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Carcinogenesis |
Department of Medicine [K. S., A. J. D.] and Surgery [K. S., A. J. D.], New York Presbyterian Hospital-Cornell, New York, New York 10021, USA; Anne Fisher Nutrition Center [K. S., A. J. D.] at Strang Cancer Prevention Center, New York, New York 10021, USA; and Head and Neck Service [P. M.], Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA, and Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA [M. B. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiple lines of evidence suggest that COX-2 is important in
carcinogenesis. COX-2 is overexpressed in transformed cells
(5, 6, 7)
and in various forms of cancer
(8, 9, 10, 11, 12)
, whereas levels of COX-1 remain essentially
unchanged. Moreover, Oshima et al. (13)
showed
that a null mutation for COX-2 markedly reduced the number
and size of intestinal tumors in APC
716
knockout mice, a murine model of familial adenomatous polyposis.
COX-2 deficiency also protected against chemically induced
skin papillomas (14)
. In addition to the genetic evidence
implicating COX-2 in tumorigenesis, selective inhibitors of COX-2
decrease tumor formation in experimental animals (13
, 15
, 16)
. Because targeted inhibition of COX-2 is a promising
approach to prevent cancer, chemopreventive strategies have focused on
inhibitors of COX-2 enzyme activity. An equally important strategy may
be to identify compounds that suppress the signaling pathways that
regulate COX-2 expression (17, 18, 19, 20)
.
Triterpenoids exist widely in nature and are used for medicinal
purposes in many Asian countries. Ursolic acid, a pentacyclic
triterpenoid found in rosemary, possesses anticancer and
anti-inflammatory effects (Refs. 21, 22, 23, 24
; Fig. 1
). It inhibits PMA-induced inflammation and tumor promotion in mouse
skin (21)
. These effects have been attributed, in part, to
inhibition of PG synthesis (21
, 23)
, although the
underlying mechanisms are incompletely understood. In the current work,
we have extended prior observations concerning the effects of ursolic
acid on PG synthesis by determining whether ursolic acid inhibits the
induction of COX-2 by PMA. Our data show that ursolic acid suppresses
the activation of COX-2 gene expression by inhibiting the
PKC signal transduction pathway. These data provide a mechanistic basis
for the chemopreventive and anti-inflammatory properties of ursolic
acid.
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MATERIALS AND METHODS |
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Tissue Culture.
The 184B5/HER cell line has been described previously
(25)
. Cells were maintained in MEM-KBM mixed in a ratio of
1:1 (basal medium) containing epidermal growth factor (10
ng/ml), hydrocortisone (0.5 µg/ml), transferrin (10 µg/ml),
gentamicin (5 µg/ml), and insulin (10 µg/ml). Cells were grown to
60% confluence, trypsinized with 0.05% trypsin-2 mM EDTA,
and plated for experimental use. MSK Leuk1 was established from a
dysplastic leukoplakia lesion adjacent to a squamous cell carcinoma of
the tongue in a 46-year-old nonsmoking female (26)
. Cells
were routinely maintained in keratinocyte growth medium and passaged
using 0.125% trypsin-2 mM EDTA. In all experiments,
184B5/HER and MSK Leuk1 cells were grown in basal medium for 24 h
prior to treatment. Treatment with vehicle (0.2% DMSO), ursolic acid,
or PMA was always carried out in basal medium. Cellular cytotoxicity
was assessed by measurements of cell number, release of LDH, and MTT
assay. The MTT assay was performed according to the method of Denizot
and Lang (27)
. LDH assays were performed according to the
manufacturer’s instructions. There was no evidence of toxicity in any
of our experiments.
PGE2 Production by Cells.
Cells (5 x 104 per well) were
plated in six-well dishes and grown to 60% confluence in growth
medium. The cells were then treated as described below. Levels of
PGE2 released by the cells were measured by
enzyme immunoassay. Amounts of PGE2 production
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 x g for 10 min to sediment the particulate material. The protein
concentration of the supernatant was measured by the method of Lowry
et al. (28)
. SDS-PAGE was performed under
reducing conditions on 10% polyacrylamide gels as described by Laemmli
(29)
. The resolved proteins were transferred onto
nitrocellulose sheets as detailed by Towbin et al.
(30)
. 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 Inc. Ten 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
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 radiolabeled cDNA probes for human COX-2 and 18 S rRNA.
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. COX-2
and 18 S rRNA probes were labeled with
[32P]CTP by random priming.
Nuclear Run-off Assay.
Cells (2.5 x 105) were plated in
four T150 dishes for each condition. Cells were grown in growth medium
until they were approximately 60% confluent. Nuclei were isolated and
stored in liquid nitrogen. For the transcription assay, nuclei
(1.0 x 107) were thawed and
incubated in reaction buffer (10 mM Tris, pH 8, 5
mM MgCl2, and 0.3 M KCl)
containing 100 µCi of uridine
5'[
-32P]triphosphate and 1 mM
unlabeled nucleotides. After 30 min, labeled nascent RNA transcripts
were isolated. The human COX-2 and 18 S rRNA cDNAs were
immobilized onto nitrocellulose and prehybridized overnight in
hybridization buffer. Hybridization was carried out at 42°C for
24 h using equal cpm/ml of labeled nascent RNA transcripts for
each treatment group. The membranes were washed twice with 2x SSC
buffer for 1 h at 55°C and then treated with 10 mg/ml RNase A in
2x SSC at 37°C for 30 min, dried, and autoradiographed.
Plasmids.
The COX-2 promoter constructs (–1432/+59, –327/+59,
–220/+59, –124/+59, –52/+59, KBM, ILM, CRM, and CRM
plus ILM) were a generous gift of Dr. Tadashi Tanabe (National
Cardiovascular Center Research Institute, Osaka, Japan; Refs.
31
and 32
). The human COX-2 cDNA was
generously provided by Dr. Stephen M. Prescott (University of Utah,
Salt Lake City, UT). The AP-1 reporter plasmid (2xTRE-luciferase),
composed of two copies of the consensus TRE ligated to luciferase, was
kindly provided by Dr. Joan Heller Brown (University of California, La
Jolla, CA; Ref. 33
). pSV-ßgal was obtained from Promega.
Transient Transfection Assays.
184B5/HER cells were seeded at a density of 5 x 104 cells/well in six-well dishes and grown to
50–60% confluence. For each well, 2 µg of plasmid DNA were
introduced into cells using 8 µg of LipofectAMINE as per the
manufacturer’s instructions. After 7 h of incubation, the medium
was replaced with basal medium. The activities of luciferase and
ß-galactosidase were measured in cellular extract as described
previously (34)
.
Electrophoretic Mobility Shift Assay.
Cells were harvested and nuclear extracts were prepared as described
previously (35)
. For binding studies, oligonucleotides
containing the CRE of the COX-2 promoter were used:
5'-AAACAGTCATTTCGTCACATGGGCTTG-3' (sense) and
5'-CAAGCCCATGTGACGAAATGACTGTTT-3' (antisense; Genosys Biotechnologies,
Inc., The Woodlands, TX). The complementary oligonucleotides were
annealed in 20 mM Tris, pH 7.6, 50
mM NaCl, 10 mM
MgCl2, and 1 mM DTT.
The annealed oligonucleotide was phosphorylated at the 5'-end with
[
-32P]ATP and T4 polynucleotide kinase. The
binding reaction was performed by incubating 5 µg of nuclear protein
in 20 mM HEPES, pH 7.9, 10% glycerol, 300 µg
of BSA, and 1 µg of poly(dI·dC) in a final volume of 10 µl for 10
min at 25°C. The labeled 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% nondenaturing polyacrylamide gel.
The gel was then dried and subjected to autoradiography at –80°C.
PKC Assay.
The activity of PKC was measured according to directions from Life
Technologies, Inc. Briefly, cells were plated in 10 cm dishes at
106 cells/dish and grown to 60% confluence.
Cells were then treated with fresh basal medium containing vehicle
(0.2% DMSO), PMA (50 ng/ml), or PMA (50 ng/ml) plus ursolic acid (15
µM) for 30 min. Total PKC activity was measured in cell
lysates. To determine cytosolic and membrane-bound PKC activity, cell
lysates were centrifuged at 100,000 x g for 30 min. The
resulting supernatant contains cytosolic PKC; membrane-bound PKC
activity is present in the pellet. Subsequently, DEAE cellulose columns
were used to partially purify PKC enzymes. PKC activity was then
measured by incubating partially purified PKC with
[-32P]ATP (3000–6000 Ci/mmol) and the
substrate myelin basic protein for 20 min at room temperature. The
activity of PKC is expressed as cpm incorporated/µg of protein.
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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. 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. Fig. 3A
shows that PMA induced COX-2 in human mammary epithelial
cells. Treatment with ursolic acid caused a dose-dependent decrease in
PMA-mediated induction of COX-2. Neither PMA nor ursolic acid altered
amounts of COX-1 (data not shown). To confirm that these effects of
ursolic acid were not unique to mammary epithelial cells, we determined
whether ursolic acid inhibited PMA-mediated induction of COX-2 in a
premalignant, oral leukoplakia cell line. As shown in Fig. 3B
, ursolic acid also suppressed the induction of COX-2 by
PMA in this cell line. The maximal effect was observed when 30
µM ursolic acid was used.
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. Nuclear run-offs were performed to determine whether
differences in amounts of COX-2 mRNA reflected altered rates of
transcription. As shown in Fig. 4B
, we detected
3-fold higher rates of synthesis of nascent COX-2 mRNA after
treatment with PMA, consistent with the differences observed by
Northern blotting. This effect was suppressed by ursolic acid.
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that responded to PMA and ursolic acid, transient
transfections were performed with a series of human COX-2
promoter deletion constructs. As shown in Fig. 5B
, PMA
increased COX-2 promoter (–1432/+59) activity about 3-fold,
an effect that was suppressed by ursolic acid. Both the inductive
effect of PMA and the suppressive effect of ursolic acid were detected
with all COX-2 promoter deletion constructs except the
–52/+59 construct. A CRE is present between nucleotides –59 and –53,
suggesting that this element may be responsible for mediating the
effects of PMA. To test this notion, transient transfections were
performed using COX-2 promoter constructs in which specific
enhancer elements including the CRE were mutagenized. As shown in Fig. 5C
, mutagenizing the CRE site caused a decrease in basal
promoter activity and a loss of responsiveness to PMA. By contrast,
mutagenizing the NF-IL6 or NF
B sites had little effect on
COX-2 promoter function (Fig. 5C)
.
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, ursolic acid completely inhibited PMA-induced translocation of PKC
activity from cytosol to membrane. PKC signaling increases MAPK
activity, which, in turn, regulates COX-2 expression (32)
.
Treatment with PMA induced the activities of ERK1/2, JNK, and p38 MAPK;
these steps were blocked by ursolic acid in a dose-dependent manner
(Fig. 7)
.
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. By contrast, treatment with PMA did not increase binding
when the CRE site was mutagenized (data not shown). The DNA binding
complex induced by PMA was removed by treatment with a blocking
antibody to c-Jun, a component of the AP-1 transcription factor
complex. This antibody prevents c-Jun from binding to DNA rather than
causing a supershift (36)
. Transient transfections were
performed to further investigate the anti-AP-1 activity of ursolic
acid. Ursolic acid suppressed PMA-mediated activation of an AP-1
reporter plasmid (2xTRE-luciferase; Fig. 8B
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several of the known properties of ursolic acid can be explained, in part, by its ability to inhibit COX-2 and PG synthesis. For example, overexpression of COX-2 promotes angiogenesis (38) and suppresses apoptosis (39) , whereas ursolic acid inhibits both of these effects (40 , 41) . Moreover, because PGs are proinflammatory and chronic inflammation predisposes to malignancy (42) , the inhibition of COX-2 by ursolic acid helps to explain both its anti-inflammatory (21, 22, 23) and chemopreventive activities (21 , 24) .
With regard to the mechanism by which ursolic acid modulates gene expression, it suppressed PMA-mediated activation of COX-2 transcription by inhibiting the PKC signal transduction pathway. Ursolic acid blocked PMA-induced translocation of PKC activity from cytosol to membrane and the activation of ERK1/2, JNK and p38 MAPKs. These results are significant because PKC activity is increased in some cancers (43) and is considered a potential target for cancer therapy. Moreover, there is recent evidence that compounds that block the activation of ERK1/2 MAPK inhibit tumor growth (44) .
The inductive effects of PMA were mediated by the CRE of the COX-2 promoter. Interestingly, PMA increased the binding of c-Jun to the CRE, an effect that was suppressed by ursolic acid. Previously, Xie and Herschman (45 , 46) showed that c-Jun was important for activation of the murine COX-2 promoter via the CRE. To the best of our knowledge, this is the first time a similar observation has been made with the human COX-2 promoter. JNK induces the expression and phosphorylation of c-Jun (47) . It is possible, therefore, that ursolic acid blocks PMA-mediated induction of c-Jun binding to the CRE by inhibiting the stimulation of JNK activity. Ursolic acid also inhibited PMA-mediated activation of ERK1/2 and p38 MAPKs. ERK1/2 stimulates AP-1 activity by inducing c-Fos, which heterodimerizes with c-Jun (47) . p38 MAPK induces AP-1 by phosphorylating ATF-2. A heterodimer composed of phospho-ATF-2 and c-Jun can induce c-Jun expression (48) . Future studies are warranted to determine whether c-Jun heterodimerizes with c-Fos or ATF-2 after treatment with PMA.
The suppressive effects of ursolic acid on PMA-mediated activation of COX-2 transcription are explained by inhibition of AP-1 activity. Because AP-1 activity has been linked to carcinogenesis (49 , 50) , the results of this study help to explain the anticancer properties of triterpenoids. Previously, we reported that retinoids blocked PMA-mediated induction of COX-2 by antagonizing AP-1-mediated transcription (34) . Ursolic acid and retinoids appear to inhibit AP-1 activity by different mechanisms. In contrast to ursolic acid, retinoids did not block the redistribution of PKC activity from cytosol to membrane mediated by PMA (data not shown). Additionally, retinoids block the activation of COX-2 expression without altering the activity of ERK1/2 MAPK (17) . Thus, chemopreventive agents can inhibit AP-1-mediated induction of COX-2 by disrupting PKC signal transduction at different levels in the pathway.
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FOOTNOTES |
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1 This work was supported in part by NIH Grant P01
CA29502 (to K. S.), the Cancer Research Foundation of America (to
K. S.), the American Institute for Cancer Research (to A. J. D.),
and the Lawrence A. Miller Memorial Foundation. 
2 To whom requests for reprints should be
addressed, at New York Presbyterian Hospital-Cornell, 525 East 68th
Street, Room F-206, New York, NY 10021. Phone: (212) 746-4403; Fax:
(212) 746-4885; E-mail: ajdannen{at}mail.med.cornell.edu
3 The abbreviations used are: COX, cyclooxygenase;
CRE, cyclic AMP response element; PG, prostaglandin; PGE2,
prostaglandin E2; PKC, protein kinase C; AP-1, activator
protein-1; PMA, phorbol 12-myristate 13-acetate; ERK, extracellular
signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK,
mitogen-activated protein kinase; KBM, keratinocyte basal medium; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH,
lactate dehydrogenase; SSPE, sodium chloride-sodium phosphate-EDTA
buffer.
Received 10/20/99. Accepted 2/29/00.
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REFERENCES |
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716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87: 803-809, 1996.[Medline]
R1-mediated induction of nuclear factor of activated T-cells. J. Biol. Chem., 270: 16333-16338, 1995.This article has been cited by other articles:
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S. C. Degner, M. Q. Kemp, G. T. Bowden, and D. F. Romagnolo Conjugated Linoleic Acid Attenuates Cyclooxygenase-2 Transcriptional Activity via an Anti-AP-1 Mechanism in MCF-7 Breast Cancer Cells J. Nutr., February 1, 2006; 136(2): 421 - 427. [Abstract] [Full Text] [PDF]
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S. Shishodia, S. Majumdar, S. Banerjee, and B. B. Aggarwal Ursolic Acid Inhibits Nuclear Factor-{kappa}B Activation Induced by Carcinogenic Agents through Suppression of I{kappa}B{alpha} Kinase and p65 Phosphorylation: Correlation with Down-Regulation of Cyclooxygenase 2, Matrix Metalloproteinase 9, and Cyclin D1 Cancer Res., August 1, 2003; 63(15): 4375 - 4383. [Abstract] [Full Text] [PDF]
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K. Subbaramaiah, P. A. Cole, and A. J. Dannenberg Retinoids and Carnosol Suppress Cyclooxygenase-2 Transcription by CREB-binding Protein/p300-dependent and -independent Mechanisms Cancer Res., May 1, 2002; 62(9): 2522 - 2530. [Abstract] [Full Text] [PDF]
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 D. Boutemmine, N. Bouchard, D. Boerboom, H. E. Jones, A. K. Goff, M. Dore, and J. Sirois Molecular Characterization of Canine Prostaglandin G/H Synthase-2 and Regulation in Prostatic Adenocarcinoma Cells in Vitro Endocrinology, March 1, 2002; 143(3): 1134 - 1143. [Abstract] [Full Text] [PDF]
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R. Yu, V. Hebbar, D. W. Kim, S. Mandlekar, J. M. Pezzuto, and A.-N. T. Kong Resveratrol Inhibits Phorbol Ester and UV-Induced Activator Protein 1 Activation by Interfering with Mitogen-Activated Protein Kinase Pathways Mol. Pharmacol., July 1, 2001; 60(1): 217 - 224. [Abstract] [Full Text]
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), PMA (50 ng/ml, 
), or PMA (50 ng/ml) and ursolic acid (0–30
µM) for 4.5 h. The medium was then replaced with
basal medium and 10 µM sodium arachidonate. Thirty min
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.
). Reporter activities were
measured in cellular extract 6 h later. Luciferase activity
represents data that have been normalized with ß-galactosidase.
Columns, means; bars, SD;
n = 6.
