Jpn J Clin OncoI1998;28(6)360-363
, Original Articles
I
Effects of Soybean Isoflavones on Cell Growth and Apoptosis of
the Human Prostatic Cancer Cell Line LNCaP
Mizuki Onozawa 1,2, Kazunori Fukuda1, Mikinobu Ohtani2, Hideyuki Akaza2, Takashi Sugimura 1 and
Keiji Wakabayashi 1
1Cancer Prevention
Division, National CancerCenter Research Institute, Tokyo and 2Department of Urology,
Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan
Background: Epidemiological studies have suggested that soybean isoflavones are associated
with a lower risk of prostate cancer. However, the mechanisms of prostate cancer prevention by
soybean isoflavones have yet to be fully clarified.
Methods: Two soybean isoflavones (genistein and daidzein) and their glucosides (genistin and
daidzin) were tested for their effects on cell growth and apoptosis of the LNCaP human prostatic
cancer cell line.
Results: Among these isoflavones, genistein was found to inhibit the growth of LNCaP most
effectively, with an ICso value of 40 J.lM. The inhibition of cellgrowth by genistein was accompanied
by the suppression of DNAsynthesis andthe induction of apoptosis. Expression of prostate-specific
antigen (PSA) in LNCaP was also significantly reduced by the treatment with genistein.
Conclusions: The results suggest that genistein mightprimarily influence human prostatecancer
development by reducing tumor growth.
Key words: genistein - isoflavone - soybean - prostatecancer
INTRODUCTION
The incidence of prostate cancer in Japan is much lower than in
Western countries (1,2). A higher consumption of soybeans and
related products is speculated to be one contributory dietary factor
(3-5). An inverse association between intake of soybean products
and risk of cancer has also been suggested for cancers of other
organs such as the breast and the colon (5,6).
The average daily consumption of soybean and its products per
person in Japan in 1993 was 64.2 g, tens of times higher than that
in Americans (7,8). Soybean is a rich source of the isoflavone
genistein, which has been identified as a putative cancer
chemopreventive agent (9). The daily intake of genistein and its
~-glucoside conjugated form, genistin, in the Japanese is
estimated to be 1.5-4.1 and 6.3-8.3 mg, respectively, from the
intake of soy products (0). In contrast, the daily consumption of
total isoflavones by the British is calculated to be <1 mg (5).
Adlercreutz et al. (4) also reported plasma concentrations of
isoflavones in Japanese males to be 7-110 times higher than in
Received February 12, 1998; accepted March 30,1998
For reprints and all correspondence: Keiji Wakabayashi, Cancer Prevention
Division, National Cancer Center Research Institute, I-I, Tsukiji 5-chome,
Chuo-ku, Tokyo 104-0045, Japan
Finnish males. Urinary excretion of genistein is 40 times higher
in the Japanese than in Caucasian populations (6,11). Hence the
daily intake of genistein is significantly higher in Japanese than
Western European or American people (3).
Genistein is a protein tyrosine kinase inhibitor and it also
inhibits DNA topoisomerases and other critical enzymes involved in signal transduction 01-13). In addition, antioxidant
effects (14) and inhibition of angiogenesis have been reported
(11). Its chemical structure shows a relationship to estrogenic
steroids and genistein possesses weak estrogenic activity and has
been shown to act in animal models as an anti-estrogen (5,15-17).
It stimulates sex hormone-binding globulin (SHBG) production,
which may lower the risk of hormone-related cancers by
decreasing the amount of free and active hormones in the blood
(5,18). Genistein has been demonstrated to suppress mammary
cancer development in rats 09,20).
Although possible anti-carcinogenic properties of genistein
have been suggested, reports concerning direct effects against
prostate cancer cells and mechanisms of action are limited (21).
In the present study, two abundant soybean isoflavones (genistein
and daidzein) and their glucosides (genistin and daidzin) were
tested to compare their effectiveness at cell growth inhibition of
the LNCaP human prostatic cancer cell line. Induction of
apoptosis and suppression of prostate-specific antigen (PSA)
expression by genistein were also examined.
Jpn J Clin OncoI1998;28(6)
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NUCLEAR MORPHOLOGY
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Figure 1. Structuresof soybeanisoflavonesandtheir glucosides. Glc =glucose.
MATERIALS AND METHODS
CHEMICALS
Genistein, genistin and daidzin were purchased from WakoPure
Chemical Industries (Osaka, Japan). Daidzein was purchased
from Extrasynthese (Genay, France). The structures of the
soybean isoflavones and their ~-glucoside conjugates used in the
present study are shown in Fig. 1.
CELL CULTURE
The human prostatic cancer cell line LNCaP was obtained from
the American Type Culture Collection (Rockville, MD) and
cultured in RPM! 1640 containing 10% heat-inactivated fetal
bovine serum, 100 units/ml penicillin G and 100 ug/ml streptomycin at 37°C in 5% C02.
MEASUREMENT OF PROLIFERATION RATES
LNCaP cells were seeded at 4 x 103 cells per well in 96-well
microplates and allowed to attach for 24 h, then each isoflavone
was added to the medium at various concentrations. After 72 h,
cell growth was assessedas follows, using a WST-l assay (Wako
Pure Chemical Industries). Briefly, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-l) and
l-methoxy-5-methylphenazinium methylsulfate (l-methoxy
PMS) (WakoPure Chemical Industries) were added to the wells
and incubatedfor 2 h, thencell growthwas assessedby measuring
the absorbanceat 450 nm. Six replicatedwells were used for each
experimental condition.
MEASUREMENT OF DNA SYNTHESIS
DNA synthesis was evaluated using a kit from Boehringer
Mannheim (Mannheim, Germany) as follows. LNCaP cells were
plated and exposed to genistein as described above. After 2 h,
5-bromo-2-deoxyuridine (BrdU)was addedto the mediumto give
a fmal concentration of 10I-lM. After 2 h of additional incubation,
culture medium was removed and BrdU incorporation was
measured by an enzyme-linked immunosorbent assay (ELISA).
Cells were plated in culture plates at a concentration of 5 x 1()4
cells/ml. On the next day, genistein was added to the medium at
a final concentration of 150 I-lM and culturing was continuedfor
24 h. Then both attachedand detachedcells were collected,fixed
in 4% parafonnaldehyde, stained with 0.17 mM Hoechst 33258
and examined under a fluorescent microscope.
DNA FRAGMENTATION ASSAY
Cells were plated and treated with genistein as described above.
After 24 h, both attached and detached cells were collected and
lysed in 10 mM Tris-HCI (pH 7.4), 10 mM EDTA and 0.5%
Triton X-100 for 10 min on ice. The lysates were centrifugedat
18 000 g for 20 min and the supernatantswere incubatedwith 0.4
mg/mlRNaseA (Worthington Biochemical,Freehold,NJ)for 1 h
at 37°C, then with 0.4 mg/ml proteinase K (Merck, Darmstadt,
Germany) for 1 h at 37°C. The DNA was precipitated at -20°C
by adding an equal volume of propan-z-ol and 0.5 M NaCI,
loaded on to 2% agarose gels containing 0.5 I-lg/ml ethidium
bromide and electrophoresed in Tris-borate-EDTA (TBE)
buffer. Gels were photographed under UV light.
SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL
ELECTROPHORESIS (SDS-PAGE) AND IMMUNOBLOT ANALYSIS
Cultured cells were placed in SDS-sample buffer (62.5 mM
Tris-HCI, pH 6.8, 5% ~-mercaptoethanol, 2% SDS), sonicated
for 10 s and then heated at 100°C for 5 min. The protein lysates
were separated on 12% SDS-PAGE gels and transferred
electrophoretically on to poly(vinylidene difluoride) (PVDF)
membranes (Immobilon-P; Millipore, Bedford, MA). After
blocking with 2% non-fat skim milk in TTBS (20 mM Tris-HCI,
pH 7.5,140 mM NaCI, 0.05% Tween 20), the membranes were
incubatedwith a I:2000dilutionof anti-PSApolyclonalantibody
(Dako, Glostrup, Denmark) in TTBS containing 1% bovine
serum albumin. After washing with TTBS, they were incubated
for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbitimmunoglobulin (Amersham, UK), washedand
developed using an enhanced chemiluminescence (ECL) system
(Amersham). As an internal control, actin levels were also
determined using anti-actin polyclonal antibody (Biomedical
Technologies, Stoughton, MA).
RESULTS
LNCaP cells were cultured for 3 days in the presence of each
soybeanisoflavoneat variousconcentrationsandthen viablecells
wereevaluatedby WST-1 assay (Fig. 2). Amongfour isoflavones
tested, genisteininhibited the growth of LNCaP most effectively,
with an 1Cso value of 40 I-lM. Daidzein showed a weak inhibitory
effect on the growth of LNCaP. The ~-glucoside conjugates
genistin and daidzin exerted far less influence than their
aglycones genistein and daidzein, respectively. Treatment with
genisteinalso reducedBrdU incorporation. The amountsof BrdU
taken up by LNCaP between 2 and 4 h after the exposure to 37.5,
362
INCaP growth inhibition by genistein
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Figure 2. Effect of isoflavones and their glucosides on the growth of the LNCaP
human prostatic cancer cells. Cells were cultured in the presence of various
concentrations of genistein (0), daidzein (0), genistin (e) and daidzin (.)
After culturing for 3 days, cell viability was examined by a WST-I assay. Cell
viability is expressed as a percentage of the control cell value. Each point
represents mean ± SE data for six wells.
75 and ISO IlM genistein were 74,65 and 47% of control cells,
respectively, consistent with the effects on cell growth.
When LNCaP cells were treated as monolayer sheets with
genistein at concentrations>75 IlM, they began to detach to some
extent after 12 h and floating cells increased time dependently.
Fluorescent microscope examination revealed these floating cells
to show characteristic nuclear features of apoptosis such as
chromatin condensation and nuclear fragmentation (Fig. 3). DNA
was extracted from LNCaP cells cultured in the absence or
presence of 75 or 150 j.lM genistein for 24 h, then determined
using agarose gel electrophoresis. Fragmented DNA was observed in the cells treated with 75 or 150 IlM genistein, but not in
the cells without genistein treatment (Fig. 4). A DNA ladder
pattern was clearly observed for LNCaP cells treated with 150
IlM genistein (Fig. 4). The DNA fragments consisted of
multimers of 180-190 base pairs, consistent with intemucleosomal cleavage of chromatin DNA by an endonuclease.
Analysis by immunoblotting showed that the cellular amounts
ofPSA in LNCaP cells were reduced when cells were treated with
151lM genistein, at which dose cell growth was hardly affected,
but the levels were not altered with 1.5 IlM genistein, as shown
in Fig. 5. A further decrease in PSA level was observed when cells
were treated with ISO IlM genistein. Densitometric analysis
revealed that the amounts of PSA adjusted for the actin levels in
the cells treated with IS and ISOIlM genistein for 24 h were 46
and 7%, respectively, of the control cell value .
DISCUSSION
In the present study, genistein was shown to be the most effective
among the four soybean isoflavones tested in inhibition of the
growth of human prostate cancer LNCaP cells. The estimated IC50
Figure 3. Nuclear morphology of LNCaP cells treated with 150 J..lM genistein
for 24 h. After the treatment, cells were stained with Hoechst 33258 and
examined under a fluorescent microscope.
rk r
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812
603
310
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Figure 4. Effect of genistein on DNA fragmentation of LNCaP cells. Cells
were cultured in the absence (lane I) or presence of 75 J..lM (lane 2) or 150 J..lM
(lane 3) genistein. After 24 h, fragmented DNA was extracted from LNCaP
cells in lysis buffer and loaded on to a 2% agarose gel.
value, 40 IlM, of genistein was comparable to that reported
previously (21). Both suppression of DNA synthesis and induction
of apoptosis were observed on treatment of the cells with genistein.
These effects of genistein are not specific to LNCaP; cell growth
inhibition and induction of apoptosis were also observed in another
human prostate cancer cell line, DUI45 (data not shown). Cellular
amounts of PSA, a glycoprotein produced in the prostate having
chymotrypsin-like serine protease activity and a tumor marker for
prostate cancer (22), were also decreased dose dependently by
genistein. The suppression of PSA expression was observed at a
concentration of 15 IlM, with which no growth inhibition of the
Jpn J Cli n OncoI1998;28(6)
Genistein 0
1.5
15
150
( JlM)
PSA
Ac t i n
Figure 5. Immunoblot analysis demonstrating the effects of genistein on the
cellular amounts of PSA. lNCaP cells were treated with genistein at the
indicated concentrations for 24 h. Cell Iysates were subjected to immunob lot
ana lysis. probed with anti-PSA (top ) or anti-actin (bottom) antibodies.
cells was evident. This may be related to the effects of genistein on
protein synthesis and/or signal transduction for PSA expression.
Daidzein exhibited a weak inhibitory effect on the growth of
LNCaP cells. Genistin and daidzin, the ~-glucoside forms of
genistein and daidzein, respectively, showed much less growth
inhibition activity than their aglycones. Genistein can be absorbed
in the upper small intestine (23,2 4), whereas the ~-glucoside
conjugate, genistin, needs conversion to an aglycone through the
action of a ~-glucosidase produced by the intestinal bacteria before
being taken up (5,23). Therefore, genistin may show anti-carcinogenetic activity after absorption from the intestine in vivo.
The present results support the view that genistein is a
candidate component in soybeans accounting for the lower risk
of prostate cancer in the Japanese. However, the contribution of
other components to the preventive effects of soy-based diet'> in
prostate cancer should be investigated further, becau se soybeans
contain several other anti-carcinogens such as proteas e inhibitors,
phytosterols , saponins and inositol phosphates (9,25). It is also
possible that some other chemopreventive substances could act
additively or synergistically with genistein. Although a 6 11M
plasma level of genistein has been detected in subjects after the
consumption of soymilk (24), few reports are available concerning the possible in vivo toxicity at higher concentrations.
It should be clarified whether the inhibition of DNA synthesis
and the induction of apoptosis in pro state cancer cells are really
observed in vivo without inducing any adverse effects. As far as
colon cancer is concerned, a recent report indicated that genistein
enhanced azoxyrnethane-induced colon carcinogenesis in a rat
model (26). Further anim al experiment s and interventi on studies
are needed to determine the in vivo chemopreventive potential of
genistein for prostate cancer.
Acknowledgments
This study was supported by a grant from the Program for
Promotion of Fundamental Studies in Health Sciences of the
Organization for Drug ADR Relief, R&G Promotion and Product
Review of Japan and a Grant-in-Aid from the Ministry of Health
and Welfare for the Second-Term Comprehen sive 10-Year Strategy for Cancer Control. M. Onozawa is the recipient of a Research
Resident Fellowship from the Foundation for Promotion of Cancer
Research.
363
References
I. Yatani R, Shiraishi T, Nakakuri K, Kusano I, Takanari H, Hayashi T, et al.
Trends in frequency oflatent prostate carcinoma in Japan from 1965-1979 to
1982-1986. J Natl Cancer lnst 1988;80:683-7.
2. Carter HB, Coffey OS. Th e prostate: an increasing medical proble m. Prostate
1990;16:39-48.
3. Coward L, Barnes NC, Se tchell KOR , Bames S. Geniste in. daidzein and their
~-glycoside conjugates: antitumor isoflavo nes in soybe an foods from
American and Asian diets. J Agric Food Chern 1993;4 1:1 96 1-7.
4 . Adle rcreutz H, Markkane n H. Watanabe S. Plasma concentrat ions of
phyto-oestrogens in Japanese men . Lancet 1993;342:1 209- 10.
5. Messina MJ, Persky VP, Setc hcll KOR, Bame s S. Soy intake and cancer risk :
a review of the in vitro and in vivo data. Nutr Cancer 1994;21:113-31.
6. Adlercreutz H, Honjo H, Higashi A, Fotsis T. Hamalainen E. Hasegawa T, ct
a!. Urinary excret ion oflignans and isoflavonoid phytoestro gens in Japanese
men and women consum ing a traditional Japanese diet. Am .r Clin Nun'
199 1;54:I093-1 00.
7. Health and Welfare Statistics Association . Kokuminn Eiyou No Nenji Suii.
Kousei No Shihyou 1995 ;42(16):6-8 (in Japanese).
8. Peterson G. Evaluation of the biochemical targets of genistein in tumo r cells.
J Nutr 1995; I 25:784S-9 S.
9 . Wynder El. , Rose DP, Co hen LA. Nutrition and prostate cancer: a proposal
for dietary intervention. Nutr Cancer 1994;22:1-10.
10 . Fukutake M, Takahashi M, Ishida K, Kawamura H, Sugimu ra T, Wakabaya shi K. Quantification of genistein and genistin in soybea ns and soybean
produ cts. Food Chern ToxicolI 996;34:457-6 1.
II . Fotsis T, Pepper M, Ad lercreutz H, Hase T, Montesano R. Schweigerer L.
Genistein. a dietary ingested isoflav onoid, inhibits cell prolifer ation and in
vitro angiogenesis. J Nutr 1995;125:7905-7S.
12. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S. Itoh N, et al.
Genistein. a specific inhibitor of tyros ine-specific protein kinases . .r BioI
Chern 1987;262:5592-5.
13. Markovits J, Linassier C, Fosse P, Couprie J, Pierre J, Jacquem in-Sablon A.
et al.Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 1989;49:5111-17.
14. Wei H, Wei l . Frenkel K. Bowen R. Barnes S. Inhibition of tumor
promoter-induced hydroge n peroxid e formation ill vitro and in \'ivo by
genistein. Nutr Cancer 1993;20:1-1 2.
15. Folman Y, Pope GS. The interaction in the immature mouse of potent
oestrogens with coumestro l, genistein and other utero-v aginotroph ic compound s of low poten cy. J Endocrinol 1966;34:215-25.
16. Folman Y, Pope GS. Effect of norethisterone acetate , dimethyl stilboestrol,
geni stein and coume stroI on uptake of [3H]oestradiol by uterus, vagina and
skeletal muscle of imm ature mice. J Endocrinol 1969;44:21 3-8 .
17. Setchell KOR , Borriello SP, Hulme P, Kirk ON. Axelson M. Nonsteroid al
estrogens of dietary origin: possible roles in hormone-dependent disease. Am
.r Clin Nutr 1984;40:569-78.
18. Mousavi Y, Adlercreutz H. Genistein is an effective stimulator of sex
horm one-bindin g globulin production in hepatocarcinoma human liver
cancer cells and suppresses proliferation of these cells in culture. Steroids
1993:58:301-4.
19 . Lamarti niere CA, Moore JB, Brown NM, Thompson R, Hard in MJ , Bam es S.
Genistein suppresses mamm ary cancer in rats. Carcinogenesis
1995;16:2833-40.
20. Murrill WB , Brown NM, Zhang J-X. Manzolillo PA, Barnes S, Larna rtiniere
CA. Prepubertal genistein exposure suppresses mamm ary cance r and
enhances gland differentiation in rats. Carcinogenesis 1996;17:145 1-7.
21. Peterson G, Barnes S. Ge nistein and biocha nin A inhibit the growt h of human
prostate cancer cells but not epide rmal growth factor receptor tyrosine
autophosphorylation. Prostate 1993;22:335-45 .
22. McConnack RT, Rittenhouse HG, Finlay JA, SokoloffRL, Wang TJ, Wolfert
RL , et al. Molecular forms of prostate-specific antigen and the human
kallikrein gene fam ily: a new era. Urology 1995;45:729-44 .
23. Bam es S. Effect of genistein on in vitro and inl 'ivo models of cancer. J Nutr
I995; 125:777 S-83S.
24 . Xu X, Harris KS. Wang H-J, Murphy PA, Hendrich S. Bioavailability of soybean
isoflavones depends upon gut microflora in women. J Nutr 1995;1 25:2307-15.
25 . Kennedy AR. The eviden ce for soybean product s as cancer prev entive
agents. .r NUll" 1995;125:733S-43S.
26. Rao CV, Wang CoX, Sim i B, Lubet R. Kelloff G, Steele V,et al. Enhancement
of experimental colon cancer by genistein. Cancer Res 1997;57:3717- 22.