Influence of marketed herbal menopause preparations on MCF-7 cell proliferation.

Abstract

Given the increasing use of alternative menopause treatments, we evaluated the effect of several herbal preparations used for menopause relief on the proliferation of estrogen-sensitive breast cancer cells (MCF-7) as a means of assessing appropriateness for use in women at risk for estrogen-sensitive breast cancer.
An MCF-7 cell culture model, as described previously, was used to evaluate the estrogen-agonist and -antagonist activity of commercially available herbal menopause preparations containing red clover, soy, black cohosh, or a combination of herbs. Each test substance was evaluated for cytotoxic effects before conducting the proliferation assays.
Commercially available products containing soy, red clover, and herbal combinations induced an increase in the MCF-7 proliferation rates, indicating an estrogen-agonistic activity in the absence of estradiol. In contrast, an isopropanolic black cohosh extract (Remifemin Menopause) did not stimulate MCF-7 growth and exerted inhibitory effects on cellular proliferation. None of the tested products enhanced estradiol-induced cell proliferation. The black cohosh preparation and one of the herbal combinations exhibited strong estrogen-antagonistic effects.
The lack of proliferative effects of isopropanolic black cohosh extract on estrogen-sensitive breast cancer cells in vitro suggests a favorable safety profile for use in women with a history of breast cancer. Alternatively, preparations containing red clover, soy, and combinations of various herbal ingredients may induce cell proliferation, suggesting that such herbal preparations should be used with caution in the treatment of menopause symptoms in women at risk for, or with a history of, estrogen-sensitive breast cancer.

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Influence of marketed herbal menopause preparations on MCF-7
cell proliferation
Cornelia Bodinet, PhD, and Johannes Freudenstein, PhD
ABSTRACT
Objective:Giventheincreasinguseofalternativemenopausetreatments,weevaluatedtheeffect
of several herbal preparations used for menopause relief on the proliferation of estrogen-sensitive
breast cancer cells (MCF-7) as a means of assessing appropriateness for use in women at risk for
estrogen-sensitive breast cancer.
Design: An MCF-7 cell culture model, as described previously,1was used to evaluate the estro-
gen-agonistand-antagonistactivityofcommerciallyavailableherbalmenopausepreparationscon-
tainingredclover,soy,blackcohosh,oracombinationofherbs.Eachtestsubstancewasevaluated
for cytotoxic effects before conducting the proliferation assays.
Results: Commercially available products containing soy, red clover, and herbal combinations
induced an increase in the MCF-7 proliferation rates, indicating an estrogen-agonistic activity in
the absence of estradiol. In contrast, an isopropanolic black cohosh extract (Remifemin Meno-
pause) did not stimulate MCF-7 growth and exerted inhibitory effects on cellular proliferation.
Noneofthetestedproductsenhancedestradiol-inducedcellproliferation.Theblackcohoshprepa-
ration and one of the herbal combinations exhibited strong estrogen-antagonistic effects.
Conclusions:Thelackofproliferativeeffectsofisopropanolicblackcohoshextractonestrogen-
sensitive breast cancer cells in vitro suggests a favorable safety profile for use in women with a
historyofbreastcancer.Alternatively,preparationscontainingredclover,soy,andcombinationsof
various herbal ingredients may induce cell proliferation, suggesting that such herbal preparations
should be used with caution in the treatment of menopause symptoms in women at risk for, or with
a history of, estrogen-sensitive breast cancer.
T
mone therapy (HT), which includes estrogen and pro-
gestin supplementation, has been a popular treatment
for menopause, a naturally occurring state of hormone
depletion. The U.S. Preventive Services Task Force re-
cently published a recommendation against the routine
use of estrogen and progestin for the prevention of
Keywords: Black cohosh – Soy – Red clover – Breast cancer – Menopause – Herbal therapy.
he association between breast cancer and es-
trogen has been widely documented in the
scientificliterature.2Despitethelinkbetween
estrogen use and risk of breast cancer, hor-
chronicconditionsinpostmenopausalwomenbased,in
part, on an associated risk of breast cancer.3In light
of the recent findings regarding HT and increased
cancer risk, many women are turning to alternative
approaches.
Alternative therapies that have been used for meno-
pause symptom relief include herbal preparations of
black cohosh (Cimicifuga racemosa), red clover (Tri-
folium pratense), chaste tree berry (Vitex agnus-
castus),andotherherbs4orpreparationscontainingsoy
isoflavones. Clinical support for the effectiveness of
these ingredients varies greatly, with the most positive
evidence available for the isopropanolic extract of
black cohosh (Remifemin Menopause) and for soy-
containing products.5Despite the varying degrees of
scientific evidence supporting the efficacy of herbs,
thereisanincreaseinuseofalternativeherbaltherapies
Received March 10, 2003; revised and accepted August 13, 2003.
Schaper & Brümmer GmbH & Co. KG, Research and Development De-
partment, Salzgitter, Germany
Address correspondence to: Cornelia Bodinet, PhD, Bahnhofstr. 35,
38259 Salzgitter, Germany. E-mail: cornelia.bodinet@schaper-
bruemmer.de.
Menopause: The Journal of The North American Menopause Society
Vol. 11, No. 3, pp. 281-289
DOI: 10.1097/01.GME.0000094209.15096.2B
© 2004 The North American Menopause Society
? ? Text printed on acid-free paper.
Menopause, Vol. 11, No. 3, 2004 281

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for treatment of menopause symptoms, particularly in
women with breast cancer.6Considering such wide-
spread use, it is important to evaluate the potential of
herbal preparations to exert estrogen-like effects and
potentiallyincreasetheriskofestrogen-sensitivecancers.
Apreviousstudyconductedinourlaboratory1evalu-
ated the effects of an isopropanolic extract of black co-
hosh (Remifemin) on the proliferation of MCF-7 cells.
Results suggest the commercially available black co-
hosh extract does not exert an estrogen-agonistic effect
on estrogen receptor-positive breast cancer cells and,
therefore, might be a safe alternative to HT in meno-
pausal women with estrogen-sensitive disorders.
Based on these findings and the growing popularity
of alternative therapies for treatment of menopausal
symptoms, the current study was designed to test vari-
ouscommerciallyavailableherbalmenopauseprepara-
tionsfortheirpotentialestrogen-agonisticand-antago-
nisticactivity.Usingthesameexperimentalmethodsas
our previous study,1the experiments were performed
using MCF-7 cells, an established in vitro estrogen-
dependentmammarytumormodel.7,8Inthismodel,the
presence of estrogen or estrogen-like substances in-
creasestheproliferationrateofMCF-7cells.Usingrate
of cell proliferation as a measure of estrogenic activity,
we evaluated the activity of various commercially
available herbal preparations containing soy, black co-
hosh,redclover,andtwocombinationsofmultipleherbs.
METHODS
Herbal preparations and extracts
Commercially available products were purchased at
localretailoutlets.Thevariousherbalpreparations,ac-
tive ingredients, and lot numbers that were tested in the
MCF-7 assay are outlined in Table 1. From each herbal
preparation, an amount corresponding to the recom-
mendeddailydosewasextractedwith10mLisopropyl
alcohol(40%V/V)for30minutesinanultrasonicbath.
The extracts were then filtrated through a 0.2 µm pore-
size membrane filter and stored at 4°C.
Cells
Estrogen receptor-positive (ER+) MCF-7 cells were
purchased from American Type Culture Collection
(ATCC HTB 22, Manassas, VA). They were main-
tained in Eagle’s MEM with nonessential amino acids,
1 mM sodium pyruvate, 10 µg/mL insulin, 10% FCS,
andantibiotics.HeLacells(epithelialcervixcarcinoma
cells)usedinthetoxicityassayalsowereobtainedfrom
American Type Culture Collection.
Toxicity assay
To determine the cytotoxicity of the individual test
substances, a fluorescence assay was performed using
4-MeUH as a fluorogenic substrate for cellular ester-
ases.Thissubstrateisnonfluorescentuntiltakenupand
cleaved by esterases in living cells. The toxicity assay
was performed using MCF-7 cells (in complete as well
as estrogen-deprived medium) and HeLa cells, as de-
scribed earlier.1MCF-7 cells were plated at an initial
cell density of 1.2 × 105cells per well in Eagle’s MEM
without phenol red, supplemented with nonessential
aminoacids,1mMsodiumpyruvate,10µg/mLinsulin,
and 5% FCS or charcoal-stripped FCS (CSF, Sigma
C-1969). After incubation at 37°C and 5% CO2for 24
TABLE 1. Commercial herbal preparations
Herb LOT# Active ingredients Daily dose
SoyHW/30C201
HW/30C203
PM/9KV0442
PM/9HV0578
REM/906931
320 mg soy extract (55 mg isoflavones and >15% saponins)1 tablet
Red cloverRed clover leaf extract (40 mg isoflavone phytoestrogens) 1 tablet
Black cohosh0.018–0.026 mL isopropanolic extract (40 vol.-%)
from black cohosh root corresponding to 20 mg
black cohosh root
50 mg purified isoflavones (from pueraria
lobata root and non-GMO soybean standardized
extract; 100 mg kava kava root standardized
extract (Piper methysticum); 40 mg black
cohosh (Cimicifuga racemosa) root
Soy protein concentrate, standardized kudzu
extract (root), standardized red clover extract
(leaf, provides 80 mg isoflavone), 410 mg;
standardized chastetree extract (berry), 75 mg;
standardized black cohosh (Cimicifuga
racemosa) extract (root) 40 mg
2 tablets
Combination I EV/5004H0
EV/6540K0
1 tablet
Combination IINP/00H704
1 tablet
GMO, genetically modified organism.
BODINET AND FREUDENSTEIN
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hours, extracts were diluted in cell culture medium and
added starting at a 1/100 dilution. The microtiter plates
wereincubatedforanadditional48hoursandthencen-
trifuged at 800 rpm for 10 minutes. The supernatants
were removed and 200 µL 4-MeUH (0.1 mg/mL in
PBS) were added per well. After 120 minutes, the fluo-
rescence units per well were measured in a microtiter
plate fluorometer (Fluoroskan II).
Proliferation assay with MCF-7 cells
Toobtainestrogen-deprivedconditionsintheprolif-
eration assay, the FCS in the cell culture medium was
replaced by charcoal-stripped FCS (CSF, Sigma
C-1696). A MCF-7 cell suspension (200 µL), adjusted
to 5×104cells/mL in test cell culture medium (Eagle’s
MEM without phenol red and supplemented with non-
essential amino acids, 10 µg/mL insulin, 1 mM sodium
pyruvate, and 5% CSF), was plated in 96-microtiter
plates and incubated at 37°C and 5% CO2for 24 hours.
After incubation, the supernatants were removed, and
150 µL fresh culture medium without insulin was
added. The test extracts were diluted (6 dilution steps
1:10) in test cell culture medium without insulin and
pipetted in six parallels at 50 µL/well. After 2 days of
incubation(asdescribedabove),cellswerepulsedwith
25 µL/well [6-3H]Thymidine (spec. activity 2
Ci/mMol, 0.25 µCi/well) for 8 hours. Cells were har-
vested according to standard methods (Cell Harvester
Inotech) onto glass fiber filters and counted in a liquid
scintillation counter (Wallac). This method of cell
quantificationisanestablishedmethodofmeasuringde
novo DNA synthesis.9As control substances, the cell
culture medium and corresponding solvent dilutions
were tested simultaneously. An estrogen-agonistic
control substance, 17?-estradiol, was dissolved in di-
methyl sulfoxide and diluted in cell culture medium to
10−7M.
For evaluation of estrogen-antagonistic effects of
each herbal preparation, the test design was altered by
addition of constant amounts of estradiol (10−7M) to
each test extract dilution. Tamoxifen, a known estro-
gen-antagonist, was added to the cell culture at a con-
centration of 10−5M as a positive control for estrogen-
antagonistic effects.
Tofullyunderstandtheeffectofblackcohoshoncell
proliferation,isopropanol,theliquidusedintheextrac-
tion process, was evaluated in the MCF-7 model. Iso-
propanol was prepared and filtered in the same way as
the black cohosh extract and tested with and without
estradiol.
Statistical analysis
The results were expressed as mean ± SD. The phar-
macological data from the proliferation assays were
analyzed by the Student’s t test, and significance was
assumed at P < 0.05.
RESULTS
Toxicity assay
The results of the cytotoxicity assays on HeLa and
MCF-7 cells are summarized in Table 2. The extracts
were tested at a dilution series from 1:100-1:51,200.
Solvent controls were performed in each assay. Isopro-
pyl alcohol at the maximal concentration tested had no
cytotoxic effect on either cell type.
The soy (HW/30C201; HW/30C203) and black co-
hosh (REM/906931) extracts showed no cytotoxic ac-
tivity on MCF-7 and HeLa cells in the tested dilution
range from 1:100-1:51,200. Red clover (PM/9KV0442;
TABLE 2. Cytotoxicity of tested extracts
Noncytotoxic dilution rangea
Test extractb
HeLa cells
MCF-7 cells
+ Estradiol − Estradiol
HW/Lots: 30C201; 30C203
PM/Lot 9KV0442
PM/Lot 9HV0578
NP/Lot 00H704
REM/Lot 906931
EV/Lot 5004H0
EV/Lot 6540K0
HeLa cells, epithelial cervix carcinoma cells; MCF-7 cells, estrogen-sensitive breast cancer cells.
aDilutions given in the table represent the noncytotoxic dilution range.
bTested dilution range, 1:100–1:51,200.
cLower or higher dilutions have not been tested.
dLower dilutions are cytotoxic.
1:100-1:51,200c
1:200-1:51,200d
1:400-1:51,200d
1:100-1:51,200c
1:100-1:51,200c
1:100-1:51,200c
1:100-1:51,200c
1:100-1:51,200c
1:100-1:51,200c
1:100-1:51,200c
1:100-1:51,200c
1:100-1:51,200c
1:200-1:51,200d
1:100-1:51,200c
1:100-1:51,200c
1:400-1:51,200d
1:400-1:51,200d
1:200-1:51,200d
1:100-1:51,200c
1:400-1:51,200d
1:200-1:51,200d
HERBAL PREPARATIONS AND MCF-7 CELLS
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PM/9HV0578) was cytotoxic at the highest concentra-
tions (dilutions 1:100 and 1:200), and Combination II
(NP/00H704)exertedtoxiceffectsatthe1:100dilution
in MCF-7 cells only. Similarly, Combination I extract
(EV/5004H0; EV/6540K0) showed no toxic effects on
HeLacells,butexertedmild,althoughinconsistent,cy-
totoxiceffectsonMCF-7cells.OnelotofCombination
I(EV/5004H0)showedaslightlystrongercytotoxicac-
tivity than the other lot of the same combination
(EV/6540K0).
Proliferation assay
The various herbal extracts were tested at a dilution
series ranging from 10−2to 10−7in the MCF-7 prolif-
eration assay.
Soy (HW/30C201; HW/30C203) induced signifi-
cantstimulationoftheMCF-7cells.Bothlotsexhibited
a strong proliferation increase overall dilutions from
10−3to 10−6, reaching incorporation rates of about
18,000 cpm (control rates: medium: 5,700 cpm; estra-
diol10−7M:23,000cpm)(Fig.1a).Dilutionof10−2did
notinfluenceproliferationineitherlot,andthedilution
of 10−7exhibited decreased proliferation-enhancing
activity. At the 10−5dilution, the two soy lots exerted
different proliferative effects (data not shown), an ex-
ceptionthatwasinterpretedasatestartifactbecausethe
incorporation rates induced by the other dilutions of
both lots were in the same range.
Red clover (PM/9KV0442; PM/9HV0578) also ex-
erted proliferation-enhancing activity. The two lots in-
duced significant enhancement of MCF-7 proliferation
at dilutions from 10−4to 10−6(Fig. 1b).
Herbal Combination I induced significant stimula-
tion of the MCF-7 cell proliferation at dilutions of
10−3to 10−6(Fig. 1c). For both lots (EV/5004H0;
EV/6540K0), there was a decrease in the proliferation
rate at the 10−2dilution, a result that could be contrib-
uted to the cytotoxic activity observed for both lots in
this dilution range.
The herbal Combination II (NP/00H704) also dem-
onstrated proliferation-stimulating activity on MCF-7
cells in dilutions of 10−5and 10−6; however, dilutions
of 10−3, 10−4, and 10−7had no significant influence on
FIG. 1. Tested herbals inducing MCF-7 cell proliferation. A: Soy (HW/30C203); B: Red clover (PM/9KV0442); C: Herbal Combination I
(EV/5004H0); D: Herbal Combination II (NP/00H704). * P < 0.05, ** P < 0.01, *** P < 0.001 versus medium controls (Student’s t test). n.s., not
significant.
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the proliferation rates (Fig. 1d). At a dilution of 10−2,
Combination II (NP/00H704) induced a highly signifi-
cant decrease in the incorporation rates, a result that
may be due to a cytotoxic effect, as observed in the
toxicity assays.
In contrast to soy, red clover, and the herbal combi-
nations, the isopropanolic extract of black cohosh
(REM/906931) did not increase cell proliferation (Fig.
2).Thisextractnotonlyfailedtoincreasetheprolifera-
tion of MCF-7 cells but actually significantly inhibited
proliferation at dilutions between 10−2and 10−5. A cy-
totoxic effect can be excluded because the preparation
showed no toxic effects in the toxicity assay. Higher
extractdilutionshadnofurthersignificantinfluenceon
the proliferation rates. The isopropanol control did not
influence the proliferation rate of the cells nor the pro-
liferation-inducing effect of estradiol, suggesting the
notedeffectsareduetotheactivityofblackcohoshand
not the extraction solvent.
Tests for estrogen-antagonistic effects demonstrate
that none of the tested extracts acted synergistically
with estradiol on MCF-7 proliferation and all extract
preparations reduced the estrogen-induced prolifera-
tion at a dilution of 10−2. The strongest estrogen-
antagonistic activity was demonstrated in assays of
Combination II (NP/00H704) and the isopropanolic
black cohosh product (REM/906931). Combination II
(NP/00H794) reduced the estrogen effect by 93% and
59% in dilutions of 10−2and 10−3, respectively. In di-
lutionsof10−4to10−7,therewasalsoaslightreduction
in the estrogen-induced proliferation rates observed
(Fig. 3). Comparable results were found for the black
cohosh extract (REM/906931). Dilutions from 10−2to
10−6reduced the estrogen-induced proliferation rates.
The estradiol-induced increase (21,000 cpm) was re-
duced by 92% (to 1,700 cpm) at a dilution of 10−2, and
by 60% (to 8,535 cpm) at a dilution of 10−3(Fig. 4).
Soy showed a strong estrogen-antagonistic activity
only at the 10−2dilution. The estrogen effect was an-
tagonized by about 65% (data not shown). Similar re-
sults were found for red clover, with the estrogen-
induced incorporation rate reduced by 77% at the 10−2
dilution (data not shown). Likewise, neither extract of
CombinationI(EV/5004H0;EV/6540K0)actedsyner-
FIG. 2. Tested herbals inhibiting MCF-7 cell proliferation: black cohosh (Remifemin). IPA, isopropanol. * P < 0.05, ** P < 0.01, *** P < 0.001 versus
medium controls (Student’s t test). n.s., not significant.
HERBAL PREPARATIONS AND MCF-7 CELLS
Menopause, Vol. 11, No. 3, 2004 285