Mutation Research 551 (2004) 245254

Effect of flavonoids and Vitamin E on cyclooxygenase-2

(COX-2) transcription
Karen A OLeary a,b,c , Sonia de Pascual-Tereasa a,d , Paul W Needs a ,
Yong-Ping Bao a , Nora M OBrien b , Gary Williamson a,
a Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK
b Department of Food Science and Nutritional Sciences, University College, Cork, Ireland
c Department of Health Toxicology Unit, Section on Clinical Pharmacology, Imperial College, Hammersmith Campus,

9th Floor Commonwealth Building, DuCane Road, London W12 0NN, UK

d Department of Plant, Food Science and Technology, Instituto del Frio Consejo Superior de Investigaciones Cientificas,

Jose Antonio Novais 10, E-28040 Madrid, Spain

Received 22 October 2003; received in revised form 27 January 2004; accepted 27 January 2004

Abstract
Cyclooxygenase-2 (COX-2)-catalysed synthesis of prostaglandin E2 plays a key role in inflammation and its associated
diseases, such as cancer and cardiovascular disease. There are numerous reports demonstrating that flavonoids inhibit COX-2
activity. However, transcriptional regulation of COX-2 can also be important. Nobiletin, amentoflavone, quercetin, quercetin
penta-acetate, flavone, resveratrol, apigenin, chrysin, kaempferol, galangin, and genistein have been reported to modulate
COX-2 transcription in a wide variety of systems. Here, we briefly review the literature on regulation of COX-2 transcrip-
tion by flavonoids, and report some new preliminary data on Vitamin E and quercetin conjugates. Quercetin, quercetin
3-glucuronide, quercetin 3 -sulfate and 3 methylquercetin 3-glucuronide reduced COX-2 mRNA expression in both un-
stimulated and interleukin-1 stimulated colon cancer (Caco2) cells. Quercetin and quercetin 3 -sulfate, unlike quercetin
3-glucuronide and 3 methylquercetin 3-glucuronide, also inhibited COX-2 activity. In contrast, tocopherols (-tocopherol,
-tocopherol acetate, and -tocopherol at 10 M) did not affect COX-2 mRNA expression in unstimulated Caco2 cells. How-
ever, the tocopherols inhibited COX-2 activity showing that the tocopherols act post-transcriptionally on activity, whereas
quercetin and some quercetin conjugates affect both the transcription and activity of COX-2. Flavonoid modulation of COX-2
transcription may therefore be an important mechanism in anti-carcinogenesis.
2004 Elsevier B.V. All rights reserved.

Keywords: Flavonoids; Vitamin E; Cyclooxygenase; Quercetin

1. Introduction

Corresponding author. Present address: Nestle Research Centre,
P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
Tel.: +41 21 785 8546; fax: +41 21 785 8544.
E-mail address: gary.williamson@rdls.nestle.com (G. Williamson).
There are two major isoforms of cyclooxygenase,
constitutive (COX-1) and inducible (COX-2) [1]. The
COX-1 isoenzyme is a house-keeping protein in
most tissues, and does not change in response to

0027-5107/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2004.01.015
246 K.A. OLeary et al. / Mutation Research 551 (2004) 245254

stimuli. COX-2 is induced significantly in vivo during
inflammation. COX-2 is rapidly induced by tumour
promoters, growth factors, cytokines, and mitogens
[2,3]. Over-expression of COX-2 has been implicated
in the pathogenesis of cancer, and increased COX lev-
els have been detected in many colorectal and gastric
tumors [46].
Antioxidants, such as fat-soluble Vitamin E and
the more hydrophilic flavonoids, possess free radical
scavenging properties. The interaction of these natural
antioxidants with reactive oxygen species implicated
in inflammation has prompted a number of studies
on their effects on the formation of proinflammatory
eicosanoids derived from the cyclooxygenase path-
way of arachidonic acid metabolism. Flavonoids and
flavonoid containing foods have been investigated as
selective COX-2 inhibitors. The 5 ,7-dihyroxy flavone,
galangin, with an IC50 value of 5.5 M, was found
to be the most active flavonoid [7]. Flavonoids with
an ortho-dihydroxy (catechol moiety) in rings A or
B were stronger inhibitors of COX-2 than those with
a free 3-OH group [7,8]. The presence of a C2C3
double bond appears to be a major determinant of
COX activity. It has previously been shown that Vita-
min E supplementation can inhibit COX-2 activity in
a variety of different model systems [911], with the
different isomers of Vitamin E exhibiting different
potencies of inhibition.
COX-2 is an important contributor to colon can-
cer formation and candidates that have an effect on
the expression level of the enzyme are potentially
of great interest as chemopreventive agents. Interfer-
ence of signal transduction pathways or modulation
of the inflammatory pathway through transcription
factors are potential mechanisms of COX-2 tran-
scription. Flavonoids have been reported to modulate
COX-2 transcription in a number of different cell
models (Table 1). The peroxisome proliferator acti-
vated factor (PPAR) transcription factor has been
implicated in the anti-inflammatory response. PPARs
bind to specific response elements as heterodimers
with the retinoid X factor and activate transcription

Table 1
Inhibition of basal or induced-COX-2 transcription by flavonoids
Flavonoid No effect Cell system Concentration Reference
Apigenin, chrysin, LPS-activated macrophages IC50 = 50 M, [17]
kaempferol dose-dependent
Quercetin A549 cells 50 M [18]
Quercetin DLD-1 cells IC50 = 10.5 M [19]
Epigallocatechin, DLD-1 cells IC50 = 415 M [19]
Quercetin, apigenin myricetin LPS-treated J774A.1 macrophage cells 0.550 M, [20]
dose-dependent,
significant at 5 and
50 M
Quercetin penta-acetate Quercetin, rutin l-NAME/LPS and NLA/LPS-treated 20, 40 M [21]
RAW 264.7 cells (mouse macrophage)
Oroxylin A LPS-treated J774.1 macrophage cells 20 g/ml [22]
Apigenin, genistein, LPS-activated RAW 264.7 macrophages IC50 < 50 M [23]
kaempferol
Amentoflavone TNF--treated A549 cells [24]
Quercetin Cooking oil fumes-treated human lung [25]
adenocarcinoa CL-3 cells
Flavone HT29 cells 150 M [26]
Quercetin, kaempferol, With and without TNF- stimulation of 20 M quercetin, [27]
genistein, resveratrol human colon cancer DLD-1 cells 40 M kaempferol,
40 M genistein,
100 M resveratrol
Resorcinol 500 M resorcinol
Resveratrol Phorbol ester induced human 2.530 M [32]
mammary/oral epithelial cells
Nobiletin IL-1 induced human synovial fibroblasts Dose-dependent < [33]
64 M
 K.A. OLeary et al. / Mutation Research 551 (2004) 245254
in response to a variety of different exogenous or
endogenous ligands such as NSAIDS, arachidonic
acid metabolites and some drugs [1214]. NSAIDS
such as aspirin, sodium salicylate, and indomethacin
exert their anti-inflammatory effects by inhibiting the
inhibitor B (IKB) kinase IKK-), thereby prevent-
ing activation by NF-B of genes in the inflammatory
response [15]. Other NSAIDS such as ibuprofen are
PPAR ligands and block production of inflammatory
cytokines in human monocytes [16].
Several flavonoids (apigenin, chrysin, and kaemp-
ferol) can bind to PPAR in vitro, acting as possi-
ble PPAR ligands [17]. Structurally these flavonoids
possess lipophilic backbone characteristics similar to
those of other PPAR ligands but lack the acid moi-
ety that is common to other PPAR ligands. The fla-
vanones and flavan-3-ol were inefficient at activating
PPAR, indicating that the C2C3 double bond of the
C ring was essential for activation of PPAR [17].
The flavones, flavonols, and isoflavones were able
to activate PPAR, but this activation was dependent
on the number and position of the hydroxyl groups.
Important residues were the hydroxyl groups at posi-
tion 5 and 7 on the A ring and the 4 -position on the
B ring such as in kaempferol, apigenin, and chrysin.
The presence of the 3 hydroxyl group on the B ring
resulted in a decrease in PPAR activation such as in
luteolin and quercetin [17].
There have been several reports that suggest a
role for nitric oxide as a regulatory molecule in the
eicosanoid pathway. Banerjee et al. in [18] showed
that the production of PGE2 and Cox-2 by A249 cells
in response to cytokine stimulation was accompa-
nied by an increase in NO and corresponding iNOS
enzyme. It has been shown that pre-treatment with
either quercetin or amentoflavone almost completely
decreased NO generation in A549 cells, without in-
terference with iNOS at the transcriptional level.
Quercetin at 50 M had little effect on COX-2 MRNA
and protein level [18]. However, flavanones neither
affected COX-2 expression and PGE2 production nor
NO generation in A549 cells. The different effects
seen with the flavonoids may again be related to their
structure. The potent COX-2 inhibitor, quercetin, has
a C2C3 double bond in the B ring and a 3 and 4
hydroxyl group in the B ring. However, flavanones
lack the C2C3 double bond in the B ring. Flavonoids
with 3 , 4 , and 5 hydroxyl groups in the B ring such
247
as myricetin and epigallocatechin have been shown
not to suppress the transcriptional activity of COX-2
in human adenocarcinoma cell line DLD-1 [19].
Also, it has been reported that quercetin, galangin,
and apigenin can down regulate iNOS expression by
modulating enzyme activity related to signal trans-
duction [20]. These flavonoids all decreased PGE2
release and COX-2 expression in a concentration
dependent manner in a lipopolysaccharide (LPS) in-
duced macrophage cell line J774A.1 [20]. Quercetin
penta-acetate, but not quercetin or rutin, showed
strong inhibitory activity on PGE2 production and
COX-2 protein expression in RAW 264.7 cells treated
with nitric oxide synthase inhibitors nitro-l-arginine
(NLA) and N-nitro-l-arginine methyl ester (l-NAME)
co-treated with LPS [21].
Another potential mechanism by which flavonoids
mediate their inhibition of COX-2 gene expression
is by alteration of the NF-B pathway. Oroxylin A
also has been shown to be an effective inhibitor of
LPS-induced COX-2 gene expression by blocking
NF-B activation in RAW264.7 macrophages [22].
Apigenin, genistein, and kaempferol were active in-
hibitors of transcriptional activation of COX-2 in
LPS-activated RAW 264.7 macrophages [23]. Api-
genin was the most potent inhibitor of transcriptional
activation of both COX-2 and iNOS [23], blocking
the LPS-induced activation of NF-B and therefore,
suppressing the promoter activity of COX-2 [23].
Banerjee et al. in [24] reported that amentoflavone,
a biflavonoid, down-regulated COX-2 expression
in -TNF-activated A549 cells with a concomitant
inhibition of NF-B mediated signalling cascades.
Similarly, Lin et al. in [25] showed that addition of
quercetin to human lung adenocarcinoma cells CL-3
cells exposed to cooking oil fumes significantly de-
creased COX-2 mRNA and protein levels. It was also
shown that NF-B DNA binding capacity was sig-
nificantly inhibited by quercetin [25]. The effects of
flavone in HT29 cells were associated with changes
in mRNA levels of COX-2 and NF-B [26].
Additionally, flavonoids can suppress COX-2 tran-
scriptional activity by inhibition of phosphorylation
signal transduction pathways. Structurally, the num-
ber of hydroxyl groups on the B ring may be related
to the molecular conformation that influences the
interactions between the flavonoids and enzymes such
as tyrosine kinase and protein kinase C which are
248 K.A. OLeary et al. / Mutation Research 551 (2004) 245254
involved in COX-2 transcriptional activity. Quercetin,
kaempferol, genistein, resveratrol, and resorcinol sup-
pressed both TGF-stimulated and non-stimulated
COX-2 promoter activities in colon cancer cells
DLD-1 [27]. It is well established that TGF acti-
vates protein-tyrosine kinases (PTK) [28] and it has
been shown that quercetin, kaempferol, genistein,
and resveratrol can inhibit tyrosine kinases [2931].
All five flavonoids that exhibited inhibitory effects
on COX-2 promoter activity have a common resor-
cin moiety, and so it is possible that this moiety is
critical for COX-2 inhibition. Similarly, Subbarama-
iah et al. in [32] reported that resverstrol blocked
phorbol ester-induced translocation of PKC activity
from the cytosol to the membrane in both human
mammary and oral epithelial cells and also blocked
the several-fold increase in COX-2 promoter activity
mediated by PKC.
Lin et al. (2003) reported that nobiletin (5,6,7,8,3 ,
 tained from the European Collection of Cell Cul-
4 -hexamethoxy flavone) suppressed interleukin-1 in-
duced production of PGE2 in human synovial cells
in a dose-dependent manner (64 M) [33]. Further,
nobiletin selectively downregulated COX-2, but not
COX-1, mRNA expression [33].
To date, there has been no comparative study in-
vestigating the differences in effect of Vitamin E and
the flavonoids on activity and expression of COX-2
in colon cancer cells. In this study, we compare the
ability of Vitamin E (-tocopherol, -tocopherol ac-
etate, and -tocopherol) and that of the quercetin
conjugates found in human plasma (quercetin,
quercetin-3-glucuronide, quercetin-3 -sulfate, and
3 methylquercetin-3-glucuronide) to inhibit COX-2
activity and transcription. We address these questions
by investigating the effect of these antioxidants on
COX activity in vitro and their effect on expression
of COX-2 at the RNA level in colorectal cancer cells
using Taqman RT-PCR.
2. Materials and methods
2.1. Materials
All reagents were obtained from Sigma (Poole,
UK) unless otherwise stated and were of analyti-
cal or HPLC grade where applicable. Tissue culture
plastics were supplied by Life Technology (Paisley,
UK). Water was purified via a Millex Q plus system
(Millipore, Watford, UK). Oligonucleotide primers
and fluorogenic probes were purchased from PE
Biosystems (Warrington, UK). Human recombinant
COX-2 protein was obtained from Cayman Chemicals
(Alexis Corporation, UK). Quercetin 3-glucuronide
was purified from green bean tissue [34]. Quercetin
3 -sulfate was kindly provided by Prof. Denis Bar-
ron [35]. Isorhamnetin 3-glucuronide was chemically
synthesised as described [36]. These compounds have
previously been confirmed by MS and 1 H and 13 C
NMR. All flavonoids and conjugates were checked
for purity, prior to use, by HPLC and were found to
be >98% pure.
2.2. Cell culture
Caco2 cells (human adenocarcinoma cells), ob-
ture were maintained in Eagles Minimal Essential
Medium (EMEM), supplemented with 10% foetal calf
serum (FCS), 1% (v/v) non-essential amino acids and
2 mM l-glutamine, penicillin (100 U/mL) and strepto-
mycin (100 g/mL). Cells were grown in a humidified
incubator of 5% CO2 , 95% air at 37 C and passaged
every 810 days. Cells were seeded at a density of ap-
proximately 2 104 cells/cm2 in 56-cm2 dishes and
allowed to adhere overnight. The cells were to grown
to confluency over a period of 8 days and the medium
was changed every second day. After this time, the
medium was removed and replaced with DMEM sup-
plemented with 10% FCS containing test compounds.
The final concentrations in growth medium of these
compounds were 0.1, 1 or 10 M. The samples were
incubated for a further 16 h. Control samples not
treated with either Vitamin E or flavonoids were in-
cubated with the equivalent volume of carrier vehicle
(water, methanol or ethanol). For experiments involv-
ing stimulated COX-2, a cytokine, interleukin-1,
was added simultaneously to the medium with test
compounds at a concentration of 10 ng/ml.
2.3. RNA extraction
Total RNA was extracted from exponentially grow-
ing cells using a commercially available RNA extrac-
tion kit (Qiagen RNeasy Mini Kit for cell lines).
 K.A. OLeary et al. / Mutation Research 551 (2004) 245254
2.4. RNA quantification
The concentration and purity of the extracted iso-
lated RNA were determined by measurement of the
optical densities at 260 and 280 nm and using Ri-
bogreen RNA Quantitation Kit (Molecular Probes,
USA, R11490) against a standard curve of ribosomal
RNA (16S and 23S rRNA from E. coli).
2.5. Taqman RT-PCR
Oligonucleotide primers and Taqman probes for
COX-2 were designed using Primer express software
(Applied Biosystems), using an ABI Prism 7700
Sequence Detection System (Applied Biosystems).
The TaqMan probe consists of an oligonucleotide
with a 5 reporter dye (FAM) and a downstream 3
quencher dye (TAMRA). The forward primer was:
GCC CTT CCT CCT GTG CC, the reverse primer
was: AAT CAG GAA GCT GCT TTT TAC and the
probe was: ATG ATT GCC CGA CTC CCT TGG
GT GT. The PCR reaction mixture (25 L) contained
17.2 L of Universal PCR Master Mix, 300 nM for-
ward and reverse primers, 150 nM Taqman probe and
7.8 L of the RNA sample and water. All wells were
sealed with a coversheet following complete loading
of the reagents (25 L). The thermal cycling condi-
tions comprised of an initial step at 48 C for 30 min
followed by 95 C for 10 min. Subsequent PCR am-
plifications consisted of 40 cycles of denaturation at
95 C for 15 s and annealing and extension at 60 C
for 1 min. The standard curve was constructed with
serial dilutions of untreated Caco2 cell RNA.
2.6. COX-2 activity assay
The COX activity assay utilizes the peroxidase com-
ponent of cyclooxygenases. The peroxidase activity is
assayed colorimetrically by monitoring the appearance
of oxidized N,N,N ,N -tetramethyl-p-phenylenediamine
(TMPD) at 610 nm. Hydroperoxidase activity was
determined by spectrophotometry. Reaction mix-
tures contained 0.1 M TrisHCl (pH 8.5), 1.2 M
hematin, 0.24 mM TMPD, COX-2 (45 g of pro-
tein), and inhibitory test compounds. Reactions were
initiated by adding 1.2 mM arachidonic acid to a
mixture and changes in absorbance at 610 nm were
249
measured. Inhibitory activity was calculated by com-
paring the initial rate of change in absorbance in
the presence of test compounds with that observed
with a potent COX-2 inhibitor DuP-697 (5-bromo-
2-[4-fluorophenyl]-3-[4-(methylsulfonyl)phenyl]thio-
phene) [37].
3. Results
Sufficient total RNA (1050 g) was obtained from
approximately four million Caco2 cells for quan-
titation of COX-2 mRNA using real time RT-PCR.
None of the treatments produced any visible toxic
effects (cell death and loss of cell adhesion) in Caco2
cells as assessed microscopically. The effect of com-
pounds on both basal (unstimulated) and interleukin 1
(IL-1)-induced COX-2 transcription was measured.
Salicylic acid was used as a positive control, since sal-
icylic acid decreases COX-2 expression in both basal
and stimulated cells in a number of different models
such as human A549 cells, HUVEC cells and murine
macrophages [3840]. Salicylic acid (104 to 107 M)
suppressed both basal and IL-induced COX-2 tran-
scription in Caco2 cells in a concentration dependent
manner (data not shown), consistent with other reports
on different cells. This concentration range (104 to
107 M) is consistent with plasma salicylate levels in
individuals taking therapeutic doses of aspirin.
Basal COX-2 expression was inhibited by quercetin
and its metabolites (Fig. 1). The quercetin conju-
gates inhibited in a dose-dependent manner with
isorhamnetin-3-glucuronide exerting the most potent
effect. At the lowest concentration, a small poten-
tial activation of transcription was observed, and if
real, this effect requires more investigation. Quercetin
showed a much smaller non-significant reduction,
which was not dose-dependent.
In the presence of IL-1 and methanol (the car-
rier vehicle for the metabolites of quercetin), Cox-2
mRNA was induced, as measured using Taqman
RT-PCR. Cytokine-stimulated COX-2 expression was
inhibited by quercetin and its conjugates (Fig. 2),
with the greatest inhibition seen with quercetin
3-glucuronide and quercetin-3 sulfate. However, the
effect was not dose-dependent (data not shown).
We also investigated the effects of the different to-
copherol isomers on COX-2 mRNA expression in
250 K.A. OLeary et al. / Mutation Research 551 (2004) 245254

Fig. 1. Effect of quercetin plasma metabolites (0.1, 1.0, 10 M) on the gene expression of COX-2 in unstimulated Caco-2 cells following
16-h treatment.

Caco2 cells. In comparison to flavonoids, the to-
copherols (-tocopherol, -tocopherol acetate, and
-tocopherol) did not down regulate mRNA expres-
sion of COX-2 in unstimulated Caco2 cells at any
concentration investigated (Fig. 3). These preliminary
results indicate that the flavonoids but not the toco-
pherols inhibit COX-2 transcriptional expression in
Caco2 cells.
We also tested the flavonoids and tocopherols
for their effect on COX-2 activity. (Fig. 4). A
dose-dependent inhibition of COX-2 activity was
exhibited by quercetin. Quercetin was the most po-
tent inhibitor of COX-2, resulting in an inhibition
of 85%. Quercetin 3 -sulfate also exhibited inhibi-
tion, a maximum inhibition of 50% at 10 M. There
was no suppression of COX-2 activity by quercetin
3-glucuronide or 3 methylquercetin 3-glucuronide at
the concentrations tested. COX-2 activity was inhib-
ited by all the tocopherols tested (Fig. 5). All con-
centrations of -tocopherol exhibited an equipotent

Fig. 2. Effect of quercetin plasma metabolites (10 M) on the mRNA expression of COX-2 in IL-1 stimulated Caco2 cells following
16-h treatment.
 K.A. OLeary et al. / Mutation Research 551 (2004) 245254 251

Fig. 3. Effect of -tocopherol, -tocopherol acetate, and -tocopherol on Cox-2 gene expression in Caco2 cells as measured by Taqman
RT-PCR.

Fig. 4. Effect of quercetin plasma metabolites on Cox-2 measured using a purified human standard Cox-2 protein.

inhibition of COX-2 activity, with a maximum in-
hibition of 33% observed at 10 M -tocopherol.
-Tocopherol was more effective than -tocopherol
at inhibiting COX-2 activity with a maximum in-
hibition of 56% at 10 M -tocopherol. Similarly,
-tocopherol acetate also inhibited COX-2 activity
in a dose-dependent manner, but was less effective
than -tocopherol and -tocopherol with a maxi-
mum inhibition of only 12% at 10 M -tocopherol
acetate.
4. Discussion
Cytokines, such as IL-1, act through distinct re-
ceptors to induce specific cellular responses [41,42].
252 K.A. OLeary et al. / Mutation Research 551 (2004) 245254
Fig. 5. The effect of -tocopherol, -tocopherol acetate, and -tocopherol on COX-2 activity, using a purified human COX-2 protein.
Incubation of Caco2 cells with interleukin-1 (IL-1)
resulted in a dose-dependent increase in expression
of COX-2 mRNA as expected [43,44]. In addition,
sodium salicylate and aspirin, at pharmacological con-
centrations, inhibited COX-2 transcription induced by
phorbol-12 myristate (PMA), interleukin-1 (IL-1),
and liposaccharide (LPS) in HUVEC cells [40]. Sal-
icylic acid is also an effective inhibitor of COX-2
activity [45]. Previous structure-activity studies have
indicated that >2 hydroxyl groups on the B ring and
the presence of the oxo group at the 4-position of the
C ring are important for suppression of COX-2 tran-
scriptional activity [27]. Quercetin conjugates found
in plasma, substituted either in the B ring or on the
3-position, all decreased basal and induced COX-2
expression in Caco2 cells, although the extent of
inhibition varied between the metabolites.
With respect to suppression of post-translational
activity by the flavonoids, quercetin, and quercetin
3 -sulfate, which exhibited inhibitory effects on
COX-2 activity, have a common free hydroxyl
group in the C ring. Quercetin 3-glucuronide and
isorhamnetin-3-glucuronide were less potent at in-
hibiting COX-2 activity and they share a common sub-
stitution in the 3-position of the C ring. Letan in [46]
proposed that flavonoids with a free hydroxyl group
at the 3- or 7-position act as antioxidant or possess
anti-inflammatory properties. This is consistent with
our observation that substitution of the 3-position with
a glucuronide diminished the inhibitory COX-2 activ-
ity observed with quercetin. However, Robak et al. in
[47] demonstrated that the lack of the free hydroxyl
group at the 3-position did not influence the COX-2
inhibitory effects of flavonoids such as luteolin. This
may indicate that it is not the free hydroxyl group
that is necessary for the inhibition of COX-2 activity
but large substitutions such as sugars or glucuronic
acid in the 3-position sterically hinder binding of the
flavonoid to the enzyme. Therefore, flavonoids, which
lack a substitution in the 3-position or possess a free
hydroxyl group in this 3-position, may play a criti-
cal role in the inhibition of COX-2 activity, although
other physicochemical factors may also be involved.
Unlike the flavonoids, the inhibitory effect of
the tocopherols on COX-2 was only at the level
of activity. -Tocopherol at physiologically relevant
concentrations (10 M) inhibited COX-2 activity by
40%. -Tocopherol and -tocopherol acetate also in-
hibited COX-2 activity but were not as effective as
-tocopherol. -Tocopherol reduced COX-2 activity
in both RAW264.7 macrophages and A549 human
epithelial cells, whereas -tocopherol was much less
potent [11]. However, Wu et al. in [10] reported
that in vitro supplementation of -tocopherol and
-tocopherol in old mice cells caused an equivalent
inhibition of COX-2 activity. Although we observed
that the tocopherols inhibited COX-2 activity, they
had no effect on COX-2 mRNA expression in Caco2
cells. Similarly, Wu et al. in [9] observed that Vita-
min E caused a decrease in COX-2 activity in murine
macrophages but it had no effect on mRNA and pro-
tein expression of COX-2. These results indicate that
Vitamin E exerts its effect post translationally rather
than transcriptionally.
 K.A. OLeary et al. / Mutation Research 551 (2004) 245254
Acknowledgements
The work was funded by the European Union
(POLYBIND, QLK1-1999-00505), the Biotechnology
and Biological Research Council, UK, by a fellow-
ship to KOL (National University of Ireland) and by
a Marie Curie Individual Fellowship to SdPT.
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