ORIGINAL PAPER

Jpn J Reprod Endocrinol ⓒ2004 by
Japan Society of Reproductive Endocrinology

Preventive Effects of Indole-3-carbinol on
Endometrial Carcinogenesis in Mice
Kenji NIWA1), Keiko TAGAMI1), Zenglin LIAN1), Jingchun GAO1), Yun Wu1), Kyoko ONOGI1),
Hideki MORI2) and Teruhiko TAMAYA1)
1) Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu 501-1194, Japan
2) Department of Pathology, Gifu University School of Medicine, Gifu 501-1194, Japan

Abstract. [Object] The short- and long-term experiments were designed to determine the effects of indole-3-carbinol (I3C) on estrogen-related endometrial carcinogenesis in mice, associated with the expression of c-fos and c-jun.
[Methods] In the short-term assay (2 weeks), ovarectomized mice were examined for the expression of c-fos andc-jun mRNAs under estogenic condition [5 ppm estradiol-17β (E2) in the diet]. In the long-term experiment (30weeks), mice were administered of I3C (500 ppm) under estogenic condition (5 ppm E2 in the diet) after single exposure of a direct carcinogen, N-mehtyl-N-nitrosurea. The uteri were pathologically examined at the termination of theexperiment.
[Results] In the short-term experiment, dietary I3C significantly reduced E2-stimulated expression of c-fos mRNA
(P<0.05). c-Jun--Jun mRNA showed a decreasing tendency by the I3C treatment. In the long-term experiment, administration of I3C in the diet reduced the incidence of MNU- and E2-induced endometrial atypical or simple hyperplasia(P<0.01).
[Conclusion] Present results suggest that dietary exposure of I3C prevents estrogen-related endometrial tumorigenesis in mice, through the inhibition of estrogen-related c-fos and c-jun expression.

Key words: Indole-3-carbinol, Endometrial carcinogenesis, Prevention, c-fos, Mice

Introduction

CH2OH

Several natural products in fruits and vegetables are reported to possess anti-mutagenic and anti-carcinogenicNH

properties [1-3]. Cruciferous vegetables, such as cabbage,broccoli, cauliflower, are known to contain indole deriva-

Fig. 1. Chemical structure of indole-3-carbinol

tives, dithiolthiones and isothiocyanates, and shown to exert anti-carcinogenic potentials in several rodent carcinogenic models [4, 5]. Phytochemical indole-3-carbinol (I3C,

the prevention of I3C on the mammary [6,8], uterine cervi-

Fig. 1), one of the indole derivatives, is reported to be anti-

cal [9] and endometrial [10] tumorigenesis in rodent mod-carcinogenic and anti-estrogenic. Dietary I3C functions as aels.

potent inducer of 2-hydroxylation of estradiol in rodents [6]The transient expression of the immediate early gene,and humans [7], and increases the anti-proliferative metab-c-fos/jun, is considered to be necessary for cellular prolifer-olite 2-hydroxyestrone and decreases 16α-hydroxyestrone.

ation and differentiation [11-13]. c-fos/jun mRNA in theThis effect on the estrogen metabolism may be related touterine corpora of ovarectomized mice is overexpressed by
the treatment of estrogens [14-16].

Accepted: August 30, 2004
Correspondence: Kenji NIWA, Department of Obstetrics andGynecology, Gifu University School of Medicine, 1-1 Yanagido,
Gifu-city, Gifu 501-1194, Japan
TEL: +81-58-230-6349
FAX: +81-58-230-6348
E-mail: kniwa@cc.gifu-u.ac.jp

These circumstances prompted us to determine if I3C
exerts inhibitory effects on mouse endometrial carcinogenesis induced by MNU and E2. Furthermore, the effects of
I3C on the expression of c-fos/jun mRNAs were also investigated.

Japanese Journal of Reproductive Endocrinology（2004）9：61-65

61Kenji NIWA et al.

Materials and methods
１）Animals and chemicals

phate dehydrogenase (GAPDH, a house-keeping gene)mRNA (995 bp) as an internal standard were performed at
the same time.

Female ICR mice, 10 weeks of age, were purchased fromThe following oligodeoxynucleotides were synthesized as

Japan SLC Co. (Shizuoka). The basal diet (Oriental MF,

specific primers in PCR according to the published informa-

Oriental Yeast Co., Tokyo) and filtered tap water were avail-tion (cDNA for c-fos [20], c-jun [21] and GAPDH [22]:able ad libitum throughout the experiment. I3C, E2 andsense for c-fos, 5 -CTTACGCCAGAGCGGGAATG-3 ; an-

MNU were purchased from Sigma Chem Co. (St. Louis,

ti-sense for c-fos, 5 -AAGCCTCAGGCAGACCTCCA-3 ;MO).

sense for c-jun, 5 -AGCGTGTTCTGGCT-3 ; anti-sense２）Experimental protocol for short-term effects of I3C
Female ICR mice, 12 weeks-of-age, were ovarectomized
under general anesthesia with diethylether. Two weeks later, the ovarectomized mice were divided into four groups.
Group 1 was given a diet containing 500 ppm I3C and 5 ppm
E2 (n=5); group 2 was given 5 ppm E2 (n=5); group 3 wasfor c-jun, 5 -CTGGGAAGCGTGTTCTGGCT-3 ; sense for
GAPDH, 5 -TGAAGGTCGGTGTGAACGGATTTGG-3 ;anti-sense for GAPDH, 5 -CTCCTTGGAGGCCATGTAGGCCAT-3 .
４）Semi-quantitative analysis of I3C mRNA expression in the mice uterine corpus by PCR products

fed on a diet containing 5 ppm E2 alone (n=6); group 4

PCR products were applied on 1.5% agarose gel electro-

served as a non-treatment control (n=5). After two weeks

phoresis at 50-100 V. The quantification of the products wason the above diet, the mouse uteri were resected and cut incarried out with Bio image (Nihon Millipore Corp., Tokyo).

half longitudinally. One half was quickly frozen in liquid ni-

The intensity of specific bands was standardized with thattrogen for the following experiments, and the other wasof GAPDH mRNA.

subjected to pathological examinations.
３）Reverse transcriptase-PCR (RT-PCR)５）Experimental protocol for long-term effects of I3C
A total of 90 female ICR mice, 10 weeks of age, under-

Total RNA was isolated from the frozen tissues by a gua-went laparotomy under general anesthesia with diethyle-nidium thiocyanate-phenol-chloroform extraction method

ther. MNU solution (total volume: 0.1 ml) at a dose of 1[19]. Total RNA (3 µg) was reverse transcribed with Mo-

mg/100 g body weight was injected into the left uterine

loney murine leukemia virus reverse transcriptase

tube and normal saline into the right. One week after the(MMLV-RTase, 200 units, Gibco BRL, Gaitherburg, MO)

exposure to MNU, the animals were divided into the follow-

in 20 µM Tris-HCl buffer (PH 8.4) with 50 µM KCl, 2.5 µM

ing four experimental groups. Group 1 (20 mice) was given

MgCl2, 0.1 µg/ml bovine serum albumin, 10 µM dithiothrei-

a diet containing 500 ppm I3C and 5 ppm E2. Group 2 (24tol, and 0.5 µM deoxynucleotides to generate cDNAs, usingmice) was treated with 5 ppm E2 alone. Group 3 (20 mice)

random hexamers (50 ng, Gibco BRL) at 37℃ for 60 min.

were fed with a diet containing 500 ppm I3C. The dose of E2

RT reaction was carried out at 94℃ for 5 min to inactivate

was the same as in the short-term assay, and the above di-

MMLV-RTase. For c-fos or c-jun mRNA expression, treat-ets were given throughout the experiment. Group 4 (26ment included 40 cycles of PCR consisting of 1 min at 94℃mice) served as a control group. Thirty weeks after thefor denaturation, 1 min at 55℃ for annealing and 1 min atstart of the experiment, all major organs, especially the re-

72℃ for extension. The reactions were carried out in re-productive organs, were grossly inspected. The uterus,verse transcribed cDNA and 0.1 mM specific primers de-ovaries, vagina, and other lesions suspected of being hyper-

scribed below, using an IWAKI thermal sequencer TSR-300

plastic or neoplastic were cut in half. After being fixed in(IWAKI Glass, Tokyo) with Vent DNA polymerase (New

10% formaline, tissues were sectioned at 3 µm and stained

England Biolabs, Bervely, MA) in 20 µM Tris-HCl buffer

with hematoxyline and eosin.

(PH 8.8) with 10 µM KCl, 10 µM (NH4)2SO4, 2 µM MgSO4,
0.1% Triton X-100, and 0.15 µM dexynucleotide phosphates. Twenty cycles of PCR for glyceraldehyde-3-phos-

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Japanese Journal of Reproductive Endocrinology Vol. 9 2004

６）Histology of the uterine lesions
Uterine endometrial lesions were divided into 4 types ac-

Preventive Effects of Indole-3-carbinol on Endometrial Carcinogenesis in Micecording to the WHO criteria [23]; a) endometrial hyperplasia, simple; b) endometrial hyperplasia, complex; c) atypi-７）Statistical analysis
Statistical analysis was done according to the κ2 test or

cal endometrial hyperplasia; d) adenocarcinoma.

c-jun mRNA level (AU/GAPDH mRNA)

2.0

c-fos mRNA level (AU/GAPDH mRNA)

student s t test.

2.0

Results
１）Short-term experiment

1.0

The levels of c-fos mRNA expression are shown in Fig.
2. I3C treatment significantly reduced the E2-induced ex-

0pression of c-fos (P<0.05). The levels of c-jun mRNA exP<0.05

pression are also shown in Fig. 2. I3C treatment generated
a decreasing tendency, but the effect was not significant.
P<0.05

1.0２）Long-term experiment
Two mice in group 1 and three in group 3 died within 15
weeks, yet no pathological abnormalities other than pneu-

0E2 + I3C

E2 alone

I3C

control

Fig. 2. Expressions of c-fos mRNA treated with E2 plus I3C, E2 alone,
I3C alone and the control. I3C treatment significantly reduced
the expressions of c-fos (P<0.05). Each column shows themean and each bar expresses S.D. Data are calculated fromeach triplicated samples of the treated animals in each group.
Expressions of c-jun mRNA treated with E2 plus I3C, E2 alone,
I3C alone and the control. I3C treatment showed a decreased
tendency of the expressions of c-jun. Each column shows themean and each bar expresses S.D. Data are calculated fromeach triplicated samples of the treated animals in each group.

monia were found. The remaining animals survived until
the termination of the experiment and were enrolled as effective animals (group 1, 18; group 2, 17; group 3, 25; andgroup 4, 25). Histological properties of endometrial hyperplasias and adenocarcinoma in the present study were basically as the same as in our previous report [14,16-18]. All
adenocarcinomas seen in the endometria were well- or
moderately-differentiated. The incidence of the preneoplastic and neoplastic lesions of the left utreine endometria issummarized in Fig. 3. The incidence of atypical hyperplasia(%)
100
P<0.0175

P<0.015025EE H H, s, c imp
om le
pl
e
At x
H
AD
E
C
EHH, s
i
m
,c p
om le
pl
e
At x
H
A
EH D C
EH , s, c imp
om le
pl
e
At x
H
A
EH D C
EH , s, c imp
om le
pl
At ex
H
AD
C

0

Group 1
(MNU + E2 + I3C)

Group 2(MNU + E2)

Group 3
Group 4
(MNU + I3C) (MNU alone)

Fig. 3. Incidence of the preneoplastic and neoplastic endometrial lesions of the treated (left) side with MNU. I3C treatment significantly reduced theincidences of atypical hyperplasia and endometrial hyperplasia, simple compared with the group 2 (P<0.01). Treatment of E2 increased theincidence of (pre)neoplastic endometrial lesions (groups 1 vesus 3; 2 versus 4).

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Kenji NIWA et al.

(4/18, 22%) on the left side of the uterine corpus of group 1

could be effective for prevention of human endometrial car-was significantly lower than that of group 2 (16/25, 67%,

cinoma.

P<0.01). The incidence of endometrial hyperplasia, simple(9/18, 50%) was also significantly lower than that of group2 (22/25, 92%, P<0.01). The incidences of adenocarcinoma and endometrial hyperplasia, complex on the left side of
the uterine corpus in group 1 tended to be lower than thoseof group 2, although the differences were not significant.
The incidences of endometrial hyperplasias and adenocarcinoma of the left side of the uterine corpus in group 3 werealso rather lower than those of group 4, but the differenceswere not significant.

Discussion
In our previous experiment, dietary I3C had a suppressive effect on E2-related endometrial tumorigenesis in
mice, possibly through suppression of estrogen-induced
c-fos/jun expression [15,16]. The incidence of endometrial
hyperplasia, simple, being considered to be affected by estrogens in this study, was more prominently decreased by
I3C compared with our previous reports [16-18]. I3C issuggested to possess an anti-estrogenic activity more potently than other agents, such as Glycyrrhizae radix, Juzentaiho-to and toremifene, which were examined by us previously.
The chemopreventive effects of I3C on estrogen-relatedendometrial tumorigenesis thus probably relate to the antiestrogenic function of indole derivative. Acid condensation
products of I3C are known to be ligands for the aryl hydrocarbon receptor [24]. This interaction is suggested to be thereason why I3C alters expression of some cytocrome P-450
that regulates the estrogen metabolism [25,26]. I3C is also
reported to increase 2-hydroxyestrone [26]. Both of I3C
and 2-hydroxyestrone are found to function as an anti-estrogen, and compete with E2 for the estrogen receptor [27].
Other possible anti-cancer effects of I3C are assumed torelate to G1-arrest [28] or apoptosis [29, 30].
Results in this study suggest that dietary I3C acts as achemopreventive agent on the estrogen-related endometrial tumorigenesis. This indole compound, I3C is already regarded as a promising agent for therapy as well as prevention against lar yngeal papillomatosis [31,32]. The
attractiveness of I3C is the possibility that a diet enrichedwith cruciferous vegetables or a supplementation of I3C

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Japanese Journal of Reproductive Endocrinology Vol. 9 2004

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