely regulated by PPAR ligands in that these compounds up-regulated PPAR but down-regulated COX-2. In contrast, epidermal growth factor (EGF), a potent activator of COX-2, decreased PPAR mRNA levels. This down-regulation of PPAR mRNA by EGF was blocked in the presence of NS-398, a selective COX-2 inhibitor. PPAR ligands suppressed the binding activities of AP-1 (binding to CRE) and NFB but not AP-2. Transient transfection results indicated that EGF stimulated whereas PPAR ligands inhibited COX-2 promoter (-327/+59) activity. This effect by PPAR ligands on the COX-2 promoter was blocked when the CRE, but not the NFB, binding site was mutagenized.
Conlcusion: Cervical cancer cells express readily detectable levels of PPAR. There is reciprocal negative regulation between COX-2 and PPAR signaling in human cervical cancer cells. The ability of PPAR ligands to inhibit COX-2 appears to be mediated predominantly through inhibition of AP-1 protein binding to the CRE site in the COX-2 promoter.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PPARs are ligand-dependent transcription factors belonging to the steroid hormone nuclear receptor superfamily (7
, 8)
. The expression of PPAR has been reported in several organs and tissues, such as liver, lung, skeletal muscle, and monocyte/macrophages, and at high levels in adipose tissue (9, 10, 11)
. Although PPAR is expressed at low levels in normal colonic and breast ductal epithelium, it is increased significantly in both colon and breast carcinoma (12, 13, 14)
. The expression of this receptor in cervical tissue has heretofore not been reported.
PPAR has been implicated in the regulation of critical aspects of development and homeostasis, including cell cycle control and tumor growth inhibition (8
, 15)
. The PGD2 metabolite 15-deoxy-12, 14 PGJ2 (PGJ2) has been identified as a potent natural ligand for PPAR and can be produced by overexpression of COX-2 (16)
. This fact suggests that PPAR signaling may be related to COX-2 gene expression. To this end, there are several reports suggesting a reciprocal interaction between COX-2 expression and PPAR signaling. Thus, induction of COX-2 by 15d-PGJ2 was reported in immortalized epithelial and colorectal cancer cells (16
, 17)
, although 15d-PGJ2 suppressed COX-2 expression in fetal hepatocytes (18)
. The molecular mechanism(s) that underlie PPAR regulation of COX-2 expression remains to be elucidated. In this study, we investigated whether PPAR signaling was involved in COX-2 regulation in human cervical cancer. Our data showed that PPAR ligands can down-regulate COX-2 gene expression and that this regulation is mediated predominately through inhibition of the function of the AP-1 nuclear transcription factor.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Chemicals.
The CaSki human cervical cancer cell line obtained from American Type Culture Collection (Manassas, VA) was grown in RPMI 1640 (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 µg amphotericin (complete medium) as described previously (19)
. Murine 3T3-L1 cells obtained from American Type Culture Collection were grown in complete medium and were induced to differentiate into adipocytes by standard procedures (20)
. These "adipocyte-differentiated" 3T3-L1 cells were used for comparing PPAR expression in adipocytes versus cervical cancer cells. 15d-PGJ2 and Cig were purchased from Alexis Biochemical (San Diego, CA) dissolved in methyl acetate and ethanol, respectively. GW1929 and GW9662 (21
, 22)
were synthesized by the Medicinal Chemistry Department at Glaxo Wellcome Research and Development and were generous gifts from Dr. Timothy M. Willson at same Institute (Glaxo Wellcome Co., Research Triangle Park, NC). The selective COX-2 inhibitor NS-398 (23)
was obtained from Cayman Chemical (Ann Arbor, MI). Recombinant human EGF purchased from R&D systems (Minneapolis, MN) was dissolved in 10 µM acetic acid. Poly(deoxyinosinic-deoxycytidylic acid) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). NFB, AP-1, CRE, and AP-2 oligouncleotides were purchased from Santa Cruz Biotechnology (San Diego, CA); 5' DNA Terminus Labeling System was purchased from Life Technologies, Inc. (Rockville, MD). [-32P]dATP was purchased from Perkin-Elmer Life Sciences, Inc. (Boston, MA). All of the RT-PCR kit components were from Perkin-Elmer Co. (Foster City, CA). All of the other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated.
RT-PCR.
Total RNA was prepared from primary human cervical tissues and cultured CaSki cells by using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. To amplify 474-bp PPAR, 282-bp COX-2, and 200-bp GAPDH cDNA fragments, the sequences of PCR primers synthesized by Genosys (The Woodlands, Texas) were: for PPAR sense (5'-TCTCTCCGTAATGGAAGACC-3'), antisense (5'-GCATTATGAGACATCCCCAC-3'); for COX-2 sense (5'-CTGTATCCCGCCCTGCTGGTG-3'), antisense (5'-ACTTGCGTTGATGGTGGCTGTCTT-3'); and for GAPDH sense (5'-CCATGGAGAAGGCTGGGG-3'), antisense (5'-CAAAGTTGTCATGGATGACC-3') as according to the published data (24
, 25)
. The RT-PCR was carried out as described (24)
. The samples were first denatured at 95°C for 30 s, followed by 32 PCR cycles, each with temperature variations as follows: 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The last cycle was followed by an additional extension incubation of 7 min at 72°C. Analysis of amplicons was accomplished on 1% agarose gel containing 0.2 µg/µl ethidium bromide and visualized under UV transiluminator. The densitometric analysis of PCR products was performed by the computer software (Bio-Rad Quantity One), GS-700 Imaging Densitometer (Bio-Rad, Hercules, CA), and standardized by the GAPDH product. Ratio of PPAR:GAPDH or COX-2:GAPDH density bands in control group was considered as 100%. Values of treatment group PPAR:GAPDH or COX-2:GAPDH ratios are given as a percentage of controls. A 100-bp ladder (Life Technologies, Inc.) was used as a size standard.
For real-time PCR, the treatment and total RNA preparations were the same as those for the RT-PCR procedures described above. All of the PCR reactions using LightCycler-FastStart DNA Master SYBR Green I kit were performed in the Cepheid Smart-Cycler real-time PCR cycler (Sunnyvale, CA). The cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 5 s, and 72°C for 10 s. Primers for PPAR and GAPDH were the same as shown above. Primers used for detection of PPAR in mouse 3T3-L1 cells were sense (5'-CGACTGCTGGTTGACACAGAGATGC-3') and antisense (5'-CGAGATCTGGCCATGAGGGAGTTAG-3'). Quantitative analysis for determining threshold cycle for each sample was performed according to the vendor guidelines. Experiments were performed in triplicate for each data point. For all of the experiments, controls without templates were included.
EMSA.
Nuclear protein extracts from CaSki cells were prepared for EMSA as described earlier (26)
. The protein content of the nuclear extract was determined using the bicinchoninic acid protein assay kit (Sigma). EMSA experiments were performed using double-stranded oligonucleotides comprising the consensus sequences (italicized) for NFB (5'-AGTTGAGGGGACTTTCCCAGGC-3'), CRE (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'), AP-1 (5'-GCTTGATGACTCAGCCGGAA-3'), and AP-2 (5'-GATCGAACTGACCGCCCGCGCCCCGT-3'). The binding site for CRE comprised the cyclic AMP-responsive element. The NFB, CRE, and AP-2 oligonucleotides were end labeled with [-32P]dATP using T4 polynucleotide kinase as recommended by the manufacturer. In the mutated NFB oligonucleotide, the consensus motif was changed to GGcGACTTTCCC. For mutated CRE and AP-2 oligonucleotides, the consensus motifs were changed to TGACTtg and CGCttGCGC, respectively. Five µg nuclear proteins from control and treated cells were incubated with 32P-labeled oligonucleotide probe under binding conditions [10 mM HEPES, Tris-HCL (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 12% (v/v) glycerol, and 2 µg poly(deoxyinosinic-deoxycytidylic acid)] for 20 min at room temperature in a final volume of 20 µl. For cold competition, a 100-fold excess of the respective unlabeled consensus oligonucleotides was added with the probe. The same amount of mutated oligonucleotides added with the probe was used as another control. All of these were in the same binding conditions as described before. After binding, protein-DNA complexes were electrophoresed on a native 4.5% polyacrylamide gel at 120 V using 1x Tris-Glycine buffer [10x Tris-glycine: Tris-base 30.28 g, glycine 142.7 g, EDTA 3.92 g, and H2O added up to 1 liter (pH 8.5)]. Each gel was then dried and subjected to autoradiography at -80°C for up to 72 h. All of the vehicle controls were considered as 100%. Value of treatment groups was given as percentage of controls.
Plasmids.
The COX-2 promoter constructs ligated to luciferase (-327/+59, KBM, CRM, and KBM+CRM) has been reported previously (27)
. Among those, KBM represents the -327/+59 COX-2 promoter construct in which the NFB site (-223/-214) was mutagenized; CRM refers to the -327/+59 COX-2 promoter construct in which the CRE site (-59/-53) was mutagenized; KBM+CRM represent the -327/+59 COX-2 promoter construct in which the both NFB and CRE sites were mutagenized. Synthetic Renilla Luciferase Report Vector (phRL-TK) was obtained from Promega.
Transient Transfection Assays.
CaSki cells were seeded at a density of 4 x 104 cells/well in six-well dishes and grown to 50–60% confluence. For each well, 2 µg of plasmid DNA of the above constructs and 0.3 µg of the internal control plasmid pRL-TK (renilla luciferase gene) were cotransfected into the cells using 3 µl of FUGENE 6 lipofection reagent (Roche Molecular Biochemicals, Indianapolis, IN) as performed in our earlier work (28)
. After 24 h of incubation, cells were treated with PPAR ligands (GW1929 and Cig), EGF, or solvent control for another 24 h. The preparation of cell extracts and measurement of luciferase activities were carried out using the Dual-Luciferase Reporter kit according to recommendations of the manufacturer (Promega). The assays for firefly luciferase activity and renilla luciferase activity were performed sequentially using one reaction tube in a luminometer with one injector. Changes in firefly luciferase activity were calculated and plotted after normalization with changes in renilla luciferase activity in the same sample. All of the vehicle controls were considered as 100%. Values of treatment group firefly luciferase:renilla luciferase ratio were given as percentage of controls.
Statistical Analysis.
All of the experiments were repeated a minimum of three times. All of the gel shift assays, luciferase activity assays, and RT-PCR data were expressed as a mean ± SE. The data in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined with Student’s t test (two-tailed) comparison between two groups of data set. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition (P 
0.05; see figure legends).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genes in Human Primary Cervical Cancer Cells and Cell Lines. in this malignancy. As exemplified in Figs. 1
and 2
, RT-PCR analysis indicated that PPAR was readily detected in both human primary cervical cancer as well as the CaSki cervical cancer cell line. In addition, Fig. 1
shows relatively high expression of both COX-2 and PPAR in malignancies, whereas COX-2 expression was low in nonmalignant samples. Similar results were obtained in a total of 8 cervical cancer and 13 normal tissue samples. To compare PPAR mRNA levels in cervical cancer to that in adipose tissue, which are known to express very high levels of PPAR (29)
, real-time RT-PCR was performed on CaSki cells and adipocyte-differentiated mouse 3T3-L1 cells (30)
. Results showed that the steady-state levels of PPAR mRNA in adipocyte-differentiated 3T3-L1 was 
1000-fold greater than that found in CaSki. These findings represent the first report that PPAR is expressed in either normal or malignant cells of the cervix.
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Signaling. ligands at concentrations of 10–50 µM. Preliminary experiments demonstrated that modulation of COX-2 by PPAR ligands in CaSki cervical cancer cells showed a similar dose dependency (data not shown). Therefore, 20 µM of PPAR ligands were used in many of the following experiments. Fig. 2A
shows that treatment of CaSki cells with 15d-PGJ2 and GW1929 (20 µM each) for 5 h significantly induced PPAR mRNA levels, whereas EGF (20 ng/ml) remarkably inhibited the expression of PPAR as compared with the controls. In contrast, the PPAR ligands decreased COX-2 mRNA levels, whereas EGF, a known activator of COX-2 (4)
, enhanced COX-2 expression (Fig. 2)
. Next we determined whether COX-2 signaling was directly involved in the inhibition of PPAR expression caused by EGF treatment. As shown in Fig. 2B
, this was indeed the case; down-regulation of PPAR mRNA levels induced by EGF was blocked in the presence of NS-398, a selective COX-2 inhibitor, at a concentration of 100 µM. In other experiments, PPAR ligands GW1929 and 15d-PGJ2 at a concentration of 20 µM blocked EGF induction of COX-2 mRNA and suppression of PPAR mRNA (Fig. 3)
, additionally suggesting that suppression of COX-2 could be mediated by PPAR activation. Taken together, these findings suggest a reciprocal interaction between PPAR and COX-2 regulation in cervical cancer cells.
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B Binding Activities Were Inhibited by PPAR Ligands. ligands and EGF. The human COX-2 promoter region contains multiple transcription factor binding sites including a cyclic AMP response element (CRE), and binding motifs for NFB and nuclear factor interleukin-6 (27
, 33)
. Reports showed that AP-1 protein could bind to the CRE site located in the COX-2 promoter region (31)
. In agreement with other studies (34, 35, 36)
, CaSki cells treated with EGF (20 ng/ml) for 24 h slightly increased AP-1 and NFB binding activity as compared with solvent controls. There was no binding activity when the CRE or NFB sites were mutagenized (Figs. 4
and 5
). In contrast, 15d-PGJ2 and GW1929 (20 µM each) inhibited AP-1 and NFB binding as compared with controls. Inhibitory effects on AP-1 by these ligands were blocked in the presence of the PPAR antagonist GW9662 (20 µM), whereas GW9662 alone had no effect on AP-1 binding activity (Fig. 6)
. These findings additionally indicated that the inhibitory effects of 15d-PGJ2 and GW1929 were mediated through the PPAR signaling pathway. Additional experiments were performed to investigate the specificity of these PPAR ligands on AP-1 and NFB binding by assessing their effects on AP-2 binding activity. An AP-2 binding motif is also known to be located in the COX-2 promoter (37)
. Fig. 7
shows that 15d-PGJ2 and GW1929 had little effect on AP-2 binding. As a result of competition assays, specific bands for AP-1 (Fig. 6)
, NFB (Fig. 5)
, and AP-2 (Fig. 7)
were attenuated by a 100-fold molar excess of unlabeled oligonucleotides. These results confirmed that the nuclear protein binding to CRE is AP-1 and that the PPAR ligands (15d-PGJ2 and GW1929) specifically inhibited the binding levels of both AP-1 and NFB transcription factor complexes in human cervical cancer cells.
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Ligands. ligands, transient transfections were performed with human COX-2 promoter constructs. As shown in Fig. 8
, EGF (20 ng/ml) treatment caused 4–5-fold increase in COX-2 promoter (-327/+59) activity, whereas the activity was suppressed to 35–40% of control in the presence of GW1929 or Cig. To additionally test for specific elements that are responsible for mediating the effect of EGF and PPAR ligands, transient transfections were performed using COX-2 promoter constructs in which the CRE, NFB, or both binding sites were mutagenized. As shown in Fig. 9
, mutagenizing the NFB site (KBM construct) caused some decrease in responsiveness to EGF, but no significant effect on GW1929 or Cig inhibitory responses. However, mutagenizing the CRE site (CRM) caused a marked inhibition of responses to GW1929 and Cig, as well as to induction by EGF. Mutations of both the CRE and NFB binding sites almost completely blocked the EGF-dependent promoter activation and totally blocked the PPAR ligand-suppressive effect on COX-2 promoter activity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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genes are expressed in both primary human cervical cancer tissues and the cervical cancer cell line CaSki. The expression of PPAR in cells derived from the cervix has heretofore not been reported. Our results demonstrated reciprocal negative regulation between PPAR and COX-2 gene expression, because up-regulation of COX-2 was coincident with down-regulation of PPAR, and vice versa. The ability of PPAR ligands to down-regulate COX-2 has also been reported in breast cancer cells and macrophages (31
, 32)
, but was shown to up-regulate COX-2 expression in monocytes (38)
, synovial fibroblasts (39)
, and some colon cancer cells (17
, 40) . Taken together, these divergent results indicate that the qualitative nature of the interaction(s) between PPAR and COX-2 (negative or positive) is tissue-specific and extrapolation of findings to different cell types cannot be made.
In light of our demonstration that PPAR ligands negatively regulate COX-2 in cervical cancer cells, the strong expression of both PPAR and COX-2 in primary cervical cancer is surprising and suggests aberrant interaction between these pathways in malignant versus normal tissue. However, it should be remembered that down-regulation of COX-2 by PPAR was dependent on activation with PPAR ligands, and it is not known whether the availability of endogenous ligands (e.g. certain polyunsaturated fatty acids as well as 15d-PGJ2) is different between normal and cancerous tissue. In addition, the fact that the activity of PPAR is also dependent on its phosphorylation status (41)
could provide another potential mechanism for the aberrant functioning of this receptor. Similar conflicting observations have also been reported in the case of colon cancer; that is high tissue expression of both PPAR and COX-2, in the face of their inverse regulation by PPAR and COX-2 activators (42)
. The cause(s) for the simultaneous high expression of PPAR and COX-2 in colon cancer, as in cervical cancer, remains unknown.
In experiments to shed light on the mechanisms(s) responsible for the PPAR-induced inhibition of COX-2 mRNA, electrophoretic mobility shift assays were performed to identify the binding activities in CaSki cells of the nuclear transcription factors AP-1 and NFB. The human COX-2 promoter contains multiple transcription factor binding sites including NFB, CRE, and NF-IL6. Recent reports demonstrated that AP-1 nuclear protein can bind to the CRE site located in the COX-2 promoter region in human mammary cells (31)
. Our results showing that CRE binding activity was attenuated in the presence of 100-fold excess of unlabeled AP-1 oligonucleotides demonstrated that this phenomenon is also true in cervical cancer cells. We showed that PPAR ligand treatment of CaSki cervical cancer cells reduced the binding activities of both AP-1 and NFB nuclear proteins. On the other hand, PPAR ligands had little effect on AP-2, another transcription factor binding motif located in the COX-2 promoter, indicating that PPAR signaling affects only certain transcription factors involved in COX-2 regulation. The blocking effect on AP-1 binding was inhibited in the presence of GW9662, a specific PPAR antagonist, confirming that PPAR signaling was involved in this activity. To determine whether these effects were responsible for PPAR-mediated inhibition of COX-2, transient transfection experiments were performed with a series of COX-2 promoter reporter constructs in which the binding sites for NFB and/or AP-1 nuclear protein were mutagenized. The results demonstrated that down-regulation of COX-2 by PPAR ligands was predominantly mediated by antagonizing the transactivating activity of AP-1 nuclear proteins on the COX-2 promoter. This finding is consistent with that found in normal and malignant human mammary cells where this AP-1 binding to CRE site was shown to play a major role in mediating PPAR ligand regulation of COX-2 expression (31)
. In contrast, it was found that modulation of COX-2 by PPAR ligands in macrophages was largely mediated by NFB (32)
. It is clear that regulation of COX-2 through PPAR signaling is cell-type specific and may also be dependent on developmental processes.
The present data and previous findings have demonstrated that alteration of COX-2 levels can be mediated by a diverse group of seemingly unrelated natural, dietary, and synthetic compounds that have been shown to bind to and activate PPAR (43, 44, 45)
. These include long chain PUFAs such as that found in fish oil (e.g. 
3-PUFA), aromatic small chain fatty acids (e.g. phenylacetate), various eiconsanoids (e.g. 15d-PGJ2), lipid hydroperoxides [e.g. 9(s)-HODE], oxidatively modified lipoproteins (e.g. oxidized low-density lipoprotein), linoleic acid, thiazolidinediones (e.g. rosiglitazone), and a variety of recently synthesized compounds (e.g. GW1929). Studies in our laboratory have used a number of these agents in showing that 15d-PGJ2, GW1929, fish oil, and linoleic acid increased PPAR while decreasing COX-2 mRNA levels. No differences have been seen between oleic acid, which is not a PPAR ligand, and vehicle-treated control groups.3
Because routine screening and diagnostic procedures for cervical cancer have become so well developed, this disease may represent the ideal cancer for chemopreventive intervention by dietary means or long-term pharmaceutical administration. As such, routine screening by Pap smear can detect dysplasia long before micro or frank invasive cancer develops. Thus, it is conceivable that dietary modification that includes high PPAR ligand-containing foods might be able to prevent certain dysplastic progressions and cervical cancer occurrence. This hypothesis is supported by reports showing that populations having frequent intake of foods rich in 3 and 6 PUFA, such as found in boiled or broiled fish, show decreased risk of cervical cancer (46
, 47)
.
In conclusion, our study indicates that there is cross-regulation between COX-2 and PPAR gene expression in human cervical cancer cells. The ability of PPAR ligands to inhibit COX-2 appears to be mediated predominantly through inhibition of AP-1 protein binding to the CRE binding site in the COX-2 promoter. Because COX-2 can act as a promoter of several cancers, the ability of PPAR activators to inhibit COX-2 expression in human cervical cancer suggests that PPAR signaling may be a useful target for therapeutic intervention and/or chemoprevention of this disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Department of Gynecology and Obstetrics, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, GA, 30322. Phone: (404) 727-9155; Fax: (404) 727-8615; E-mail: Nsidell{at}emory.edu 
2 The abbreviations used are: COX, cyclooxygenase; PPAR, peroxisome proliferator-activated receptor; AP, activator protein; RT-PCR, reverse transcription-PCR; 15d-PGJ2, 15-deoxy-
prostaglandin J2; Cig, ciglitazone; NFB, nuclear factor B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; EGF, epidermal growth factor; PUFA, polyunsaturated fatty acid.
Received 10/25/02; revised 4/28/03; accepted 5/ 9/03.
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) or COX-2/GAPDH (
) band densities of at least three independent experiments; bars, ±SD. * indicates significant differences as compared with the vehicle control. ** indicates a significant difference after combination treatment as compared with treatment with EGF alone. NS-398 is known to block COX-2 activity, not COX-2 expression as reflected by no decrease in COX-2 mRNA levels in cells treated with this inhibitor.
