Int. J.
Cancer: 122, 2689–2698 (2008)
' 2008 Wiley-Liss, Inc.
b-ionone suppresses mammary carcinogenesis, proliferative activity and
induces apoptosis in the mammary gland of the Sprague-Dawley rat
Jia-Ren Liu1, Xiang-Rong Sun2, Hong-Wei Dong1, Chang-Hao Sun2, Wen-Guang Sun2, Bing-Qing Chen2*,
You-Qiang Song3 and Bao-Feng Yang4
1
Department of Environmental Health, Public Health College, Harbin Medical University, NanGang District,
Harbin, People’s Republic of China
2
Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, NanGang District,
Harbin, People’s Republic of China
3
Department of Biochemistry, Hong Kong University, Hong Kong, China
4
Department of Pharmacology, Harbin Medical University, NanGang District, Harbin, People’s Republic of China
b-Ionone demonstrates potent anticancer activity both in vitro and
in vivo. We determined tumor incidence and the number of rats
bearing tumors as well as cell proliferation and apoptosis in a rat
mammary cancer model induced by 7, 12-dimethylbenz[a]anthra-
cene (DMBA). Rats were fed an AIN-76A diet containing b-ionone
(0, 9, 18 or 36 mmol/kg), starting 2 weeks before DMBA adminis-
tration and continuing for 24 weeks. A dose-dependent inhibition
of mammary carcinogenesis by dietary b-ionone was observed.
Corresponding tumor incidence values were 82.1, 53.3, 25.9 and
10.0% (p < 0.01 or 0.05). Time to tumor appearance increased and
tumor multiplicity decreased with increasing dietary b-ionone.
Histopathological and immunohistochemical evaluations of
tumors were performed on the 64, 31, 15 and 3 tumors, respec-
tively, identified in rats from the respective groups of 30. The pro-
portions of adenocarcinomas, adenomas and benign masses were
equally distributed in the latter group. In proportions within the
other groups, the proportions of adenocarcinomas and benign
masses decreased and increased with increasing dietary b-ionone.
Proliferating cell nuclear antigen (PCNA), cyclin D1 and Bcl-2
expression decreased, and Bax expression and nuclear fragmenta-
tion increased with increasing dietary b-ionone. These results
demonstrate the potent capacity of dietary b-ionone to suppress
DMBA-initiated mammary cancer in rats.
' 2008 Wiley-Liss, Inc.
Key words: b-ionone; mammary carcinogenesis; cell proliferation;
apoptosis
Epidemiological studies have generally shown that the con-
sumption of vitamin-rich fruits, vegetables and whole grains is
inversely associated with risks for cancer and other chronic dis-
eases.1–4 Failing to demonstrate an inverse relationship between
cancer incidence and consumption of the vitamins prominent in
these plant products, investigators have turned attention to the
many classes of nutrient phytochemical constituents also present
in these foods.5 Specific isoprenoids, culled from a group of some
22,000 products of secondary plant metabolic pathways sharing a
common precursor, mevalonic acid,6 have demonstrated anticar-
cinogenic and chemoprotective activities. One phytochemical
compound, b-ionone, an end ring analog of b-carotenoid, repre-
sents a subclass of cyclic isoprenoids. Physiological functions
attributed to b-ionone include the inhibition of the growth of
fungi7 and the regulation of the synthesis of mevalonate-derived
constituents.8 In previous studies, b-ionone inhibited the growth
of breast-, gastric-, colon- and HL-60- cancer cells in a dose-
dependent manner,9–13 affected the MAPK pathway of MDA MB
435 cells,14 and induced apoptosis of SGC-7901 gastric cancer
cells and B16 murine tumor cells.10,12 In addition, b-ionone could
also affect metastasis in B16 and SGC-7901 cancer cell
lines.11,12,15 Dietary studies reveal the chemopreventive16 and
antitumor17 activities of b-ionone.
This study confirms and extends observations of the chemopre-
ventive impact of, and actions triggered, by dietary b-ionone dur-
ing DMBA-initiated mammary carcinogenesis in Sprague-Dawley
rats.
Publication of the International Union Against Cancer
Material and methods
Chemicals
b-ionone, [95% purity, 4-(2,6,6-trimethyl-l-cyclohexen-1-yl)-3-
buten-2-one, FW 192.30) and 7,12-dimethylbenz[a]anthracene
(DMBA) were purchased from Sigma Chemical Co. (St. Louis,
MO). All other chemicals used in this study were of analytical grade.
Animals and treatment
Pathogen-free weaning female Sprague-Dawley rats, 30 days of
age, were purchased from Sino-British SIPPR/BK Lab Animal
Ltd. (Shanghai, China). The rats adapted immediately to the AIN-
76A diet and were housed in a room with a 12-hr light/12-hr dark
cycle. The rats were acclimated to the surroundings in the animal
room for 1 week prior to the initiation of the experiment. Care and
treatment of rats followed the recommended guidelines of the
National Research Council (1985). The rats were randomly
assigned to 5 groups (n 5 30). Four groups of rats were given 10
mg of DMBA dissolved in 1 ml of corn oil by gavage, which
served as the negative control, was given 1 ml of corn oil. Rats
were administered the control or b-ionone diet, starting 2 weeks
before DMBA administration continuing for 24 weeks until the
end of the experiment. The 3 treatment of rats were given: 9.0
(low), 18.0 (medium) and 36.0 mmol/kg (high) b-ionone in the
AIN-76A diet. Animals were weighed weekly and palpated twice
a week to check for the development of palpable mammary
tumors. The time of tumor appearance was also recorded.
Histological evaluation of mammary tissue
Twenty-four weeks after the carcinogen or vehicle treatment,
all animals from each group were killed under anesthesia. All ani-
mals were subjected to complete autopsy. At the time of necropsy,
all tumors were removed and weighed, and the tumor volume was
recorded.18 The tumors were fixed in 10% buffered formol saline,
and subsequently dehydrated and embedded in wax. The tissue
wax was cut into 3- to 5-lm sections, fixed on slides and proc-
essed for microscopy examination [stained with hematoxylin and
eosin (HE)] or immunohistochemistry analysis. Histopathological
evaluation was carried out on coded slides following the Interna-
tional Agency for Research on Cancer classification of mammary
rodent tumors.19 Tumor cells in cancer tissues were easily identi-
fied in HE sections by their basophilic properties and polymorphic
Grant sponsor: National Natural Science Fund, People’s Republic of
China; Grant number: 30200229.
Jia-Ren Liu’s current address is: Department of Food Science, Cornell
University, 127 Stocking Hall, Ithaca, NY 14853-7201, USA.
*Correspondence to: Department of Nutrition and Food Hygiene,
Public Health College, Harbin Medical University, 157 BaoJian Road,
NanGang District, Harbin, 150081 PRC. Fax: 186-8750-2885.
E-mail: jiarl@ems.hrbmu.edu.cn
Received 6 November 2007; Accepted after revision 8 January 2008
DOI 10.1002/ijc.23453
Published online 3 April 2008 in Wiley InterScience (www.interscience.
wiley.com).
 10970215, 2008, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.23453 by Jeonbuk national university library, Wiley Online Library on [04/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2690 LIU ET AL.

FIGURE 1 – The effect of b-ionone on body weight over 24 weeks.

Thirty-day-old female Sparague-Dawley rats were supplemented with
9 (low), 18.0 (medium) and 36.0 mmol / kg (high) of b-ionone in the
AIN-76A diet starting 2 weeks before DMBA administration and con-
tinuing for 24 weeks until the end of the experiment. Control 2: the
negative control group, Control 1: the positive control group.

appearance. There are 4 types of histologies in mammary cancer

of rat including benign (hyperplasia and adenolipoma), fibroade-
noma, adenoma and adenocarcinoma.19,20 Microscopic examina-
tion of the control group indicated that the mammary tumors
showed the characteristics of malignant tumors, described in detail
by Papotti et al.20
Immunohistochemical staining of proliferating cell nuclear
antigen, Cyclin D1, Bcl-2 and Bax
The sections of mammary cancer tissues were deparaffinized in
xylene and rehydrated through graded alcohol. The sections were
incubated for 10 min at 95–100°C in 10 mmol/l sodium citrate
buffer (pH 6.0). Endogenous peroxidases were inactivated by
immersing the sections in hydrogen peroxide for 10 min, and were
then incubated for 10 min with 10% normal goat serum to block
nonspecific binding. The sections were subsequently incubated at
4°C overnight with anti-PCNA antibody (monoclonal mouse, PC10,
IgG, 1:50 dilution, Calbiochem Lab, Delaware, CA) anti-cyclin D1
antibody (monoclonal mouse, DCS-6, IgG, 1:25 dilution, Calbio-
chem Lab), anti-Bcl-2 antibody (rabbit polyclonal, N-9, IgG, 1:15
dilution, Santa Cruz Biotechnology, Temecula, CA) or anti-Bax
antibody (polyclonal rabbit, AB-1, IgG,1:20 dilution, Calbiochem
Lab). Then, the sections were incubated with biotinylated anti-
mouse IgG or anti-rabbit IgG (ZYMED Lab, Carlsbad, CA) for 30
min, followed by peroxidase-conjugated streptavidin (ZYMED Lab)
for 30 min. The chromogenic reaction was developed with 3,30 -dia-
minobenzidine tetrahydrochloride (DAB) for 3 min, and all sections
were counterstained with hematoxylin. The same protocol was FIGURE 2 – The effect of b-ionone on survival without tumor (a),
applied to the controls with the omission of the primary antibody. tumor incidence (b) and the number of rats bearing tumors (c) in
Microscopic images were measured at a magnification of 4003. female Sprague-Dawley rats exposed to DMBA. Rats (n 5 30) were
Two thousand cells were counted in 5 visual fields in each section, started on AIN-76A diet (controls) or diets supplement with 9.0 (low),
and an average of over 10 tissue masses randomly chosen from at 18.0 (medium) and 36.0 mmol/kg (high) of b-ionone 2 weeks prior to
least 5 rats in each group (except for the high-dose group) were ana- the administration of DMBA. Rats were maintained on the diet
lyzed to determine the cells positively stained for specific protein throughout the experiment. Tumors were detected by palpation during
the study and verified after removal. Control 2: the negative control
expression (mean 6 SD). group, Control 1: the positive control group.

Immunohistochemical staining by TUNEL

The effect of b-ionone on apoptosis in rat mammary cancer tis-
sues was evaluated by the TUNEL assay (Promega Corporation,
Madison, WI). The slices of mammary cancer tissues were depar-
affinized in xylene and rehydrated through graded alcohol, then
washed in 2% NaCl in phosphate-buffered saline (PBS) buffer,
fixed in 4% paraformaldehyde, treated with proteinase K (20 lg/
ml) for 30 min, refixed with 4% paraformaldehyde and labeled
with TdT reaction mix in a humidified box for 1 hr at 37°C. The
slices were washed in 23 SSC (0.15 mol/l sodium chloride and
0.015 mol/l trisodium citrate, SSC solution) for 15 min and then
blocked in 3% H2O2 for 5 min. The slides were then inculated
with streptavidin HRP and colorized with DAB. All sections were
counterstained with hematoxylin. Controls were subjected to the
same protocol with the omission of the TdT reaction mix. Micro-
 10970215, 2008, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.23453 by Jeonbuk national university library, Wiley Online Library on [04/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
b-IONONE PREVENTS MAMMARY CARCINOGENESIS IN SD RATS 2691
TABLE I – THE CLASSES OF MAMMARY TUMORS IN THE POSITIVE CONTROL AND b-IONONE TREATED GROUPS
Group Number of tissue (n) Adenocarcinoma (%) Adenoma (%) Fibroadenoma (%) Benign masses (%)

High 3 1 (33.33) 1 (33.33) 0 (0.00) 1 (33.34)

Medium 15 3 (20.00)* 4 (26.67) 1 (6.66) 7 (46.67)*
Low 31 14 (45.16) 2 (6.45) 5 (16.13) 10 (32.26)**
Control 1 64 38 (59.38) 11 (17.19) 7 (10.93) 8 (12.50)
*p < 0.01, **p < 0.05, compared to the corresponding positive control group. Control 1, the positive control group.

FIGURE 3 – Histopathological
findings in mammary tumors
stained with HE. (a,b) H&E
stained section of a hyperplasia
(103 and 403). (c,d) H&E stained
section of a fibroadenoma (103
and 403). (e,f) H&E stained sec-
tion of an adenoma (103 and
403). (g,h) H&E stained section
of a moderately differentiated ade-
nocarcinoma (103 and 403). The
neoplastic cells show great diver-
sity in size and arrangement
around the lumens at sites becom-
ing multilayered, palisaded and
showing papillary or even disor-
ganized or solid growth. Their
nuclei are enlarged, euchromatic
and contain multiple nucleoli. The
cytoplasm is scant and mitotic
figures are common. [Color figure
can be viewed in the online issue,
which is available at www.
interscience.wiley.com.]
 10970215, 2008, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.23453 by Jeonbuk national university library, Wiley Online Library on [04/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2692 LIU ET AL.

FIGURE 4 – Effect of b-ionone

on expression of PCNA in the
mammary cancer tissues in vivo.
High power view (4003) of the
PCNA expression: the positive
control (a) (control 1, n 5 10),
and 3 levels of 9.0 (b) (low, n 5
11), 18.0 (c) (medium, n 5 10) and
36.0 mmol/kg (d) (high, n 5 3) of
b-ionone in the AIN76A diet. The
expression of PCNA was visual-
ized with DAB; positive cells are
reddish-brown in color. Bars with
no letters in common are signifi-
cantly different (p < 0.05 and p <
0.01). [Color figure can be viewed
in the online issue, which is avail-
able at www.interscience.wiley.
com.]

scopic images were measured at a magnification of 4003. Two Results

thousand cells were counted in 5 visual fields randomly in the sec-
tion, and an average of over 10 tissue masses randomly chosen
from at least 5 rats in each group (except for the high-dose group)
was examined to determine the number of apoptotic cells which
were identified by a brown stain over the nuclei (mean 6 SD).
Statistical analysis
Data were expressed as mean 6 SD. A one-way analysis of var-
iance was used to analyze body weight.21 Fisher’s exact test22 was
used to compare the percentage of rats with tumors and tumor his-
tology in each group. The expression of proliferating cell nuclear
antigen (PCNA), TUNEL, cyclin D1, Bcl-2 and Bax in mammary
gland tumors were analyzed using the Student’s or Welch’s t-test
following the F-test. For the few rats that were killed early
because of tumor burden, we assumed that the number of tumors
remained constant until the end of the study. Data analyses were
generated, and plots were constructed using SPSS for Windows
version 13.0 (SPSS Inc., Chicago, IL) and SigmaPlot version 10
for Windows (Systat Software, San Jose, CA). Statistical signifi-
cance was set at p < 0.05 and p < 0.01, and all p values were
unadjusted for multiple comparisons.
Effect of b-ionone on body weight
Body weights of animals in each group were monitored weekly.
Throughout the experiment, there were no significant differences
in body weight in animals fed dietary b-ionone in comparison
with the control groups (p > 0.05, Fig. 1). This suggested that
there was no observed toxicity in animals fed on b-ionone in the
AIN76A diet.
Effect of b-ionone on the prevention of mammary carcinogenesis
In this study, SD rats were given low, medium and high doses
of b-ionone in the AIN76A diet, starting 2 weeks before DMBA
and continuing for 24 weeks. The positive control group, given a
10 mg dose of DMBA, developed the tumors by 10 weeks. The
treatment groups, given the low, medium and high doses of b-io-
none, developed the tumors 12, 15 and 17 weeks after carcinogen
administration, respectively. The survival of rats without tumors
in each group fed dietary b-ionone was higher than the positive
control group (Fig. 2a). Sequential observation data for incidences
of palpable mammary tumors in the positive control and b-io-
none-treated groups and the total number of rats bearing tumors
 10970215, 2008, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ijc.23453 by Jeonbuk national university library, Wiley Online Library on [04/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
b-IONONE PREVENTS MAMMARY CARCINOGENESIS IN SD RATS 2693

FIGURE 5 – Effect of b-ionone

on expression of cyclin D1 in the
mammary cancer tissues in vivo.
The expression of cyclin D1 in cell
nucleus was observed by immuno-
histochemical stain in mammary
cancer tissues of positive control
(a) (control 1, n 5 14), and 3 lev-
els of 9.0 (b) (low, n 5 20), 18.0
(c) (medium, n 5 10), and 36.0
mmol/kg (d) (high, n 5 3) of b-io-
none in the AIN76A diet, respec-
tively, (4003, counterstained with
hematoxylin). Bars with no letters
in common are significantly differ-
ent (p < 0.05). [Color figure can be
viewed in the online issue, which
is available at www.interscience.
wiley.com.]

are presented in Figures 2b and 2c. Data for final incidences of
mammary tumors in animals, including those which died or were
killed during the experiment, are also calculated and were signifi-
cantly decreased by the b-ionone treatment (incidence: 36.0
mmol/kg, 10.0%, and 18.0 mmol/kg, 25.9%, and 9.0 mmol/kg
b-ionone, 53.3%; control, 82.1%, respectively) and a clear dose
dependence was observed.
Mammary tumor histopathology
Mammary tumor histology data and morphology are presented
in Table I and Figure 3, respectively. At the termination of the
study 59.38% (38/64), 45.16% (14/31), 20.00% (3/15) and
33.33% (1/3) of adenocarcinoma in the rat’s neoplasm in the con-
trol and b-ionone treated groups, respectively, had been examined
by HE. A significant difference was observed between the control
group and the medium dose of b-ionone treatment group (p <
0.01) and an increasing trend of benign masses was observed
among the control and b-ionone treated groups, except for the
high-dose group (p < 0.05 and p < 0.01). The other types of
tumors in mammary tissue were not different between the control
and b-ionone treatment groups. There was an increasing trend of
adenoma and decreasing trend of fiboadenoma in the mammary
tissues of the groups supplemented with the low-, medium- and
high-dose b-ionone in the AIN-76A diet.
Expression of PCNA and Cyclin D1 in mammary cancer tissues
To determine whether b-ionone affected cell proliferation of
the mammary gland, the expression of PCNA was analyzed by
immunohistochemistry in mammary cancer tissues from the con-
trol and b-ionone treated groups. As shown in Figure 4, the
expression of PCNA in mammary cancer tissues of b-ionone
treated groups (434 6 119, 122 6 44, and 50 6 5, per 2,000 cells,
in the low, medium, and high doses in the AIN-76A diet, respec-
tively) had a significant decrease in comparison with the control
group (575 6 241 per 2,000 cells). Expression of PCNA in both
the positive group and the low-dose b-ionone group were signifi-
cantly different compared to the medium- and high-dose groups (p
< 0.01). In comparison with the control group, the inhibitory rates
of PCNA expression were 25, 79, and 91% in the groups fed the
low, medium and high doses of b-ionone in the AIN-76A diet,
respectively. A dose response was observed.
The expression of cyclin D1 in the mammary cancer tissues was
also determined by immunohistochemistry. As shown in Figure 5,

2694 LIU ET AL.
the expression of cyclin D1 in mammary cancer tissues in the con-
trol group (45 6 29 per 2,000 cells) was significantly different
from those of b-ionone treated groups except for the low-dose
group (p < 0.05). Clear demonstrating a dose response, the low,
medium and high doses of b-ionone inhibited cyclin D1 expres-
sion by 0, 36, and 58%, respectively, compared to the positive
control group.
Expression of apoptosis, Bcl-2 and Bax in mammary
cancer tissues
Apoptosis in mammary cancer tissues was detected by the
TUNEL assay. As shown in Figure 6, occurrence of apoptosis in
mammary cancer in the groups given the medium and high doses
of b-ionone was significantly higher than the control and low-dose
groups (p < 0.05 and p < 0.01). Apoptosis expression was
increased by 71 and 55 times in the groups given the medium and
high doses of b-ionone, respectively, compared to the control
group. Although a dose response was not observed, an increasing
trend of apoptosis was obtained in the experimental and control
groups.
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FIGURE 6 – Effect of b-ionone
on apoptosis in the mammary can-
cer tissues in vivo. Nuclear frag-
mentation and apoptotic cells were
observed by the TUNEL assay
with immunohistochemistry. The
expression of apoptotic cell in
mammary cancer tissues was
observed as brown color in nucleus
of positive control (a) (control 1,
n 5 10), and 3 levels of 9.0 (b)
(low, n 5 11), 18.0 (c) (medium, n
5 10), and 36.0 mmol/kg (d)
(high, n 5 3) of b-ionone in the
AIN76A diet, (4003, counter-
stained with hematoxylin). Bars
with no letters in common are sig-
nificantly different (p < 0.05 and
p < 0.01). [Color figure can be
viewed in the online issue, which
is available at www.interscience.
wiley.com.]
The expression of Bcl-2 and Bax in mammary cancer tissues
was also examined by immunohistochemistry. The expression of
Bcl-2 in the low-, medium-, and high-treatment groups (45 6 46,
32 6 35, 12 6 21 per 2,000 cells, respectively) was significantly
different from the control group (93 6 80 per 2,000 cells, p <
0.05, Figure 7). The inhibitory rate of Bcl-2 expression was 52,
66, and 87% in the 3 treatment groups given the low, medium, and
high doses of b-ionone, respectively, in comparison with the con-
trol group (p < 0.05). The expression of Bax in the high dose of
b-ionone group (326 6 193 per 2,000 cells) was significantly dif-
ferent from the other groups including the control group (p < 0.05
and p < 0.01, Figure 8), and was 19 times greater than the control
group. In this animal model, b-ionone treatments inhibited the
expression of Bcl-2 and induced the expression of Bax in a dose-
dependent manner (Figs. 7 and 8).
Discussion
b-Ionone, a pure isoprenoid, has been shown to possess potent
antiproliferative activity9,13,17 and to induce apoptosis10,12 in
cancer cells. Liu’s study9 reported that b-ionone at doses of
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b-IONONE PREVENTS MAMMARY CARCINOGENESIS IN SD RATS 2695

FIGURE 7 – Effect of b-ionone

on Bcl-2 expression in the mam-
mary cancer tissues in vivo. The
expression of Bcl-2 in mammary
cancer tissues was observed by im-
munohistochemical stain in cyto-
plasm in positive control (a) (con-
trol 1, n 5 15), and 3 levels of 9.0
(b) (low, n 5 22), 18.0 (c) (me-
dium, n 5 10), and 36.0 mmol/kg
(d) (high, n 5 3) of b-ionone in
the AIN76A diet, (4003, counter-
stained with hematoxylin). Bars
with no letters in common are sig-
nificantly different (p < 0.05).
[Color figure can be viewed in the
online issue, which is available at
www.interscience.wiley.com.]

25–200 lmol/l inhibited cell proliferation and DNA synthesis in
human mammary cancer MCF-7 cells in a dose-dependent man-
ner. In a previous study,13 b-ionone also inhibited the cell prolif-
eration of MCF-7 cells via inhibition of Cdk2 activity and down-
regulation of cylins D1 and E. These results indicate that mecha-
nisms other than impaired mevalonate synthesis mediate the
antiproliferative and cell-cycle regulatory effects of b-ionone in
human breast cancer cells. As a note, b-ionone could also inhibit
the cell proliferation of the estrogen receptor negative cell line
MDA MB 435 in the MAPK pathway by downregulation of ERK
and MEK-1 expression and upregulation of JNK and MKP-1
expression.14 In another study, b-ionone also suppressed the
growth of other cancer cells—human colon fibroblasts CCD-
18Co cells, HL-60 cells, and human colon adenocarcinoma
Caco-2 cells.12 Apoptosis morphology was observed in SGC-
7901 cells induced by b-ionone using transmission electron
microscopy and hoechst-33258 staining. Apoptosis was induced
in SGC-7901 cells at doses of 25–200lmol/l of b-ionone using
the TUNEL assay and showed a dose- and time-dependent
response.10 Mo and Elson12 also showed that b-ionone initiated
apoptosis and, concomitantly arrested cells in the G1 phase of
the cell cycle in human and murine tumor cells. In addition,
b-ionone also affected the progression of metastasis in SGC-
7901 cells11 and B16 cells.12,17
In this study, female Sparague-Dawley rats were fed 3 levels of
9.0 (low), 18.0 (medium) and 36.0 mmol/kg (high) of b-ionone in
the AIN-76A diet, starting 2 weeks before DMBA administration
and continuing for the 24 weeks until the end of the experiment.
The positive control group, given the DMBA, developed mammary
tumors with 82.1% tumor incidence during the 24-week study, and
no tumors were detected in the negative control group without
DMBA. A dose-dependent inhibition of mammary carcinogenesis
by b-ionone was observed (Fig. 2). These results demonstrate for
the first time that supplementation with b-ionone has potent anticar-
cinogenic activity in mammary cancer induced by DMBA in a rat
model. Tumor incidence was decreased in a dose-dependent manner
during the 24-week study. Yu et al.16 reported that the potency of
b-ionone in suppressing HMGR activity exceeds these of other
monoterpenes. In parallel trials, the efficacy of b-ionone was greater
than that of equal intakes D-linmonene and geraniol in mammary
cancer chemoprevention. The monoterpenes decreased tumor multi-
plicity by 45% (p < 0.001). The impact of b-ionone on tumor multi-
plicity was 2 3 that of the monoterpenes (p < 0.001). b-Ionone

2696 LIU ET AL.
possessed chemopreventive activity, which was observed to pre-
vent DNA damage in the initial phases of hepatocarcinogenesis
induced in rats by diethylnitrosamine and 2-acetyaminofluorene.23
He et al.17 reported that 2 mmol/kg b-ionone in the AIN-76A diet
prolonged host survival by 30% compared to the control group in a
melanoma model of C57BL female mice (p < 0.005). Thus, b-io-
none is a potent cancer chemopreventive agent.
The 4 types of histopathological differential diagnoses in the
mammary cancer tissues of rats include benign masses (hyper-
plasia and adenolipoma), fibroadenomas, adenomas and adenocar-
cinomas.19 The adenocarcinoma diagnosis is a type of highly ma-
lignant tumor that is the main cause of death in mammary cancer
patients and also a main target for chemoprevention and chemo-
therapy. Adenocarcinoma is always accompanied by occurrence
of distant metastasis and areas of necrosis, ulceration and hemor-
rhage. During this 24-week study, 59.38% of the tumors in the
control group had adenocarcinoma. In the groups supplemented
with the low, medium and high doses of b-ionone in the AIN-76A
diet, 45.16, 20.00, and 33.33% had adenocarcinoma, respectively.
The occurrence of adenocarcinoma in the medium-dose group of
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FIGURE 8 – Effect of b-ionone
on Bax expression in the mammary
cancer tissues in vivo. The expres-
sion of Bax protein in cytoplasm
was observed by immunohisto-
chemical stain in mammary cancer
tissues of positive control (a) (con-
trol 1, n 5 20), and 3 levels of 9.0
(b) (low, n 5 18), 18.0 (c) (me-
dium, n 5 13) and 36.0 mmol/kg
(d) (high, n 5 3) of b-ionone in
the AIN76A diet, (4003, counter-
stained with hematoxylin). Bars
with no letters in common are sig-
nificantly different (p < 0.05 and
p < 0.01). [Color figure can be
viewed in the online issue, which
is available at www.interscience.
wiley.com.]
b-ionone treatment was significantly different from the control
group, and a decreasing trend was observed when rats were fed di-
etary b-ionone. An increasing trend of the benign tumors and a
decreasing trend of fibroadenomas in the mammary cancer tissues
were observed. The ratio of adenocarcinoma was higher in the
high dose of b-ionone group compared to the medium-dose b-io-
none group, because only 3 tumor tissues out of the 4 types were
observed in the high-dose group. Interestingly, we observed an
increased incidence of adenoma in that the b-ionone treated
groups. A possible explanation may be that the b-ionone treatment
reduced the occurrence of invasive adenocarcinoma while the less
invasive adenoma took its place.
Cell proliferation and cell cycle distribution were determined in
mammary cancer tissues of rats fed b-ionone in the AIN-76A diet.
Cell proliferation in mammary cancer tissues was measured with
PCNA as a proliferative marker and cyclin D1 as a key factor
related to the cell cycle. PCNA is a nuclear protein which func-
tions as an auxiliary protein for DNA polymerase d, an absolute
requirement for DNA synthesis and a key protein in DNA replica-
tion and DNA damage-repair. A component of the DNA replica-
tion process, PCNA is also involved in growth regulation. PCNA

b-IONONE PREVENTS MAMMARY CARCINOGENESIS IN SD RATS
is expressed during the cell cycle and its rate of synthesis is corre-
lated directly with the proliferative rate of cells. Its cellular levels
increase through G1, peak in G1/S, decline in G2 and are virtually
absent in the M phase.24 PCNA, as an early marker of cell prolif-
eration in cancer, is expressed in cells that have entered the cell
cycle and can be used to assess the proportion of the cells from a
tumor that are proliferating. The cyclin-dependent kinase (Cdk)
family has received special attention because of their function as
sensors of the mitogenic signals and their central role in cell pro-
liferation.25 Cdks are heterodimeric complexes composed of a cat-
alytic kinase subunit and a regulatory cyclin subunit, and comprise
a family divided into 2 groups based on their roles in cell cycle
progression and transcriptional regulation.26,27 Cyclins are pro-
teins originally defined by their periodic occurrence, correlating
with transition from one cell-cycle phase to another. They associ-
ate with and activate Cdks, which as monomeric kinase subunits,
are inactive. The orderly progression through the G1 phase (which
is driven by cyclin D/Cdk4 or D/Cdk6 in response to growth factor
stimulation) and to the S phase (which is driven by cyclin E/
Cdk2), leads to phosphorylation of the retinoblastoma susceptibil-
ity protein (Rb). Phosphorylation of Rb prevents it from repressing
the E2F family of transcription factors and leads to the transcrip-
tion of several genes required for the G1-to-S phase transition,
thereby promoting cellular proliferation.28,29 Cyclin D1 is overex-
pressed in 35–50% of breast cancers. Because of its pivotal role in
promoting cell cycle progression and, hence, cell division and pro-
liferation, such overexpression might be expected to coincide with
poor prognosis.30,31 Overexpression of cyclin D1 has also been
linked to the development of endocrine resistance in breast cancer
cells.32 Cyclin D1 overexpression is a common event in cancer,
but does not occur solely as a consequence of gene amplification.
Rather, increased level of cyclin D1 frequently result from its de-
fective regulation at the posttranslational level.33 A number of
therapeutic agents have been observed to induce cyclin D1 degra-
dation in vitro.34,35 These studies indicate that the induction of
cyclin D1 degradation may offer a useful avenue for therapeutic
intervention.36 In this study, b-ionone has been observed to
potently inhibit proliferative activity and to affect the cell cycle in
mammary cancer cells. The expression of PCNA and cyclin D1
were significantly decreased in mammary cancer tissues among
the b-ionone treated groups compared to the positive control
group (p < 0.01 and p < 0.05, Figs. 4 and 5) in a clear dose-de-
pendent manner. This at least partly explains why cell prolifera-
tion in mammary cancer tissues was inhibited by downregulation
of the cylin D1 expression, which affected the cell cycle of
tumors. A further study would determine how b-ionone treatment
affects other cell cycle components in mammary cancer.
The TUNEL assay is widely accepted as an indicator of apopto-
sis, and it can be used simultaneously with morphologic observa-
tions or antibody staining of secondary biomarkers.37,38 Cell apo-
ptosis in mammary cancer tissues was detected by the TUNEL
assay with immunohistochemistry. In this study, our findings
showed that cell apoptosis was induced in mammary cancer tissues
of rats fed dietary b-ionone, and an increasing trend was observed
between the positive control and b-ionone treated groups.
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