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1999;59:597-601. Cancer Res
Toshihiko Kawamori, Ronald Lubet, Vernon E. Steele, et al.
Stages of Colon Cancer
Anti-Inflammatory Agent, during the Promotion/Progression
Chemopreventive Effect of Curcumin, a Naturally Occurring
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[CANCER RESEARCH 59, 597–601, February 1, 1999]
Chemopreventive Effect of Curcumin, a Naturally Occurring Anti-Inflammatory
Agent, during the Promotion/Progression Stages of Colon Cancer
1
Toshihiko Kawamori, Ronald Lubet, Vernon E. Steele, Gary J. Kelloff, Robert B. Kaskey, Chinthalapally V. Rao,
and Bandaru S. Reddy
2
Division of Nutritional Carcinogenesis, American Health Foundation, Valhalla, New York 10595 [T. K., C. V. R., B. S. R.]; Chemoprevention Branch, National Cancer Institute,
Bethesda, Maryland 20892 [R. L., V. E. S., G. J. K.]; and Gene Print, Inc., Bala Cynwyd, Pennsylvania [R. B. K.]
ABSTRACT
Curcumin, derived from the rhizome of Curcuma longa L. and having
both antioxidant and anti-inflammatory properties, inhibits chemically
induced carcinogenesis in the skin, forestomach, and colon when it is
administered during initiation and/or postinitiation stages. This study was
designed to investigate the chemopreventive action of curcumin when it is
administered (late in the premalignant stage) during the promotion/pro-
gression stage of colon carcinogenesis in male F344 rats. We also studied
the modulating effect of this agent on apoptosis in the tumors. At 5 weeks
of age, groups of male F344 rats were fed a control diet containing no
curcumin and an experimental AIN-76A diet with 0.2% synthetically
derived curcumin (purity, 99.9%). At 7 and 8 weeks of age, rats intended
for carcinogen treatment were given s.c. injections of azoxymethane
(AOM) at a dose rate of 15 mg/kg body weight per week. Animals destined
for the promotion/progression study received the AIN-76A control diet for
14 weeks after the second AOM treatment and were then switched to diets
containing 0.2 and 0.6% curcumin. Premalignant lesions in the colon
would have developed by week 14 following AOM treatment. They con-
tinued to receive their respective diets until 52 weeks after carcinogen
treatment and were then sacrificed. The results confirmed our earlier
study in that administration of 0.2% curcumin during both the initiation
and postinitiation periods significantly inhibited colon tumorigenesis. In
addition, administration of 0.2% and of 0.6% of the synthetic curcumin in
the diet during the promotion/progression stage significantly suppressed
the incidence and multiplicity of noninvasive adenocarcinomas and also
strongly inhibited the multiplicity of invasive adenocarcinomas of the
colon. The inhibition of adenocarcinomas of the colon was, in fact, dose
dependent. Administration of curcumin to the rats during the initiation
and postinitiation stages and throughout the promotion/progression stage
increased apoptosis in the colon tumors as compared to colon tumors in
the groups receiving AOM and the control diet. Thus, chemopreventive
activity of curcumin is observed when it is administered prior to, during,
and after carcinogen treatment as well as when it is given only during the
promotion/progression phase (starting late in premalignant stage) of colon
carcinogenesis.
INTRODUCTION
Colorectal cancer, one of the leading causes of cancer deaths in
both men and women in the United States, accounts for ;56,000
deaths annually (1). Although several epidemiological and laboratory
studies suggest a relationship between large bowel cancer risk and
dietary factors (2–4), there is increasing evidence that a high con-
sumption of fruits and vegetables and intake of certain nonnutrients
that are present in foods reduce the risk of colon carcinogenesis (5).
Although risk reduction by nutritional intervention may not be suffi-
cient to protect high-risk individuals against colon cancer develop-
ment, an alternative or complementary effective approach for second-
ary prevention has been to identify the agents with chemopreventive
potency and to evaluate them in high-risk individuals in combination
with nutritional intervention (6–8).
It is noteworthy that the use of medicinal plants or their crude
extracts in the prevention and/or treatment of several chronic diseases
has been traditionally practiced in various different ethnic societies
worldwide. Turmeric, the powdered rhizome of Curcuma longa L.,
has been used to treat a variety of inflammatory conditions and
chronic diseases (9, 10); it is also used as coloring and flavoring
additive to foods. Curcumin [Fig. 1; diferuloylmethane; 1,7-bis-(4-
hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], which has
been identified as the major pigment in turmeric, possesses both
anti-inflammatory (11–13) and antioxidant properties (14, 15). It has
been demonstrated that topical application of curcumin inhibits ben-
zo(a)pyrene-induced DNA adduct formation, and development of skin
tumors as well as TPA
3
-induced epidermal DNA synthesis and tumor
promotion in mouse skin (16–18). Curcumin has a strong inhibitory
effect on cell proliferation in the HT-29 and HCT-15 human colon
cancer cell lines (19). Importantly, dietary administration of curcumin
during initiation and/or postinitiation periods significantly suppresses
development of chemically induced forestomach, duodenal, and colon
tumors in CF-1 mice (20); it also reduces formation of focal areas of
dysplasia and aberrant crypt foci in the colon that are early preneo-
plastic lesions in rodents (21, 22). Pereira et al. (23) have reported that
administration of 0.8 and 1.6% curcumin continuously during the
initiation and postinitiation phases significantly inhibited develop-
ment of AOM-induced colonic adenomas in rats. We have shown that
continuous dietary administration of 0.2% curcumin during the initi-
ation and postinitiation stages significantly inhibited the incidence and
multiplicity of AOM-induced colon adenocarcinomas and the tumor
burden in F344 rats (24). Although all of the above studies clearly
demonstrate the potential chemopreventive activity of curcumin dur-
ing the initiation and postinitiation periods of colon carcinogenesis,
there were no studies on the efficacy of this agent during the promo-
tion/progression stage when the premalignant lesions would have
developed. We deemed it important to show that curcumin treatment
can be delayed after the carcinogen administration in experimental
carcinogenesis and still be effective, so as to provide baseline knowl-
edge for possible clinical use of this agent in secondary prevention of
colon cancer in high-risk individuals, such as patients with colonic
polyps.
Curcumin was shown to inhibit colon carcinogenesis during the
postinitiation stage through the modulation of COX activity in the
tumor tissue (24). COXs are involved in the synthesis of PGs, which
have been shown to affect tumor growth (24), suggesting that effects
on the arachidonic acid cascade by curcumin may play a role in its
tumor-inhibitory activity. We and others have shown previously that
several inhibitors of PG synthesis, such as aspirin, ibuprofen, sulin-
dac, and piroxicam suppress colon carcinogenesis in laboratory ani-
mal model assays (25–28). Inhibition of colon carcinogenesis was
consistently associated with a decrease in the activity of COX in colon
Received 8/24/98; accepted 12/3/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported in part by United States Public Health Service Grant CA17613 and
NO1-CN-55150 from the National Cancer Institute.
2
To whom requests for reprints should be addressed, at the American Health Foun-
dation, Valhalla, NY 10595.
3
The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; AOM,
azoxymethane; COX, cyclooxygenase; PG, prostaglandin; NSAID, nonsteroidal anti-
inflammatory drug; LOX, lipoxygenase; HETE, hydroxyeicosatetraenoic acid.
597
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tumors (25, 26, 28). Evidence also suggests that curcumin acts on
pathways that may inhibit cell proliferation (19) and enhance apop-
tosis (29). In vitro studies by Hanif et al. (19) suggest that curcumin
inhibits colon cancer cell proliferation, independent of its ability to
inhibit PG synthesis. Furthermore, transformation of colorectal epi-
thelium into adenomas and adenocarcinomas has been shown to be
associated with progressive inhibition of apoptosis, suggesting that
inhibition of apoptosis in colon carcinogenesis may contribute to
tumor growth and promote neoplastic progression (30).
This study was designed to specifically investigate the chemopre-
ventive efficacy and dose-response effect of curcumin when it is
administered late in the premalignant stage, representing the promo-
tion/progression phase of colon carcinogenesis in F344 rats. In addi-
tion, the effect of dietary curcumin on apoptosis in colon tumors was
determined.
MATERIALS AND METHODS
Animals, Diets, and Carcinogen. Weanling male F344 rats were received
from Charles River Breeding Laboratories (Kingston, NY). AOM was pur-
chased from Ash Stevens (Detroit, MI). Synthetically derived curcumin (purity
.99.9% diferuloylmethane) was kindly provided by Gene Print, Inc. Bala
Cynwyd, PA as part of the National Cancer Institute’s project for investiga-
tional studies of this compound. All ingredients for the semipurified diet were
purchased from Dyets, Inc (Bethlehem, PA). The experimental diets were
prepared weekly in our laboratory by adding curcumin at 0.2 and 0.6% levels
instead of cornstarch (Table 1). The experimental and control diets were stored
in a cold room.
Efficacy Study. The experimental protocols followed those detailed in our
previous publications (27). Briefly, weanling male F344 rats were quarantined
for 7 days and had access to modified AIN-76A control diet (Table 1).
Following quarantine, at 5 weeks of age, all animals were randomly distributed
by weight into the various experimental groups. As shown in Fig. 2, the points
at which the animals received the test diets from 2 weeks before, during, and
after carcinogen treatment to termination of the study were designated initia-
tion and postinitiation stages, whereas promotion/progression stages represent
the point at which the animals received test diets from 14 weeks after carcin-
ogen treatment until the end of the study. Beginning at 5 weeks of age, groups
of animals in the initiation and postinitiation study had access to either control
diet or experimental diet containing 0.2% curcumin, whereas the rats for the
assays testing efficacy during the promotion/progression stage received the
control diet. At 7 weeks of age, all rats except those intended for vehicle
treatment received s.c. injections of AOM at a dose rate of 15 mg/kg body
weight, once weekly for 2 weeks. Rats in vehicle-treated control groups were
injected with an equal volume of normal saline. The rats designated for the
intervention during the promotion/progression stage and maintained on the
control diet were then transferred to experimental diets containing 0.2 or 0.6%
curcumin beginning 14 weeks after the second dose of AOM (Fig. 2). Our past
experience on AOM-induced colon carcinogenesis suggests that the premalig-
nant lesions in the colon would have developed by week 14 following carcin-
ogen administration (26). This dietary regimen was continued until termination
of the experiment 52 weeks after the last carcinogen treatment. Body weights
were recorded every 2 weeks for the first 10 weeks and then every 4 weeks. At
the scheduled termination, all animals were killed by CO
2
euthanasia. After
laparotomy, the entire gastrointestinal tract was resected and opened longitu-
dinally, and the contents were flushed with normal saline. Colon tumors were
recorded by gross observation using a dissection microscope. All other organs,
including kidney, liver, and lungs were grossly examined under the dissection
microscope for any abnormalities. For histopathological evaluation, colon
tumors were fixed in 10% neutral buffered formalin, embedded in paraffin
blocks, cut into multiple sections, and processed. The slides were stained with
H&E and examined. The histological criteria used for classification of intes-
tinal tumors were as described previously (24, 26). Upon termination of this
study, more than 90% of the colon tumors had developed into adenocarcino-
mas that were classified as invasive or noninvasive. The invasive adenocarci-
nomas were mostly signet-ring mucinous types, invading the muscularis mu-
cosa deep into the intestinal wall and beyond. The noninvasive
adenocarcinomas were those growing outward toward the intestinal lumen
without invasion of the muscularis mucosa. They were usually well-differen-
tiated adenocarcinomas.
Fig. 1. Chemical structure of curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5-dione].
Fig. 2. Experimental design for evaluation of the chemopre-
ventive activity of curcumin against colon carcinogenesis.
Groups of male F344 rats were fed the experimental diets
containing 0 or 0.2% curcumin beginning 2 weeks prior to
exposure to AOM, during treatment, and until termination (ini-
tiation and postinitiation stages). Additional groups of animals
who were on control diet (0% curcumin) 2 weeks prior to
exposure of AOM, during treatment, and until 14 weeks after
AOM treatment were transferred to experimental diets contain-
ing 0.2 and 0.6% curcumin and were on this regimen until
termination (promotion/progression stage). AOM was given to
the animals s.c. at the beginning of 7 and 8 weeks of age at 15
mg/kg body weight.
Table 1 Percentage composition of experimental semipurified diets
Ingredients
% composition
Control diet
a
Experimental diets
Casein 2.0 20.0
DL-Methionine 0.3 0.3
Cornstarch 52.0 51.8 or 51.4
Dextrose 13.0 13.0
Corn oil 5.0 5.0
Alphacel 5.0 5.0
Mineral mix, AIN 3.5 3.5
Vitamin mix, AIN revised 1.0 1.0
Choline bitartrate 0.2 0.2
Curcumin
b
0 0.2 or 0.6
a
Adopted from the AIN reference diet (AIN-76A), with modification of the source of
carbohydrate.
b
Curcumin was added to the diets instead of cornstarch.
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CHEMOPREVENTIVE EFFECT OF CURCUMIN
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Detection of Apoptosis. Although apoptosis is characterized by DNA
fragmentation, and the appearance of a “ladder” of nucleosomal-sized frag-
ments on agarose gel electrophoresis has been used as a hallmark of apoptosis,
DNA cleavage is not universally found in apoptosis (31). A ladder of DNA
fragments has also been associated with necrosis in certain types of cells (32,
33). The gold standard for determination of apoptosis has been set through
observation of characteristic morphological changes by electron microscopy
(32, 34) or alternatively, by light microscopy (29, 35, 36). In this study, we
examined the modulation of apoptosis by curcumin by quantifying the number
of apoptotic cells in H&E-stained histological sections of colon tumors using
light microscopy (29, 36). Apoptotic cells were identified by cell shrinkage,
nuclear condensation, and formation of apoptotic bodies (29, 36). The light
microscopic appearance of apoptotic bodies are quite diverse; most are round
or roughly oval in shape. Apoptotic bodies vary in size, but they are a little
smaller than the parent cells. Some apoptotic cells contain pyknotic chromatin,
and some are devoid of a nuclear component (29, 36). The apoptotic index,
which represents the percentage of cells exhibiting apoptosis, was determined
by counting at least 300 cells in randomly chosen fields. All slides were scored
by one person who was blinded to the experimental listing by means of code
numbers.
Statistical Analysis. Data on body weights were compared among the
levels of test agent using Student’s t test. The comparative colon tumor
incidence (total number of colon tumor-bearing rats with respect to the total
number of rats at risk) in the animals fed the control diet and those given
experimental diets was analyzed using Armitage’s
x
2
method. Tumor multi-
plicities (total number of colon tumors per animal) were calculated for each
dietary group; the significance of the differences between results in groups on
the control diet and experimental diets containing curcumin was analyzed
using the unpaired Student’s t test, accounting for unequal variance. The
apoptotic index, which is expressed as the percentage of cells exhibiting
apoptosis was analyzed by unpaired Student’s t test. Differences were consid-
ered statistically significant at P , 0.05.
RESULTS
General Observations. The body weights of rats who received the
experimental diets containing 0.2% of curcumin starting from 2 weeks
before, during, and after carcinogen treatment to termination of the
study (initiation and postinitiation stages) and containing 0.2 or 0.6%
curcumin beginning from 14 weeks after carcinogen treatment until
the end of the study (promotion/progression stage) were comparable
to weights of those fed the control diet only (Table 2). As expected,
vehicle-treated animals in all groups weighed slightly more than those
treated with AOM during the course of the study. In vehicle-treated
rats, experimental diets containing curcumin did not produce any
gross changes in any organs and, thus, showed no toxicity.
Tumor Data. There were no tumors among rats given vehicle only
and maintained on control or experimental diets containing curcumin.
The results, summarized in Table 3, indicate that administration of
AOM induced adenomas and adenocarcinomas of the colon in ;9%
and 82% of rats, respectively, who were fed the control diet. Because
of long-term nature of this study (52 weeks), most of the colon tumors
had become adenocarcinomas. Administration of 0.2% curcumin dur-
ing the initiation and postinitiation stages (before, during and after
carcinogen treatment) significantly inhibited the incidence of nonin-
vasive adenocarcinomas (59% inhibition; P , 0.05), multiplicities of
noninvasive adenocarcinomas (71% inhibition; P , 0.01), and total
(noninvasive plus invasive) adenocarcinomas of the colon (34% in-
hibition; P , 0.05). The incidences of adenomas could not be com-
pared among different groups because of low yield of this lesion.
Administration of 0.2% curcumin during the promotion/progression
stages (14 weeks after carcinogen treatment) also significantly inhib-
ited the incidence of invasive adenocarcinomas of the colon (54%
inhibition; P , 0.05). Although the inhibition of the incidences and
multiplicities of noninvasive adenocarcinomas had reached 54 and
44%, respectively, in the rats given 0.2% curcumin during the pro-
motion/progression stage, the differences were not statistically signif-
icantly (P . 0.05). It is noteworthy that administration of 0.2%
curcumin during the promotion/progression stage significantly sup-
pressed total colon tumor incidence and multiplicity (adenomas plus
adenocarcinomas) as compared to results with the control diet
Table 2 Effect of dietary curcumin on body weights of male F344 rats
Experimental
group
No. of
animals/group
Body weights (g) of animals on experimental diets
Week 0 Week 1 Week 4 Week 12 Week 24 Week 36 Week 44 Week 52
AOM-treated
Control diet 36 115 6 10
a
150 6 12 224 6 14 323 6 22 395 6 25 422 6 27 437 6 38 430 6 40
0.2% curcumin
b
36 115 6 8 139 6 11 219 6 17 318 6 20 384 6 24 417 6 40 428 6 55 433 6 54
0.2% curcumin
c
36 115 6 8 148 6 10 221 6 13 322 6 19 392 6 27 433 6 32 444 6 42 437 6 49
0.6% curcumin
c
36 112 6 9 145 6 12 213 6 16 314 6 22 379 6 32 414 6 30 423 6 40 419 6 40
Vehicle-treated
Control diet 6 113 6 9 147 6 11 231 6 8 340 6 13 419 6 15 463 6 12 481 6 12 484 6 14
0.6% curcumin
c
6 111 6 10 152 6 12 235 6 13 337 6 20 405 6 30 436 6 29 456 6 37 461 6 37
a
Mean 6 SD.
b
Curcumin was administered 2 weeks before, during, and after carcinogen treatment.
c
Curcumin was administered starting 14 weeks after the second dose of carcinogen treatment.
Table 3 Effect of dietary curcumin on AOM-induced colon carcinogenesis in male F344 rats
Experimental
groups
Tumor incidence (% animals with tumors) Tumor multiplicity (tumors/animal)
Adenomas
Adenocarcinomas
Total Adenomas
Adenocarcinomas
TotalNoninvasive Invasive Total Noninvasive Invasive Total
Control diet 9 41 76 82 85 0.09 6 0.28
a
0.59 6 0.73 1.35 6 1.08 1.94 6 1.37 2.03 6 1.42
0.2% curcumin
b
3 (67)
c
17 (59)
d
57 (25) 71 (13) 71 (16) 0.03 6 0.17 0.17 6 0.38 (71)
e
1.11 6 1.14 (17) 1.29 6 1.08 (34)
f
1.31 6 1.12 (35)
f
0.2% curcumin
g
3 (67) 19 (54) 50 (54)
d
64 (22) 64 (25)
d
0.03 6 0.16 0.33 6 0.78 (44) 1.00 6 1.18 (30) 1.33 6 1.25 (31) 1.36 6 1.27 (33)
f
0.6% curcumin
g
6 (33) 9 (78)
h
54 (29) 64 (22) 64 (25)
d
0.06 6 0.23 0.09 6 0.28 (85)
i
0.74 6 0.87 (45)
f
0.83 6 0.84 (57)
i
0.89 6 0.85 (56)
i
a
Mean 6 SD.
b
Animals were administered curcumin beginning 2 weeks before, during, and after carcinogen treatment until termination of the study (initiation and postinitiation period).
c
% inhibition from control diet groups is shown in parenthesis.
d
Significantly different from control diet group by
x
2
-test, P , 0.05.
e
Significantly different from control diet group by Student’s t test, P , 0.01.
f
Significantly different from control diet group by Student’s t test, P , 0.05.
g
Animals were administered curcumin beginning 14 weeks after carcinogen treatment until termination of the study (promotion/progression period).
h
Significantly different from control diet group by
x
2
test, P , 0.01.
i
Significantly different from control diet group by Student’s t test, P , 0.001.
599
CHEMOPREVENTIVE EFFECT OF CURCUMIN
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(P , 0.05). As expected, administration of 0.6% curcumin during the
promotion/progression stage also significantly inhibited the incidence
of noninvasive adenocarcinomas (78% inhibition; P , 0.01) and
multiplicities of noninvasive (85% inhibition; P , 0.001) and inva-
sive (45% inhibition; P , 0.05) adenocarcinomas of the colon. In
addition, the incidences and multiplicities of total colon tumors (ad-
enomas plus adenocarcinomas) were reduced when rats were given
0.6% curcumin (25 and 56% inhibition; P , 0.05 and P , 0.01).
These results were analyzed using the linear correlation method for a
dose-response effect. This analysis yielded the correlation coefficients
(r) for multiplicity of adenocarcinomas with increasing levels of
curcumin in the diet from 0 to 0.6%, suggesting a dose-dependent
inhibition of colon tumors (P , 0.05): noninvasive adenocarcinomas,
20.97; invasive adenocarcinomas, 20.95; total adenocarcinomas,
20.97; and total tumors, 20.96.
Apoptosis. Having established the inhibition of colon carcinogen-
esis by dietary administration of 0.2% curcumin during the initiation
and postinitiation stages and the effects by 0.2 and 0.6% curcumin
given during the promotion/progression period, we investigated
whether the inhibition of colon tumorigenesis by curcumin is associ-
ated with the modulation of apoptosis in the colon tumors. Results
summarized in Table 4 indicate that continual administration of 0.2%
curcumin during the initiation and postinitiation stages and feeding
0.2 and 0.6% curcumin during the promotion/progression period
significantly increased the apoptotic index in the colon tumors as
compared to that in tumors of rats given control diet (P , 0.05–
P , 0.002).
DISCUSSION
This study is part of a large-scale evaluation of phytochemicals that
have anti-inflammatory and antioxidant properties for their potential
chemopreventive activities against colon carcinogenesis. The primary
mission of these studies is to identify effective and safe chemopre-
ventive agents that will facilitate the development of cancer-preven-
tive strategies and their application in a clinical setting. Curcumin, a
naturally occurring anti-inflammatory agent and antioxidant, has been
shown to inhibit tumors in several organs, including 7,12-dimethyl-
benz[a]anthracene-induced and TPA-promoted skin tumors, benzo-
(a)pyrene-induced forestomach tumors, and AOM-induced intestinal
tumors in mice (16, 17, 20), to cite a few. Recent studies from our
laboratory and elsewhere that demonstrated an inhibitory effect of
dietary curcumin when administered continuously during the initia-
tion and postinitiation phases (20–24) provided a rationale for eluci-
dating the efficacy of this agent against premalignant lesions during
the promotion/progression stage of colon carcinogenesis.
The results of this study are in agreement with earlier investigations
showing that dietary curcumin inhibits colon carcinogenesis when
administered during the initiation and postinitiation periods (20, 23,
24). Our results also demonstrate for the first time that curcumin, a
naturally occurring anti-inflammatory agent and antioxidant, given as
a dietary supplement during promotion/progression period still inhib-
its tumorigenesis in the colon, suggesting that administration of cur-
cumin may retard growth and/or development of existing neoplastic
lesions in the colon. This also suggests the potential usefulness of this
agent as a chemopreventive agent for individuals at high risk for colon
cancer development, such as patients with polyps. This study further
extends our earlier observations that synthetic NSAIDs, such as pi-
roxicam and sulindac, given during the promotion/progression period
protect against colon tumorigenesis in F344 rats (26, 37). Importantly,
unlike synthetic NSAIDs curcumin does not produce any gastrointes-
tinal toxicity, even at very high doses, which may provide advantage
over synthetic agents.
With regard to the mode of chemopreventive action, curcumin
exhibits a diverse array of metabolic, cellular, and molecular activities
including inhibition of arachidonic acid formation and its further
metabolism to eicosanoids. Studies from our laboratory have demon-
strated that dietary curcumin significantly inhibits phospholipase A
2
in colonic mucosa and tumors leading to the release of arachidonic
acid from phospholipids, alters COX and LOX activities, and modi-
fies PGE
2
levels (24). Several lines of evidence also indicate that the
mechanism of action of curcumin is not limited to PG inhibition. We
had observed earlier that dietary curcumin inhibits LOX activity, and
the production of the LOX metabolites, 5(S)-, 8(S)-, 12(S)-, and
15(S)-HETEs, in the colonic mucosa and in tumors (24). Importantly,
LOX metabolites such as 12(S)-HETE have been shown to promote
tumor cell adhesion, stimulate the spreading of tumor cells, and
augment metastatic potential (38–40). Also, a positive correlation
was observed between the levels of 8(S)-HETE and hyperproliferation
and tumor development induced by TPA (41). Moreover, curcumin
inhibits several mediators and enzymes involved in cell mitogenic
signal transduction pathways (42) and activator protein-1 and nuclear
factor
k
B activation (43). Hanif et al. (19) provided evidence that
curcumin inhibits cell proliferation and induces cell cycle changes in
the colonic adenocarcinoma cell lines, HT-29 and HCT-15, and that
this effect is independent of its ability to inhibit PG synthesis. Here,
the inhibitory effects of curcumin administered during the promotion/
progression stage of chemically induced carcinogenesis is associated
with increased apoptosis, suggesting that increased cell death through
apoptosis may be one of the mechanisms by which dietary curcumin
affects this inhibition. The results of this and other studies support the
concept that the capacity to induce apoptosis may be common to many
chemopreventive agents (28, 44, 45). This had certainly been docu-
mented for NSAIDs and other agents that inhibit colon carcinogene-
sis, suggesting that cellular responses to these agents may contribute
to chemopreventive effects (29, 35). The effects of curcumin demon-
strated here resemble those of NSAIDs and thus seem to act strongly
via inhibition of arachidonate metabolism and through reducing cell
proliferation and inducing apoptosis.
In conclusion, the study described here demonstrates for the first
time that dietary administration of curcumin during the promotion/
progression stage of AOM induced colon carcinogenesis significantly
inhibits tumor development in a dose-dependent manner and increases
apoptosis in the colonic tumors. Similar levels of inhibition of colon
tumorigenesis were achieved when 0.2% curcumin was administered
either during initiation and postinitiation periods or promotion/pro-
gression stage, suggesting indirectly that most of chemopreventive
efficacy of this agent is achieved during the promotion/progression
phase in this model. Although the exact mechanisms of its chemo-
preventive action of curcumin remain to be elucidated, it would
appear that modulation of tumorigenesis by this agent is associated
Table 4 Modulating effects of dietary curcumin on apoptosis in colon
adenocarcinomas
Experimental
group
Apoptotic
index
a
(%)
Control diet 5.33 6 0.61
b
0.2% curcumin
c
9.17 6 1.04
d
0.2% curcumin
e
7.56 6 0.82
f
0.6% curcumin
e
8.40 6 0.61
d
a
Apoptotic index represents percentage of cells exhibiting apoptosis.
b
Mean 6 SE; number of adenocarcinomas examined in each group: 10.
c
Animals were administered curcumin beginning 2 weeks before, during, and after
carcinogen treatment until termination of the study (initiation and postinitiation period).
d
Significantly different from the control diet group, P , 0.01.
e
Animals were administered curcumin beginning 14 weeks after carcinogen treatment
until termination of the study (promotion/progression period).
f
Significantly different from the control diet group, P , 0.05.
600
CHEMOPREVENTIVE EFFECT OF CURCUMIN
on June 4, 2013. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
not only with the alteration of arachidonic acid metabolism through
LOX and COX pathways (24) but also through mechanisms that are
independent of eicosanoid metabolism, such as cell proliferation and
apoptosis in the colon tumors.
ACKNOWLEDGMENTS
We thank Laura Nast for preparation of the manuscript, Ilse Hoffmann for
editing the manuscript, and staff of the Research Animal Facility and Histopa-
thology Facility for expert technical assistance. We thank Bob Kaskey of Gene
Print (Bala Cnwyd, PA) for kindly providing curcumin.
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