Curcumin inhibits cyclooxygenase-2 transcription in bile acid- and phorbol ester-treated human gastrointestinal epithelial cells
Fan Zhang1,4,
Nasser K. Altorki1,4,
Juan R. Mestre3,4,
Kotha Subbaramaiah2,4 and
Andrew J. Dannenberg2,4,5
1 Department of Cardiothoracic Surgery and
2 Departments of Medicine and Surgery, New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, NY 10021,
3 Head and Neck Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 and
4 Anne Fisher Nutrition Center at Strang Cancer Prevention Center, New York, NY 10021, USA
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Abstract
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We investigated whether curcumin, a chemopreventive agent, inhibited chenodeoxycholate (CD)- or phorbol ester (PMA)-mediated induction of cyclooxygenase-2 (COX-2) in several gastrointestinal cell lines (SK-GT-4, SCC450, IEC-18 and HCA-7). Treatment with curcumin suppressed CD- and PMA-mediated induction of COX-2 protein and synthesis of prostaglandin E2. Curcumin also suppressed the induction of COX-2 mRNA by CD and PMA. Nuclear run-offs revealed increased rates of COX-2 transcription after treatment with CD or PMA and these effects were inhibited by curcumin. Treatment with CD or PMA increased binding of AP-1 to DNA. This effect was also blocked by curcumin. In addition to the above effects on gene expression, we found that curcumin directly inhibited the activity of COX-2. These data provide new insights into the anticancer properties of curcumin.
Abbreviations: AP-1, activator protein-1; CD, chenodeoxycholate; COX, cyclooxygenase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylltetrazolium bromide; NSAIDs, non-steroidal anti-inflammatory drugs; PG, prostaglandin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate.
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Introduction
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Cyclooxygenases (COX) catalyze the synthesis of prostaglandins (PGs) from arachidonic acid. There are two isoforms of COX, designated COX-1 and COX-2. COX-1 is expressed constitutively in most tissues and appears to be responsible for housekeeping functions (1). In contrast, COX-2 is not detectable in most normal tissues but is induced by oncogenes, growth factors, carcinogens and tumor promoters (24).
Multiple lines of evidence support the idea that COX-2 is important in carcinogenesis. Thus, COX-2 is up-regulated in transformed cells (2,5,6) and in malignant tissues (710). A null mutation for COX-2 in APC
716 knockout mice, a murine model of familial adenomatous polyposis, markedly reduces the number and size of intestinal tumors (11). Furthermore, treatment with a selective inhibitor of COX-2 caused nearly complete suppression of azoxymethane-induced colon cancer (12). Several different mechanisms could account for the link between the activity of COX-2 and carcinogenesis. For example, enhanced synthesis of PGs occurs in a variety of tumors (1317); PGs can promote angiogenesis (18), inhibit immune surveillance (19) and increase cell proliferation (20). Overexpression of COX-2 also inhibits apoptosis (21) and increases the invasiveness of malignant cells (22).
Bile acids are known promoters of colorectal cancer and possibly cancer of the upper gastrointestinal tract (2326). The precise mechanisms by which bile acids promote carcinogenesis are unknown, but, like tumor-promoting phorbol esters, bile acids activate protein kinase C (PKC) (27,28) and induce activator protein-1 (AP-1) activity (29,30). We showed that bile acids induce COX-2 and synthesis of PGs via a PKC-dependent mechanism (30). These results provide a plausible explanation for the promotion of colon and esophageal cancer by bile acids. It hence appears that a viable approach for preventing and possibly treating gastrointestinal malignancies is to identify compounds that suppress the induction of COX-2 by tumor promoting bile acids.
Curcumin is a phenolic antioxidant responsible for the yellow color of turmeric that has anti-inflammatory (31) and anticancer properties (3235) (Figure 1
). It inhibits the development of chemically induced tumors of the oral cavity, forestomach, duodenum and colon of experimental animals (3235) and it blocks tumorigenesis in a two-stage model of skin cancer that was promoted by treatment with phorbol ester (36,37). The anti-inflammatory properties of curcumin have been attributed, at least in part, to suppression of PG synthesis (31). In the current work, we have extended prior observations concerning the effects of curcumin on PG synthesis by determining whether curcumin modulates COX-2 gene expression. Our data show that curcumin inhibited COX-2 transcription in bile acid and phorbol ester-treated gastrointestinal cell lines. Curcumin also directly inhibited the activity of COX-2. These data provide a mechanistic basis for the chemopreventive and anti-inflammatory properties of curcumin.
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Materials and methods
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Materials
Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were from Life Technologies (Grand Island, NY). Sodium arachidonate, chenodeoxycholate (CD), curcumin, phorbol 12-myristate 13-acetate (PMA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; thizolyl blue) were from Sigma (St Louis, MO). NS398 and PGE2 were from Biomol Research Labs (Plymouth Meeting, PA). Enzyme immunoassay reagents for PGE2 assays were from Cayman (Ann Arbor, MI). The AP-1 consensus oligonucleotide was obtained from Promega (Madison, WI). [32P]ATP, [32P]CTP and [32P]UTP were from NEN Life Science Products (Boston, MA). Random priming kits were from Boehringer Mannheim Biochemicals (Indianapolis, IN). Nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). Proteinase K, RNase-free DNase, RNase A for nuclear run-offs and 18S rRNA cDNA were from Ambion (Austin, TX). Rabbit polyclonal anti-human COX-2 antiserum was from Oxford Biomedical Research (Oxford, MI). Antibodies to human COX-1, c-Jun and c-Fos were from Santa Cruz Biotechnology (Santa Cruz, CA). Western blotting detection reagents (ECL) were from Amersham (Arlington Heights, IL). The human COX-2 cDNA was generously provided by Dr Stephen M.Prescott (University of Utah, Salt Lake City, UT).
Cell lines
The SK-GT-4 cell line was established from a well-differentiated adenocarcinoma arising in Barrett's epithelium of the distal esophagus (38). The SCC450 cell line was derived from a squamous cell carcinoma of the human esophagus (39). The IEC-18 epithelial cell line was established from rat ileum (40). The HCA-7 cell line was established from a moderately well-differentiated adenocarcinoma of the human colon (41). SK-GT-4, SCC450, IEC-18 and HCA-7 cell lines were maintained as in prior studies (3841). Treatments with CD or PMA were carried out in 1% FCS. The vehicles for CD and PMA were 0.1% ethanol and 0.01% dimethylsulfoxide, respectively. Cellular cytotoxicity was assessed by measurements of release of lactate dehydrogenase (LDH), trypan blue exclusion and MTT assay. Levels of LDH release were measured in the supernatants used for PGE2 analyses. LDH assays were performed according to the manufacturer's instructions. MTT assay was performed according to the method of Denizot and Lang (42). For trypan blue analysis, following treatments for 12 h, cells were combined 1:1 with 0.4% trypan blue and examined for dye exclusion. There was no evidence of toxicity in any of our experiments.
PGE2 production
Levels of PGE2 released by the cells were measured by enzyme immunoassay (30). Amounts of PGE2 produced were normalized to protein concentrations.
Western blotting
Cell lysates were prepared by treating cells with lysis buffer (150 mM NaCl, 100 mM Tris, pH 8.0, 1% Tween 20, 50 mM diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor and 10 µg/ml leupeptin). Lysates were sonicated for 20 s on ice and centrifuged at 10 000 g for 10 min to sediment the particulate material. The protein concentration of the supernatant was measured by the method of Lowry et al. (43). SDSPAGE was performed under reducing conditions on 10% polyacrylamide gels as described by Laemmli (44). The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbin et al. (45). The nitrocellulose membrane was then incubated with a rabbit polyclonal anti-COX-2 antiserum or a polyclonal anti-COX-1 antiserum. Secondary antibody to IgG conjugated to horseradish peroxidase was used. The blots were probed with the ECL western blot detection system according to the manufacturer's instructions.
Northern blotting
Total cellular RNA was isolated from cell monolayers using an RNA isolation kit from Qiagen (Valencia, CA). Aliquots of 10 µg of total cellular RNA per lane were electrophoresed in a formaldehyde-containing 1.2% agarose gel and transferred to nylon-supported membranes. After baking, membranes were prehybridized overnight in a solution containing 50% formamide, 5x sodium chloride/sodium phosphate/EDTA buffer (SSPE), 5x Denhardt's solution, 0.1% SDS and 100 µg/ml single-stranded salmon sperm DNA and then hybridized for 12 h at 42°C with a radiolabeled cDNA probe for human COX-2. After hybridization, membranes were washed twice for 20 min at room temperature in 2x SSPE, 0.1% SDS, twice for 20 min in the same solution at 55°C and twice for 20 min in 0.1x SSPE, 0.1% SDS at 55°C. Washed membranes were then subjected to autoradiography. To verify equivalency of RNA loading in the different lanes, the blot was stripped of radioactivity and rehybridized to determine the levels of 18S rRNA. COX-2 and 18S rRNA probes were labeled with [32P]CTP by random priming.
Nuclear run-off assay
Cells (1x105) were plated in 100 mm dishes. Cells were grown in DMEM containing 10% FCS until ~60% confluent. The medium was then replaced with fresh DMEM containing 1% FCS and vehicle, CD (400 µM), CD and curcumin (10 µM), PMA (50 ng/ml) or PMA and curcumin (10 µM) for 8 h. Nuclei were isolated and stored in liquid nitrogen. The run-off assay was performed as described previously (30).
Electrophoretic mobility shift assay
SK-GT-4 cells were treated with DMEM containing 1% FCS and vehicle, CD (400 µM), CD and curcumin (10 µM), PMA (50 ng/ml) or PMA and curcumin (10 µM) for 6 h. Cells were harvested and nuclear extracts were prepared as described previously (30). For binding studies, an AP-1 consensus oligonucleotide was used: 5'-CGCTTGATGAGTCAGCCGGAA-3' (sense) and 3'-GCGAACTACTCAGTCGGCCTT-5' (antisense). The complementary oligonucleotides were annealed in 20 mM Tris (pH 7.6), 50 mM NaCl, 10 mM MgCl2 and 1 mM dithiothreitol. The annealed oligonucleotide was phosphorylated at the 5'-end with [
-32P]ATP and T4 polynucleotide kinase. The binding reaction was performed by incubating 2 µg of nuclear protein in 20 mM HEPES (pH 7.9), 10% glycerol, 300 µg of bovine serum albumin and 1 µg of poly(dIdC) in a final volume of 10 µl for 10 min at 25°C. For blocking, antibodies to c-Jun or c-Fos were added to the binding reaction for 1 h. Subsequently, the reaction mixture was incubated with the labeled AP-1 consensus oligonucleotide. The labeled AP-1 consensus oligonucleotide was added to the reaction mixture and allowed to incubate for an additional 20 min at 25°C. The samples were electrophoresed on a 4% non-denaturing polyacrylamide gel. The gel was then dried and subjected to autoradiography at 80°C.
Statistics
Comparisons between groups were made by Student's t-test. A difference between groups of P < 0.05 was considered significant.
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Results
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Curcumin inhibits the induction of COX-2 by bile acids and phorbol esters
We investigated the possibility that curcumin inhibited CD- and PMA-mediated induction of PG synthesis in human esophageal adenocarcinoma cells (Figure 2
). CD and PMA caused >10-fold increases in synthesis of PGE2. The effects of CD and PMA were suppressed by curcumin in a dose-dependent manner. The stimulation of PGE2 production by CD and PMA was inhibited completely by 10 µM curcumin.

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Fig. 2. Curcumin suppresses chenodeoxycholate- and phorbol ester-mediated induction of PGE2 synthesis. (A) SK-GT-4 cells were treated with vehicle (stippled column) or CD (400 µM) and curcumin (020 µM, black columns). (B) SK-GT-4 cells received vehicle (stippled column) or PMA (50 ng/ml) and curcumin (020 µM, black columns). Twelve hours later, the medium was replaced with DMEM/F12 containing 1% FCS and 10 µM sodium arachidonate for 30 min. The medium was collected to determine the rate of synthesis of PGE2. Production of PGE2 was determined by enzyme immunoassay. Columns, means; bars, SD; n = 6.
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To determine whether the above effects on production of PGE2 could be related to differences in levels of COX, western blotting of cell lysate protein was carried out. Figure 3A
shows that CD and PMA induced COX-2 in human esophageal adenocarcinoma cells. Co-treatment with curcumin caused a dose-dependent decrease in CD- and PMA-mediated induction of COX-2; the maximal drug effect was observed at 1020 µM. The amounts of COX-1 were not altered by CD, PMA or curcumin (data not shown). The suppression of CD- and PMA-mediated induction of COX-2 by curcumin was not limited to adenocarcinoma cells from the esophagus but was also demonstrable in squamous carcinoma (Figure 3B
), small intestinal (Figure 3C
) and colon carcinoma cells (Figure 3D
).

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Fig. 3. Curcumin causes dose-dependent inhibition of chenodeoxycholate- and phorbol ester-mediated induction of COX-2 in gastrointestinal epithelial cells. Cellular lysate protein (25 µg/lane) was loaded onto a 10% SDSpolyacrylamide gel, electrophoresed and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibody specific for COX-2. Ovine Cox-2 was used as a standard. (A) Lysate protein was from SK-GT-4 cells treated with vehicle (control), CD (400 µM), CD and curcumin (120 µM), PMA (50 ng/ml) or PMA and curcumin (120 µM) for 12 h. (B) Lysates were from SCC450 cells treated with vehicle (control), CD (200 µM), CD and curcumin (120 µM), PMA (50 ng/ml) or PMA and curcumin (120 µM) for 6 h. (C) Lysates were from IEC-18 cells treated with vehicle (control), CD (200 µM), CD and curcumin (520 µM), PMA (50 ng/ml) or PMA and curcumin (520 µM) for 6 h. (D) HCA-7 cells were treated with vehicle (control), CD (200 µM), CD and curcumin (120 µM), PMA (50 ng/ml) or PMA and curcumin (120 µM) for 6 h.
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To further elucidate the mechanism responsible for the changes in amounts of COX-2 protein, we determined steady-state levels of COX-2 mRNA by northern blotting. Treatment with CD and PMA resulted in a marked increase in levels of COX-2 mRNA. This effect was suppressed by curcumin in a concentration-dependent manner (Figure 4
). Comparable effects were observed in SK-GT-4 and SCC450 cell lines.

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Fig. 4. Chenodeoxycholate- and phorbol ester-mediated induction of COX-2 mRNA is suppressed by curcumin. (A) SK-GT-4 cells were treated with vehicle (control), CD (400 µM) or CD and curcumin (110 µM) for 8 h. (B) SK-GT-4 cells were treated with vehicle (control), PMA (50 ng/ml) or PMA and curcumin (520 µM) for 8 h. (C) SCC450 cells were treated with vehicle (control), PMA (50 ng/ml), PMA and curcumin (10 µM), CD (200 µM), CD and curcumin (10 µM) or curcumin (10 µM) for 4 h. Total cellular RNA was isolated; 10 µg of RNA was added to each lane. The northern blots were probed sequentially with probes that recognized COX-2 and 18S rRNAs.
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Curcumin inhibits bile acid and phorbol ester-mediated increases in the transcription of COX-2
Differences in levels of mRNA could reflect altered rates of transcription or changes in mRNA stability. Nuclear run-offs were performed to distinguish between these possibilities. As shown in Figure 5
, higher rates of synthesis of nascent COX-2 mRNA occurred after treatment with CD (Figure 5A
) and PMA (Figure 5B
), which is consistent with the differences observed by northern blotting. These effects were suppressed by curcumin.

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Fig. 5. Curcumin inhibits chenodeoxycholate- and phorbol ester-mediated induction of COX-2 transcription. (A) SK-GT-4 cells were treated with vehicle (lane 1), CD (400 µM, lane 2) or CD and curcumin (10 µM, lane 3) for 8 h. (B) SK-GT-4 cells were treated with vehicle (lane 1), PMA (50 ng/ml, lane 2) or PMA and curcumin (10 µM, lane 3) for 8 h. The COX-2 and 18S rRNA cDNAs were immobilized onto nitrocellulose membranes and hybridized with labeled nascent RNA transcripts. Results of densitometry in arbitrary units: (A), lane 1, 32; lane 2, 213; lane 3, 46; (B), lane 1, 81; lane 2, 266, lane 3, 78.
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Defining the mechanism by which curcumin inhibits bile acid and PMA-mediated induction of COX-2
One of the ways that CD and PMA regulate COX-2 gene expression is by activating the PKC signal transduction pathway (30). A downstream target of activated PKC is the AP-1 transcription factor complex. We determined the effects of CD and PMA on AP-1 binding activity. Electrophoretic mobility shift assay revealed increased binding of AP-1 to DNA following treatment with CD and PMA (Figure 6
). Co-treatment with curcumin inhibited the increase in AP-1 binding mediated by treatment with CD and PMA (Figure 6A
). The AP-1 binding complex was removed by treatment with blocking antibodies to c-Jun or c-Fos (Figure 6B
). These antibodies prevent the binding of c-Jun and c-Fos to AP-1 sites rather than causing a supershift (46).

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Fig. 6. Chenodeoxycholate- and phorbol ester-mediated induction of AP-1 DNA binding activity is suppressed by curcumin. SK-GT-4 cells were treated with vehicle (control), CD (400 µM), CD and curcumin (10 µM), PMA (50 ng/ml) or PMA and curcumin (10 µM) for 6 h. (A) An aliquot of 2 µg of nuclear protein was incubated with a 32P-labeled oligonucleotide containing an AP-1 consensus site. (B) An aliquot of 2 µg of nuclear protein from CD- or PMA-treated cells was incubated with antibodies to c-Jun and c-Fos for 1 h. Subsequently, the reaction mixture was incubated with the 32P-labeled oligonucleotide containing an AP-1 consensus site. The proteinDNA complex that formed was separated on a 4% polyacrylamide gel.
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Curcumin inhibits COX-2 enzyme activity
Curcumin was reported previously to inhibit COX activity directly but its effects on COX-2 have not been evaluated. We investigated the effect of curcumin on the synthesis of PGs by cells in which COX-2 was induced by PMA. In this experiment, SK-GT-4 cells were treated with vehicle or PMA for 8 h. Fresh medium containing either curcumin or the selective COX-2 inhibitor NS398 was then added for 10 min prior to the addition of 10 µM sodium arachidonate. Thirty minutes later, levels of PGE2 were measured. As shown in Figure 7
, both curcumin and NS398 suppressed PMAstimulated synthesis of PGE2. These data indicate that curcumin inhibited COX-2 activity.

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Fig. 7. Curcumin directly inhibits COX-2 enzyme activity. SK-GT-4 cells were treated with vehicle (stippled column) or PMA (50 ng/ml, black columns) for 8 h. PMA was given to induce COX-2. Fresh medium containing (A) curcumin (050 µM) or (B) NS398 (020 µM) was then added for 10 min prior to the addition of 10 µM sodium arachidonate. Thirty minutes later, the medium was collected to determine the amount of PGE2 synthesized. Production of PGE2 was determined by enzyme immunoassay. Columns, means; bars, SD; n = 6. *P < 0.001 compared with PMA.
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Prostaglandins induce COX-2 in some cell lines (47). It was important, therefore, to determine whether the direct inhibitory effects of curcumin on COX-2 activity contributed to its suppression of the activation of COX-2 transcription. We found that curcumin inhibited CD-mediated induction of COX-2 even in the presence of exogenous PGE2 (Figure 8
). Moreover, neither indomethacin nor NS398, which inhibit the synthesis of PGs, blocked CD-mediated induction of COX-2. Taken together, these results indicate that curcumin suppresses the activation of COX-2 transcription via a mechanism that is unrelated to its inhibitory effects on COX enzyme activity.

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Fig. 8. The inhibitory effect of curcumin on chenodeoxycholate-mediated induction of COX-2 is independent of its effects on prostaglandin synthesis. SK-GT-4 cells were treated with vehicle (control), CD (400 µM), CD and curcumin, CD, curcumin and PGE2, CD and NS398 or CD and indomethacin or PGE2 for 12 h. Ovine Cox-2 was used as a standard. Cellular lysate protein (25 µg/lane) was loaded onto a 10% SDSpolyacrylamide gel, electrophoresed and subsequently transferred onto nitrocellulose. The immunoblot was probed with antibody specific for COX-2.
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Discussion
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There is growing evidence that inhibitors of COX-2 activity are useful for treating inflammation (48) and preventing or treating cancer (11,12,49). Therefore, agents that interfere with the signaling mechanisms governing the transcription of COX-2 should also inhibit inflammation and tumorigenesis (4951). The present work shows that curcumin suppressed bile acid- and PMA-mediated induction of PG synthesis by inhibiting the expression and activity of COX-2.
Several of the chemopreventive properties of curcumin can be explained, in part, by inhibition of COX-2. For example, curcumin suppresses the formation of adducts between metabolites of benzo[a]pyrene and DNA (52). This effect of curcumin may be explained by the present results because COX converts a broad array of xenobiotics, including benzo[a]pyrene, to mutagens (53). Treatment with curcumin also stimulates apoptosis in the colon (54) and overexpression of COX-2 in intestinal epithelial cells inhibits apoptosis (21). Moreover, since PGs are mediators of inflammation and chronic inflammation predisposes to malignancy (55), the inhibition of COX-2 by curcumin is likely to contribute to both its anti-inflammatory and chemopreventive activity.
We note that curcumin is not simply an alternative to non-steroidal anti-inflammatory drugs (NSAIDs), which also have anti-inflammatory and chemopreventive effects. This is so because COX is a bifunctional enzyme with cyclooxygenase and peroxidase activities. Aside from being important for PG synthesis, the peroxidase function contributes to the activation of procarcinogens. Therefore, the failure of NSAIDs to inhibit the peroxidase function of COX potentially limits their effectiveness as anticancer agents. In contrast, curcumin down-regulates levels of COX-2 and thereby decreases both the cyclooxygenase and peroxidase activities of the enzyme.
With regard to the mechanism of action of curcumin as a regulator of COX-2 gene expression, it is known that curcumin inhibits PKC activity (56). This is the likely explanation for curcumin-dependent inhibition of bile acid- and PMA-mediated induction of COX-2. Thus, tumor promoting bile acids and phorbol esters induce COX-2 gene expression by activating the PKC signal transduction pathway (30). More specifically, the AP-1 transcription factor is a downstream target of activated PKC that is implicated in inducing COX-2 and promoting carcinogenesis (57). We found that curcumin inhibited bile acid- and PMA-mediated induction of AP-1 binding. Future experiments are needed to confirm this as the mechanism by which curcumin inhibits bile acid and PMA-mediated activation of COX-2 gene expression. Another interesting but unanswered question is whether the same structural properties of curcumin account for inhibition of COX-2 transcription and COX-2 enzyme activity. Analogs of curcumin are needed to determine the relationship between its structure and these different functions as a basis for developing new chemopreventive agents.
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Acknowledgments
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The authors thank Dr David Zakim for his helpful review of this manuscript. This work was supported in part by Astra Merck Inc. (Wayne, PA), US Surgical Corp. (Norwalk, CT) and the Iris Cantor Research Unit. J.R.M. was the recipient of a fellowship award from the Cancer Research Foundation of America.
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Notes
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5 To whom correspondence should be addressed at: New York Presbyterian HospitalCornell Campus, Division of Gastroenterology and Hepatology, Room F-231, 1300 York Avenue, New York, NY 10021, USA Email: ajdannen{at}mail.med.cornell.edu 
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Received July 28, 1998;
revised November 3, 1998;
accepted November 18, 1998.