Stimulation of pS2 Expression by Diet-derived Compounds

Neeraja Sathyamoorthy, Thomas T. Y. Wang and James M. Phang

Cancer Res 1994;54:957-961.

Updated Version Access the most recent version of this article at:
[Link]

Citing Articles This article has been cited by 19 HighWire-hosted articles. Access the articles at:
[Link]

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@[Link].

Permissions To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@[Link].

Downloaded from [Link] on February 23, 2013

Copyright © 1994 American Association for Cancer Research
[CANCER RESEARCH 54, 957-961, February 15, 1994] "

Stimulation of pS2 Expression by Diet-derived Compounds

Neeraja Sathyamoorthy, 1 Thomas T. Y. W a n g , a n d J a m e s M . P h a n g
Laboratory of Nutritional and Molecular Regulation, National Cancer Institute, Frederick Cancer Research and Development Center, N1H, Frederick, Maryland 21702-1201

ABSTRACT MATERIALS AND METHODS

Epidemiological studies suggest a lowered risk of hormone-dependent Chemicals. /3-estradiol, NDGA, 2, and tamoxifen were obtained from
cancers among vegetarians, but the basis for this association remains Sigma chemical Co. (St. Louis, MO); kaempferol, daidzein, and quercetin were
unclear. Vegetables and fruits contain certain compounds which can be purchased from Indofine Chemical Co. (Somerville, NJ); and enterolactone
converted to biologically active hormone-like substances, such as lignans and enterodiol were synthesized by an adaptation of the method of Cooley et
and isoflavones, by intestinal flora. The interaction of these compounds al. (12). Equol was a generous gift from Dr. K. D. R. Setchell, Children's
with endogenous hormones may be a novel, diet-dependent mechanism in Hospital Medical Center, Cincinnati, OH. The cDNA coding for pS2 (13) and
cancer prevention. To explore this possibility, we developed a rapid, spe- the human breast cancer cell line MCF-7 were obtained from American Type
cific assay system to screen for compounds with estrogen-like activity in Culture Collection (Rockville, MD).
tissue culture. We utilized the estrogen receptor-positive breast cancer cell Cells and Cell Culture. MCF-7 cells were cultured in Falcon flasks (75
MCF-7 and monitored the expression of the estrogen-responsive protein cm2) and grown in the presence of 5% CO2 in air at 37~ The cells were
pS2 by Northern blots. Our results indicated that the phenolic compounds maintained in medium A [RPMI 1640 with 2 mM L-glutamine, hydrocortisone
daidzein, equol, nordihydroguaiaretic acid, enterolactone, and kaempferol (3.5 ng/ml), insulin (1.5 ng/ml), penicillin (100 units/ml), and streptomycin
were able to elicit an estrogen-like response, while quercetin and entero- (100 ~g/mi)] with 5% fetal calf serum. One week before the onset of the
diol were not. experiment, the cells were switched to medium A supplemented with 5%
charcoal-dextran-treated calf serum. Cells were grown to confluence and pas-
saged using trypsin-EDTA. Viable cells were counted by trypan blue exclusion.
INTRODUCTION One day prior to treatment, the cells were switched to medium B [phenol-red
free RPMI 1640 containing 2 mM glutamine, penicillin (100 units/ml), strep-
Epidemiological evidence suggests a lowered risk of hormone- tomycin (100/xg/ml), and 5% charcoal-dextran-treated calf serum].
dependent cancers among vegetarians (1). Whether this association is Total RNA Isolation. For total RNA isolation, the cells were grown in
causally related to phenolic c o m p o u n d s in fruits and vegetables or to 6-well Costar plates (1 • 106 cells/well in 3 ml of medium B). Twenty-four h
the accompanying lowered intake of fat and protein remains an open after plating, the test compounds were added at a final concentration of 1 •
10-6 M(dissolved in ethanol; final concentration of ethanol in the medium was
question. However, the former hypothesis is attractive and advanta-
0.1%). The medium was changed every 24 h after plating, and the various
geous because underlying mechanisms can be invoked and tested
compounds were added fresh with each change. Total RNA was isolated 72 h
experimentally. Foods such as whole grain products, fruits, berries, after plating by a modification of the method of Xie and Rothblum (14)
and soy products are the source of lignans and isoflavones. Increased adapted for 6-well plates. Briefly, the cells were lysed with 1.6 ml of lysis
consumption of these foods by humans results in the urinary excretion buffer. The lysis buffer was prepared by mixing water-saturated phenol con-
of appreciable amounts of c o m p o u n d s like enterolactone, enterodiol, taining 0.04% (w/w) hydroxyquinoline, 4 Mguanidium thiocyanate containing
equol, daidzein, and genistein (2--4), c o m p o u n d s formed by the action 25 mra sodium citrate (pH 7.0) and 2 M sodium acetate (pH 4.0) in a ratio of
of gut microflora on the aforementioned foods. Because of their 10:10:1. To 100 ml of the lysis buffer, 720 ~1 of 2-mercaptoethanol was finally
added. The lysate was recovered in a 2-ml tube, and 0.16 ml of chloroform:
structural similarity to estrogens (Fig. 1), the identification of these
isoamyl alcohol (24:1) was added. The tubes were vortexed for 10-20 s, set on
polyphenolic c o m p o u n d s in biological fluids (2-6) has attracted con- ice for 30 min, and then centrifuged at 12,000 • g for 30 min. The upper layer
siderable scientific interest. By monitoring the effect of these com- containing the RNA was then transferred to a new tube containing 0.8 ml of
pounds on the growth of estrogen-responsive cells in tissue culture, isopropanol. The tubes were vortexed and placed at -20~ for 2 h. RNA was
investigators have reported that certain lignans can be estrogenic precipitated by centrifugation at 12,000 x g for 20 min, washed twice with
(7, 8), antiestrogenic (2, 9), or antineoplastic (10). 70% ethanol, vacuum dried, and resuspended in 70-100 p~l diethyl pyrocar-
These findings led us to consider that fruits and vegetables contain bonate-treated water. Ten p3 of RNA were routinely used for Northern Blot
analysis.
many as yet unidentified estrogenic or antiestrogenic c o m p o u n d s
Probe for pS2. A 0.5-kilobase fragment cDNA coding for full length pS2
which can modulate carcinogenesis in hormone-dependent tissues. In was amplified from plasmid pS2 using PCR and subsequently cloned into
order to identify these compounds, we sought to develop a convenient plasmid PCR II using the TA cloning kit according to the manufacturer's
and specific assay which could also serve as a system for deciphering protocol (Invitrogen Corp., San Diego, CA). This construct allowed for the
specific molecular mechanisms. The transcription of the p S 2 gene in synthesis of a pS2 antisense RNA probe using the T7 polymerase promoter
h u m a n breast cancer MCF-7 cells presented a feasible experimental (Fig. 2). DIG-labeled pS2 antisense RNA probe was synthesized using the
model because this expression is directly controlled by estradiol nucleic acid labeling kit following the manufacturer's instructions (Boehringer
Mannheim Biochemicals, Indianapolis, IN).
through the estrogen receptor (11). We now report the development of
Northern Blot Hybridization and Detection. For detection of pS2 ex-
a cell culture system which monitors the expression of pS2 m R N A as
pression, 10/xl of total RNA were separated on 1% formaldehyde agarose gels
an indicator of estrogenic response. Using this assay, we have iden- and transferred onto a Nylon membrane (Boehringer Mannheim Biochemicals)
tified several new dietary c o m p o u n d s belonging to either the lignan or using the PossiBlot pressure blotter (Stratagene, La Jolla, CA). The RNA was
flavonoid family which have estrogen-like activity. fixed onto the membrane by UV cross-linking in Stratalinker (Stratagene) for
3 min at the highest power. The membrane was then hybridized with the
DIG-labeled RNA probe and detected by a chemiluminescence nucleic acid
detection method according to the manufacturer's protocol (Boehringer Mann-
Received 8/19/93; accepted 12/16/93.
The costs of publication of this article were defrayed in part by the payment of page heim Biochemicals). All Northern blots were also probed for/3-actin. The blots
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed, at Laboratory of Nutritional and
Molecular Regulation, Building434/2, NCI-FCRDC, P.O. Box B, Frederick, MD 21702- 2 The abbreviations used are: NDGA, nordihydroguaiaretic acid; cDNA, complemen-
1201. tary DNA; E2, estradioi; DIG, digoxigenin; PCR, polymerase chain reaction.
957

Downloaded from [Link] on February 23, 2013

Copyright © 1994 American Association for Cancer Research
 DIETARY COMPONENTS STIMULATE pS2 EXPRESSION

OH

HO
HO CH2--CH-CH-CH2-J~~OH OH
HO
171~--Estradiol NordihydroguaiareticAcid

HO H O ~ cH2OH

v [,,- -CH20H

Fig. 1. Structures of the various diet-derived Enterolactone Enterodiol

compounds used in the present study.

.o I

'

Equol Daidzein
OH
~

y y -o. y y -o.
OH O HO O
Kaempferol Quercetin
were quantitated using a densitometer (Analtech Uniscan, Newark, DE), and test compounds (10 -6 M) or estradiol (10 -l~ M) were added. All compounds
the data were normalized for/3-actin RNA content. were dissolved in ethanol, and the final concentration of ethanol was 0.1% in
Growth Assay. Each well of a 24-well plate (Costar, Cambridge, MA) was the medium. The medium was changed every 24 h, and the test compounds
seeded with 1 x 105 cells in 1 ml of medium B. Twenty-four h after plating, were replenished with each change. Cell growth was determined by the sul-
BamH forhodamine colorimetric assay as described by Skehan et al. (15) except for
I~~~ Hind III the fact that the cells were seeded in 24-well plates instead of 96-well microti-
ter plates. After color development, aliquots were pipeted into a 96-well
SP6 promo_._,
ter ,//~-----__ EcoRI microtiter plate, and the color was read at 570 nm using an ELISA microplate
reader (Molecular Devices, Menlo Park, CA).

pS2"~p.~_~. EcoRI RESULTS

9a T7promoter
pCR/pS2 Characterization of E s t r o g e n - d e p e n d e n t pS2 m R N A E x p r e s -
sion in M C F - 7 . We first characterized and validated our method for
4,40 Kb measuring estrogen-responsive pS2 m R N A in MCF-7 cells. Using an
Ampicillin adaptation of a published method (14), we were able to recover 25-30
txg of total R N A from 106 cells contained in a single well of a 6-well
plate. The quality and quantity of total R N A obtained using our
procedure were consistent and reproducible. To visualize m R N A in
Northern blots, we used a DIG-labeled probe, thereby avoiding po-
Kanamycin tential radiation hazards. Using this relatively simple and rapid pro-
Fig. 2. Plasmid used to synthesize the pS2 probe. The pS2 probes were synthesized as cedure, we tested the responsiveness of pS2 to estrogen and found that
described in "Materials and Methods."
the accumulation of pS2 m R N A in cells exposed to 10 -1~ M estradiol
958

Downloaded from [Link] on February 23, 2013

Copyright © 1994 American Association for Cancer Research
 DIETARY COMPONENTS S T I M U L A T E pS2 EXPRESSION

followed a time course (Fig. 3) similar to that described by Cho et al.

Control
(16). Thus, the sensitivity o f our system to estrogen stimulation is in
a g r e e m e n t with that reported by others. To validate the specificity o f 9 Estradiol
our assay, we showed that two steroid hormones, d e x a m e t h a s o n e and
progesterone, at 10 -6 M produced no increase in pS2 expression over Estradiol + Tam.
controls (data not shown) and, importantly, the response to 10 - ] ~ M
estradiol was abolished by 10 -s M tamoxifen (Fig. 4). 9 NDGA
Effect of NDGA on pS2 Expression. Having validated the assay NDGA + Tam.
system, w e sought to characterize its response to a naturally occurring
xenobiotic c o m p o u n d . N D G A , a major c o m p o n e n t o f "chaparral tea" Fig. 4. Effect of tamoxifen on pS2 expression in MCF-7 cells. MCF-7 cells were plated
prepared f r o m the desert shrub Larrea divaricata, was selected for at a density of 1 x 10 6 per well in 6-well Costar plates and exposed to either vehicle (0.1%
ethanol) or 10-j~ ME2 and 10 -6 M NDGA, with or without 10-s Mtamoxifen for 48 h.
several reasons. It is k n o w n to have antitumor activity (17), but RNA isolation, Northern blots, and quantitation of pS2 expression were performed as
w h e t h e r this activity is related to its inhibition o f arachidonic acid described in "Materials and Methods."
metabolism (18) and ornithine decarboxylase activity (19) remains
unclear. O f special interest to us was the fact that N D G A competes
with estradiol for binding to serum a-fetoprotein (20). Its recognition
m
by estrogen-binding sites suggested to us that N D G A m a y have es-
>
trogenic activity in our system. In cells exposed to various concen-
0
trations of N D G A (10 - l ~ to 10 -5 M), the expression o f pS2 increased m

with concentrations as low a s 10 -7 M and plateaued at 1 0 - 6 M (Fig. 5). <

The level of stimulation at 1 0 - 6 M N D G A was comparable to that seen Z
n-
E
A Actin
U)
Q.
Time(hrs) 0 5 10 24 48 72
0
>
= m

0
pS2 re'

Time (hrs) 0 5 10 24 48 72 0
.0001 9001 .01 .1 1 10 100

NDGA Concentration (IxM)

Fig. 5. Effect of NDGA concentration on pS2 expression in MCF-7 cells. MCF-7 cells
were plated at a density of 1 • 10 6 per well in 6-well Costar plates and exposed to either
1.5 vehicle (0.1% ethanol) or different concentrations of NDGA for 48 h. RNA isolation,
Northern blots, and quantitation of pS2 expression were performed as described in
"Materials and Methods."

with 10 - ] ~ estrogen, and the time course followed a similar pattern.

With N D G A , as with estrogen, an increase in pS2 m R N A accumula-
1.0 tion was observed as early as 5 h o f exposure (Fig. 6). The inhibition
e.l o f the effect o f 10 -6 M N D G A by 10 -5 M t a m o x i f e n supported the
interpretation that N D G A was stimulating pS2 expression through an
estrogen-dependent m e c h a n i s m (Fig. 4). Thus, w e showed that the
response to a naturally occurring c o m p o u n d is concentration depen-
dent and showed the same time course and specificity as with estro-
"~ 0.5 gen.
Stimulation of pS2 Expression by Lignans and Flavonoids. As
w e have demonstrated above, our assay could be used in the identi-
ILl
fication o f estrogen-like compounds. We then p r o c e e d e d to screen a
variety of diet-derived c o m p o u n d s belonging to the lignan, flavonol,
0.0 or isoflavonoid families for estrogenic activity. The c o m p o u n d s used
I ! !
0 20 40 60 80 in the present study were selected either because o f their structural
similarity to estradiol or based on previous publications suggesting an
estrogenic activity (8, 21). Table 1 summarizes the ability o f the
TIME ( h r s )
various tested c o m p o u n d s to stimulate expression o f pS2 m R N A as
Fig. 3. Time curve for pS2 expression in MCF-7 cells treated with estradiol. Cells were c o m p a r e d to estradiol. Also listed are the dietary sources o f each
plated at a density of 1 x 10 6 per well in 6-well Costar plates and cultured either in the
presence of vehicle (0.1% ethanol) alone or 10-1~ ME2 for the time indicated. Isolation compound. In our assay, we found that the addition o f 10 -6 M equol,
of total RNA and the procedure for Northern blotting and detection is described in daidzein, or kaempferol resulted in enhanced pS2 m R N A expression.
"Materials and Methods." The levels of pS2 were normalized to that of actin. A, repre-
sentative RNA blots probed for either actin or pS2. B, quantitative representation of pS2 Enterolactone at 10 -6 M was able to produce a w e a k response, while
expression based on densitometer readings. enterodiol and quercetin at 10 -6 M w e r e inactive. The relative effec-
959

Downloaded from [Link] on February 23, 2013

Copyright © 1994 American Association for Cancer Research
 DIETARY COMPONENTS [Link] pS2 EXPRESSION

A pS2 kaempferol, daidzein, and quercetin on the growth of MCF-7 cells.

The growth of MCF-7 cells in the presence of test compounds at a
Time(hrs) 0 5 10 24 48 72 concentration of 10 -6 Mwas compared to that of the cells treated with
10 -1~ M estradiol (Fig. 7). Cellular proliferation in the presence of
10 -6 M NDGA was 92% of that seen with 10 -l~ Mestradiol. In the case

o 9 O00 of daidzein, enterolactone, equol, and kaempferol, the growth seen

w i t h 10 -6 M of these compounds were 79, 92, 79, and 66%, respec-
tively. The addition of enterodiol and quercetin at the same concen-
tration did not result in growth stimulation. It appears that monitoring
pS2 expression correlates well with cellular proliferation. The com-
14 pounds that were estrogenic in our pS2 expression assay were also
found to support the growth of MCF-7 cells (Fig. 7).
~24
DISCUSSION

Our choice of pS2 mRNA expression as a marker of estrogen-like

activity was based on several considerations. Although the physiologi-
08 cal function of pS2 remains unknown, the stimulation of its expression
0
by E2 is well established (11, 13). Studies suggest that the molecular
events triggered by estrogen may be mediated through several path-
g 0.6
ways (22, 23). Thus, binding of E2 to estrogen receptor can result in
a direct stimulation of pS2 transcription through estrogen response
~ 0.4 elements (11, 24) or, alternatively, the stimulation may be indirect
through growth factor-mediated mechanisms (22-25). In either case,
W 0.2 pS2 mRNA can serve as a specific marker to identify estrogen-like
activity. In the present study, we have described a rapid, sensitive
assay based on pS2 expression to screen for compounds with estro-
0.0 ! ! I
gen-like activity. The validity and specificity of this assay were con-
o 20 40 60 8O
firmed by (a) the inhibition observed in the presence of tamoxifen and
TIME (hrs) (b) the fact that other steroid hormones such as dexamethasone and
progesterone were ineffective in stimulating pS2 mRNA.
Fig. 6. Time curve for pS2 expression in MCF-7 cells treated with NDGA. MCF-7 cells
were plated at a density of 1 • 10 6 per well in 6-well Costar plates and exposed to either The choice of NDGA for our preliminary studies of naturally oc-
vehicle (0.1% ethanol) or NDGA for the period indicated. RNA isolation, Northern blots, curring compounds was based on earlier observations by Garreau et
and quantitation of pS2 and actin mRNA levels were performed as described in "Materials
al. (20) that NDGA competes with estradiol for binding to serum
and Methods." A, representative RNA blots probed for pS2. B, quantitative representation
of pS2 expression based on densitometer readings. a-fetoprotein. The fact that NDGA shares structural similarity to
enterolactone and is commercially available prompted us to use this
Table 1 Effect of various dietary compounds on expression of pS2 mRNA compound in the further development of our assay. We have shown,
using NDGA, that under our assay conditions the pS2 accumulation
Relative pS2
Compound" level b Dietary source
NDGA 100 Chaparral tea (leaves of creosote bush)

Lignans
Enterolactone 82 Oilseeds, dried seaweeds, legumes,
cereal brans, vegetables, and fruits c
Enterodiol _d
Same as Enterolactone t- 2.0

Isoflavones
Equol 175 Cow's milk, soy products c O
Daidzein 220 Soy protein

Flavonols
Kaempferol 28 Fruits, vegetables
Quercetin _a Fruits, vegetables =m
a The compounds were used at a concentration of 10-6 M. '*" 1.0
t, The Northern blots probed for pS2 mRNA were quantitated using a densitometer. m
Using our assay system, estradiol gave a maximum response at 10-m M to which we
assigned a value of 100.
c Formed by the action of gut microflora from precursors present in these dietary
sources.
d No stimulation over control cells.

t i v e n e s s o f the c o m p o u n d s tested at a c o n c e n t r a t i o n o f 10 -~' M c o u l d be 0.0

summarized as equol, daidzein > NDGA > enterolactone > Control F_2 NDGA Enl End Eq D K Q
k a e m p f e r o l . U s i n g o u r assay, w e h a v e n e w l y i d e n t i f i e d t w o diet-
d e r i v e d , e s t r o g e n - l i k e c o m p o u n d s , N D G A and k a e m p f e r o l .
Treatment
E f f e c t o n t h e G r o w t h o f M C F - 7 C e l l s . To c o r r e l a t e the e f f e c t s o f Fig. 7. Effect of various compounds on the growth of MCF-7 cells. One • 105
cells/well were plated in 24-well Costar plates, and the test compounds were added at a
the c o m p o u n d s o n p S 2 e x p r e s s i o n w i t h that o n c e l l u l a r p r o l i f e r a t i o n , concentration of 10-6 M. The media and test compounds were changed daily. The sulfor-
w e e x a m i n e d the e f f e c t s o f N D G A , e n t e r o l a c t o n e , e n t e r o d i o l , e q u o l , hodamine assay was performed according to the method of Skehan et al. (15).
960

Downloaded from [Link] on February 23, 2013

Copyright © 1994 American Association for Cancer Research
 DIETARY COMPONENTS STIMULATE pS2 EXPRESSION

occurred in a dose-responsive and time-dependent fashion. The accu- 6. Axelson, M., Kirk, D. N., Farrant, R. D., Cooley, G., Lawson, A. M., and Setchell, K.
mulation of pS2 in M C F - 7 cells w a s abolished by the addition o f D. R. The identification of the weak oestrogen equol [7-hydroxy-3-(4'-hydroxyphe-
nyl)-chroman] in human urine. Biochem. J., 201: 353-357, 1982.
tamoxifen. Thus, the effects of N D G A on pS2 are likely to be medi- 7. Martin, R M., Horwitz, K. B., Ryan, D. S., and McGuire, W. L. Phytoestrogen
ated via the estrogen receptor pathway. In the present study, in addi- interaction with estrogen receptors in human breast cancer cells. Endocrinology, 103:
tion to N D G A , w e have successfully identified several other c o m - 1860-1867, 1978.
8. Weishons, W. V., Murphy, C. S., Koch, R., Calaf, G., and Jordan, V. C. Stimulation
pounds, including daidzein as well as kaempferol, not previously of breast cancer cells in vitro by the environmental estrogen enterolactone and the
s h o w n to be estrogenic. phytoestrogen equol. Breast Cancer Res. Treat., 10: 169-175, 1987.
It is possible that these estrogen-like diet-derived c o m p o u n d s m a y 9. Waiters, A. E, and Knowler, J. T. Effect of a lignan (HPMF) on RNA synthesis in the
rat uterus. J. Reprod. Fertil., 66: 379-381, 1982.
interact with the estrogen receptor and hence modulate the action of 10. Adlercreutz, H. Lignans and phytoestrogens: possible preventive role in cancer. Front.
estrogen. The concentration o f the c o m p o u n d s used in the in v i t r o Gastrointest. Res., 14: 165-176, 1988.
experiments presented here are physiologically relevant. The concen- 11. Brown,A. M. C., Jeltsch, J. M., Roberts, M., and Chambon, E Activation ofpS2 gene
transcription is a primary response to estrogen in the human breast cancer cell line
trations o f enterolactone in b o d y fluids have been reported to range
MCF-7. Proc. Natl. Acad. Sci. USA, 81: 6344-6348, 1984.
from approximately 10 nM to 1 p,M (2, 3). It has also been reported that 12. Cooley, G., Farrant, R. D., Kirk, D. N., and Wynn S. Chemical synthesis of the first
circulating levels o f lignans could be 100 to 10,000 times higher than lignans to be found in humans and animals. Tetrahedron Lett., 22: 34%350, 1981.
13. Jakowlev, S. B., Breathnach, R., Jeltsch, J. M,, Masiakowski, P., and Chambon, E
E2 in human subjects c o n s u m i n g a diet rich in grains (21). The
Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line
possible physiological significance o f a dietary source of estrogen-like MCF-7. Nucleic Acids Res., 12: 2861-2878, 1984.
activity merits further scrutiny. 14. Xie, W., and Rothblum, L. I. Rapid, small-scale RNA isolation from tissue culture
Our assay has several advantages over other assays that screen cells. Biotechniques, 11: 325-327, 1991.
15. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren,
c o m p o u n d s for estrogenic-like activity (7, 2 6 - 2 9 ) . As a screening J. T., Bokesch, H., Kenney, S., and Boyd, M. R. New colorimetric cytotoxicity assay
methodology, the monitoring o f pS2 expression is more versatile than for anticancer-drug screening. J. Natl. Cancer Inst., 82: 1107-1112, 1990.
a cell-free receptor binding assay b e c a u s e the physiological interac- 16. Cho, H., Ng, E A., and Katzenellobogen, B. S. Differential regulation of gene
expression by estrogen in estrogen growth-independent and -dependent MCF-7 hu-
tions within the cell are preserved. In addition, it is possible that the man breast cancer cell sublines. Mol. Endocrinol., 5: 1323-1330, 1991.
ability of a c o m p o u n d to bind to the estrogen receptor m a y not 17. Nakadate, T., Yamamoto, S., Ishii, M., and Kato, R. Inhibition of 12-O-tetradec-
necessarily result in a cellular response. Although pS2 expression can anoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity by phos-
pholipase A2 inhibitors and lipoxygenase inhibitor. Cancer Res., 42: 2841-2845,
be stimulated b y estrogen-independent mechanisms, these can be eas-
1982.
ily distinguished b y their insensitivity to inhibition by tamoxifen. 18. Nakadate, T., Yamomoto, S., Iseki, H., Sonoda, S., Takemura, S., Ura, A., Hosoda, Y.,
Furthermore, our choice o f a system that monitors pS2 expression in and Kato, R. Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor pro-
motion by nordihydroguaiaretic acid, a lipoxygenase inhibitor, and p-bromophenacyl
M C F - 7 cells provides greater relevance to h u m a n carcinogenesis.
bromide, a phospholipase A2 inhibitor. Gann, 73: 841-843, 1982.
Finally, our approach e m p l o y s a nonradioactive detection m e t h o d 19. Yu, A., Hashimura, T., Nishio, Y., Kanamaru, H., Fukuzawa, S., and Yoshida, O.
w h i c h eliminates the potential p r o b l e m s associated with m e t h o d s re- Anti-promoting effect of nordihydroguaiaretic acid on N-butyl-N-(4-hydroxybutyl)-
quiring isotopes. Thus, the assay w e have d e v e l o p e d and described in nitrosamine and sodium saccharin-induced rat urinary bladder carcinogenesis. Jpn. J.
Cancer Res., 83: 944-948, 1992.
the present paper is sensitive, specific, rapid, relatively easy to per- 20. Garreau, B., Vallette, G., Adlercreutz, H., Wahala, K., Makela, T., Benassayag, C., and
form, and allows the simultaneous screening of multiple samples. In Nunez, E. A. Phytoestrogens: new ligands for rat and human o~-[Link].
conclusion, our assay should provide a screening tool to evaluate Biophys. Acta, 1094: 339-345, 1991.
21. Mousavi, Y., and Adlercreutz, H. Enterolactone and estradiol inhibit each other's
extracts from various dietary sources for estrogenic activity. proliferative effect on MCF-7 breast cancer cells in culture. J. Steroid Biochem. Mol.
Biol., 41: 615-619, 1992.
22. Nunez, A. M., Berry, M., Imler, J. L., and Chambon, E The 5' flanking region of the
ACKNOWLEDGMENTS pS2 gene contains a complex enhancer region responsive to estrogens, epidermal
growth factor, a tumour promoter (TPA), the c-Ha-ras oncoprotein and the c-jun
We thank Dr. Victor Nelson, Program Resources, Inc., Frederick, MD, for protein. EMBO J., 8: 823~829, 1989.
the synthesis of enterolactone and enterodiol. We also thank Dr. Jiingjau Jeng, 23. Cavailles, V., Garcia, M., and Rochefort, H. Regulation of cathepsin-D and pS2 gene
National Institute of Alcoholism and Alcohol Abuse, for performing PCR. expression by growth factors in MCF-7 human breast cancer cells. Mol. Endocrinol.,
3: 552-557, 1989.
24. Berry, M., Nunez, A. M., and Chambon, E Estrogen-responsive element of the human
REFERENCES pS2 gene is an imperfectly palindromic sequence. Proc. Natl. Acad. Sci. USA, 86:
1218-1222, 1989.
1. Dunn, J. E., Jr. Cancer epidemiology in populations of the United States---emphasis 25. Bates, S. E., Davidson, N. E., Valverius, E. M., Freter, C. E., Dickson, R. B., Tam, J.
on Hawaii and California--and Japan. Cancer Res., 35: 3240-3245, 1975.
P., Kudlow, J. E., Lippman, M. E, and Salomon, D. S. Expression of transforming
2. Adlercreutz, H., Fotsis, T., Heikkinen, R., Dwyer, J. T., Woods, M., Goldin, B. R., and
Gorbach, S. L. Excretion of the lignans enterolactone and enterodiol and of equol in growth factor a and its messenger ribonucleic acid in human breast cancer: its
omnivorous and vegetarian women and in women with breast cancer. Lancet, 2: regulation by estrogen and its possible functional significance. Mol. Endocrinol., 2:
1295-1299, 1982. 543-555, 1988.
3. Dehennin, L., Reiffsteck, A., Joudet, M., and Thibier, M. Identification and quanti- 26. Pelissero, C., Bennetau, B., Babin, E, Le Menn, E, and Dunogues, J. The estrogenic
tative estimation of a lignan in human and bovine semen. J. Reprod. Fertil., 66: activity of certain phytoestrogens in the Siberian sturgeon Acipenser baeri. J. Steroid
305-309, 1982. Biochem. Mol. Biol., 38: 293-299, 1991.
4. Adlercreutz, H., Honjo, H., Higashi, A., Fotsis, T., H/im/il/iinen, E., Hasegawa, T., and 27. Mayr, U., Butsch A., and Schneider, S. Validation of two in vitro test systems for
Okada, H. Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese estrogenic activities with zearalenone, phytoestrogens and cereal extracts. Toxicology,
men and women consuming a traditional Japanese diet. Am. J. Clin. Nutr., 54: 74: 135-149, 1992.
1093-1100, 1991. 28. Mayr, U. E. Estrogen-controlled gene expression in tissue culture cells by zearale-
5. Setchell, K. D. R., Lawson, A. M., Borriello, S. P., et al. Lignan formation in none. FEBS Lett., 239: 223-226, 1988.
man-microbial involvement and possible roles in relation to cancer. Lancet, 2: 4-7, 29. Markiewicz, L., Garey, J., Adlercreutz, H., and Gurpide, E. In vitro bio-assays of
1981. non-steroidal phytoestrogens. J. Steroid Biochem. Mol. Biol., 45: 399~-05, 1993.

961

Downloaded from [Link] on February 23, 2013

Copyright © 1994 American Association for Cancer Research