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Food and Nutritional Sciences Programme, Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong and * U.S. Department of Agriculture, Beltsville Human Nutrition Research Center, Phytonutrients Laboratory, Beltsville, MD 20705
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • genistein • Bcl-2 • c-jun N-terminus kinase • MCF-7 cells • cell death
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The American Cancer Society (1993)
has reported a lower incidence of breast, colon and
prostate cancers in Southeast Asia than in Europe and America. One
specific dietary area that epidemiologists have considered is the
difference in soy consumption. Genistein is the most studied soy
isoflavone that exhibits cancer preventive activity. In many in vitro
studies, genistein was found to be growth inhibitory to HL-60 cells
(Traganos et al. 1992
), lung cancer cells (Lian et al. 1999
), esophageal, stomach and colon cancer cells
(Yanagihara et al. 1993
), head and neck cancer cells
(Alhasan et al. 1999
), liver cancer cells
(Mousavi and Adlercreutz 1993
), bladder cancer cells
(Theodorescu et al. 1998
), mammary cancer cells
(Peterson and Barnes 1991
, So et al. 1997
) and prostate cancer cells (Kyle et al. 1997
, Peterson and Barnes 1993
).
Apoptosis is a process that is crucial to the growth and development of
multicellular organisms. Bcl-2 was the first protein identified to be
antiapoptotic (Reed 1994
). Subsequently, proteins that
share structural homology with Bcl-2 have been described and
characterized. Bcl-x is one of the Bcl-2 family proteins and has
two forms, the long (L) and the short (S) forms. Bcl-x(L) facilitates
cell survival as Bcl-2, whereas Bcl-x(S) initiates proapoptotic signals
(Gross et al. 1999
, Reed 1998
). Proteins
such as Bax and Bak are proapoptotic in many systems, and specific
interaction among these proteins could be the determinant of cell
survival or death (Gross et al. 1999
, Reed 1998
). The cell death mechanism has been attributed to the
release of cytochrome c from mitochondria and the subsequent caspase
activation (Li et al. 1997
). The interactions among
Bcl-2 family proteins can affect the stability of the mitochondrial
membrane, which is important in confining cytochrome c to the organelle
(Gross et al. 1999
).
C-Jun N-terminus kinase
(JNK)3
is activated by different stresses such as redox potential alteration,
heat shock, osmotic shock, UV irradiation and cytokines. It is
activated by phosphorylation carried out by the upstream
mitogen-activated protein kinase. A functional JNK can
phosphorylate c-Jun, JunD, ATF-2, ATF
, ELK1 and Sap-1, and its
activity has been associated with apoptosis (Ip and Davis 1998
).
Recent studies have indicated that genistein is cytostatic to MCF-7
cells at high dosages (Breinholt and Larsen, 1998
,
Le Bail et al.,1998
, Shao et al. 1998
,
Wang et al. 1996
, Wang and Kurzer 1997
).
Nevertheless, genistein is a phytoestrogen that may bind to estrogen
receptors (ER) and induce proteins regulated by the receptor. Bcl-2 is
one of the well-defined antiapoptotic proteins regulated by the ER
(Perillo et al. 2000
). Using our established MCF-7 cell
system, we investigated the cellular changes induced by genistein at
its threshold cytostatic concentrations. In this study, we investigated
whether Bcl-2 proteins, especially Bcl-2, can be regulated by
genistein. We hypothesized that Bcl-2 family proteins were determining
factors in genistein’s effects on apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genistein was obtained from Sigma Chemical (St. Louis, MO) as were all other chemicals unless stated otherwise.
Cell culture.
We obtained the breast cancer cell line MCF-7 from American Type Cell Collection (Rockville, MD). The cells were incubated in RPMI 1640 medium with 2 mmol/L L-glutamine (Biofluids, Rockville, MD), 3.5 µg/L hydrocortisone, 1.5 µg/L insulin, 100,000 U/L penicillin and 100 g/L streptomycin, and 5% fetal bovine serum (FBS; Gibco BRL, Rockville, MD). The incubator was maintained at 37°C and 5% CO2 air. One week before the beginning of experiments, the cells were switched to the same medium with 5% charcoal dextran–treated FBS (Hyclone, Logan, UT) instead of untreated FBS. The cells were trypsinized and seeded at a density of 5 x 105 cells/well in 6-well Costar culture plates (Corning, Corning, NY) 1 d before treatment started.
Apoptotic death assay.
The assay was performed as previously described (Wang and Phang 1995
). A cell death detection ELISA kit obtained from
Boehringer Mannhein (Indianapolis, IN) was employed in this
determination. Cells with a density of 5 x 104/well
were plated in 24-well plates (4 wells/treatment). After 48 h of
treatment, the cells were washed once with PBS and lysed with 0.5 mL
lysis buffer provided in the kit. The cell lysates were then used for
the colorimetric quantitation of DNA fragments by ELISA as illustrated
in the manufacturer’s manual. An identically treated cell culture
plate was used for the cell viability determination by sulforhodamine B
(SRB) assay (Skehan et al. 1990
). In brief, the viable
cells were fixed with 3 mol/L trichloroacetic acid and stained with SRB
(1.7 mmol/L) dissolved in 0.16 mol/L acetic acid. The stained cells
were then washed with 10 mmol/L Tris and absorbance read at 570 nm. The
apoptotic index (AI) was obtained by normalizing the ELISA value with
the SRB absorbance reading.
Estrogen response element (ERE) reporter cells and luciferase assay.
MCF-7 cells with a stable transfected reporter gene of ERE-luciferase were obtained from Dr. J. C. Nicolas, INSERM, Montpellier, France. These cells were cultured in 6-well plates in triplicate as described above. After 16 h of treatment, the cells were harvested and assayed for the luciferase activity by using the luciferase substrate from Promega (Madison, WI).
Bcl-2 mRNA quantitation.
Total RNA was isolated from cells grown in 6-well Costar plates in
triplicate by a method previously described (Wang and Phang 1995
). One microgram of RNA was used for cDNA synthesis, and
the final volume was diluted to 100 µL. A competitive
reverse transcription-polymerase chain reaction (RT-PCR) assay was
used to quantitate Bcl-2 mRNA level. A PCR-MIMIC construction kit
from Clontech (Palo Alto, CA) was employed to build the MIMIC strand
for Bcl-2 quantitation. One set of primers, with previously published
sequences (Wang and Phang, 1995
), was utilized to
amplify both target cDNA and competitor DNA (MIMIC) of known
concentration. All PCR reactions consisted of 0.2 mmol/L dNTP, 2
µL MIMIC, 4 µL cDNA, 0.2
µmol/L of each primer, 1X PCR buffer and 1
U of Taq polymerase. The conditions were 35 cycles at
94°C for 45 s, 65°C for 45 s, 72°C for 2 min and a
final extension period of 7 min at 72°C. The PCR products were
separated on 1.8% agarose gel and stained with ethidium bromide. The
amount of Bcl-2 mRNA in the sample was determined by comparing its
relative intensity to the MIMIC amplified product. Employing the same
protocol, G3PDH mRNA was determined. G3PDH and its MIMIC primers were
purchased from Clontech.
Determination of JNK activity.
A nonradioactive JNK assay kit was obtained from New England Biolabs (Beverly, MA), and the assay was performed as illustrated in the kit manual. In brief, the stress-activated protein kinase (SAPK)/JNK was precipitated from cell lysates by binding to N-terminal c-Jun protein conjugated to glutathione-sepharose beads. The beads were washed three times in lysis buffer (20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerolphosphate, 1 mmol/L Na3VO4, 1 mg/L leupeptin) to avoid nonspecific binding. The ATP-mediated JNK reaction was then carried out by incubating the precipitated protein in 10 mmol/L ATP and kinase buffer (25 mmol/L Tris, pH 7.5, 5 mmol/L ß-glycerolphosphate, 2 mmol/L dithiothreitol, 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2). The protein-conjugated beads were washed three times and boiled with loading buffer for 5 min. An aliquot of supernatant was then separated by 10% SDS-PAGE. The relative activity of JNK in cell lysate can be visualized by Western blotting as described below with a phospho-c-Jun antibody.
Western analysis.
Treated cells in quadruplicate were washed once by PBS (pH 7.4) and harvested into a 1.5-mL microtube with 0.5 mL lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS). The lysis buffer contained protease inhibitors (40 mg/L phenylmethylsulfonyl flluoride, 0.5 mg/L aprotinin, 0.5 mg/L leupeptin, 1.1 mmol/L EDTA and 0.7 mg/L pepstatin). The harvested cells were then lysed with a Tekmar Sonic Disrupter on ice for 30 s. The protein concentration of cell lysate was determined by Dc protein assay (BioRad, Richmond, CA). Lysate protein (25 µg) was separated on 10% SDS-PAGE (Novex, San Diego, CA) and transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA). Anti-Bcl-2, anti-Bax, and antiactin primary and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The targeted protein was visualized by an ECL Detection Kit (Amersham, Arlington Heights, IL), and the amount was quantitated by densitometry.
Statistical methods.
A StatView SE+ Graphics (Abacus Concepts, Berkeley, CA) software package was used for statistical analysis. The results, whenever applicable, were analyzed by one-way ANOVA followed by Fisher’s Protected Least Significant Difference post-hoc procedure. The optical density results of Bcl-2 protein blots were analyzed by one-sample t test followed by Wilcoxon’s signed-rank test; the significance level was set at P < 0.05 in all cases.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Five different concentrations of genistein were used to titrate the one
that induced apoptosis. Administration of 0.5, 1.0 and 12.5
µmol/L genistein did not provoke significant cell death
compared with the control, whereas 25 and 50 µmol/L caused
a higher AI than the control (P < 0.05) (Fig. 1
). The 25 and 50 µmol/L AI were 125 and 160% of the
control, respectively.
|
Genistein induced luciferase transcription at all concentrations tested
(P < 0.05); 0.1 µmol/L genistein
initiated 
10 times the control luciferase activity, and 50
µmol/L induced 90 times the control activity
(Fig. 2
). A dose response was demonstrated, although it was not linear.
|
After the AI were obtained, we also determined the Bcl-2 mRNA levels
that were affected by genistein. Because it is a weak estrogen agonist,
we wanted to determine whether genistein could induce Bcl-2 expression
like 17ß-estradiol. All concentrations tested resulted in a Bcl-2
level significantly higher than that of the control (P
< 0.05; Fig. 3
). Nevertheless, induction was lower (P < 0.05) at 50
µmol/L than at 1.0, 12.5 and 25 µmol/L
genistein.
|
Because an induction of Bcl-2 mRNA expression was observed, we tested
Bcl-2 and Bax at the protein level (Fig. 4 a
,
b
). Genistein at both 25 and 50 µmol/L significantly
(P < 0.05) induced Bcl-2 and Bax protein (Fig. 4b
). The ratio of Bcl-2/Bax measured by densitometry was not
significantly affected (0.05 < P < 0.10). In a
separate experiment, we also found that genistein at 1
µmol/L could not induce Bax protein (data not shown).
|
), which might have contributed to the Bax induction.
|
A persistent, elevated SAPK has been linked to apoptosis. Genistein was
an inducer of JNK activity at the concentrations of 25 and 50
µmol/L after 8 h of treatment (Fig. 6
).
|
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genistein is an inhibitor of cell proliferation with a 50% inhibitory
concentration (IC50) in the mid-20
µmol/L range (So et al. 1997
), which is
comparable to this study. Our results suggested some mechanisms for the
apoptotic effects of genistein on MCF-7 cells. One of the pathways was
stress dependent. The levels of JNK remained high above the baseline
for 25 and 50 µmol/L genistein after 8 h of
administration. This illustrated that a sustained activation of JNK
could be achieved at or above 25 µmol/L genistein, and has
been suggested to be a determinant for apoptosis (Chen et al. 1996
). Moreover, p53 protein has been shown to be affected by
JNK in previous studies. Fuchs et al. (1998b)
showed
that p53 is stabilized and activated by the MEKK1-JNK signaling
pathway, and a mutant JNK facilitates the degradation of wild-type
p53 (Fuchs et al. 1998a
). The accumulation of p53 at
high concentrations in this study could be the result of JNK
activation.
The other route of inducing cell death could be Bax dependent. Previous
studies have demonstrated that a p53 DNA binding domain occurs in the
proapoptotic protein Bax promoter region (Miyashita and Reed 1995
). P53 can induce apoptosis by activating Bax
(Miyashita et al. 1994
). The outcome could be due to p53
accumulation mediated by JNK. In ER-negative cell lines, genistein
can induce cell death in a p53-independent pathway (Li et al. 1999b
) and up-regulate Bax (Li et al. 1999a
).
Genistein has been identified as an agonist of the ER in an animal
model (Makela et al. 1995
) as well as in cell culture
models (Fioravanti et al. 1998
, Miodini et al. 1999
, Wang et al. 1996
), and competes with
estradiol for ER binding. This agonistic property also contributes to
the induction of Bcl-2 protein seen in this study as illustrated by the
gene transcription of luciferase. Perillo et al. (2000)
identified two functional ERE at the bcl-2 promoter region.
Nevertheless, the correlation between ERE binding activity and Bcl-2
expression level does not hold beyond 25 µmol/L genistein.
It appears that Bcl-2 expression is regulated by factors other than ERE
binding activity at higher concentrations. Genistein may inactivate
Bcl-2 by phosphorylation at 150 µmol/L, but the
phosphorylation does not occur at 25 µmol/L
(Constantinou et al. 1998
) and the protein remains
functional.
Exposure to estrogen has been considered a risk factor for breast
cancer (Nenci et al. 1988
). 17ß-Estradiol enhances
cell survival by increasing Bcl-2 (Wang and Phang 1995
)
and decreasing Bak (Leung et al. 1998
) levels in MCF-7
cells. Recent studies have shown that genistein induces MCF-7 cell
proliferation at concentrations of 1 µmol/L
(Breinholt and Larsen 1998
, Le Bail et al. 1998
, Shao et al. 1998
, Wang and Kurzer 1997
) and is cytostatic at higher concentrations with an
IC50 in the mid-20 µmol/L range
(Peterson and Barnes 1991
, So et al. 1997
). It is likely that the estrogenic nature of genistein
prevails at low concentrations.
Apoptosis is an important process in cancer development and therapy as
reviewed by Lowe and Lin (2000)
. Compounds inducing
apoptosis can affect cancer initiation, progression and metastasis.
This study demonstrated that the machinery of tumor cell death could be
initiated when the genistein concentration was higher than 25
µmol/L. Although a maximum plasma level of 23
µmol/L in Taiwanese people was observed in a survey
published in 1991 (Soyatech Survey and Estimates 1991
),
a recent study (Arai et al. 2000
) found that the median
plasma concentration fell in the submicromolar range in Japan, a
country with soy consumption similar to that in Taiwan. Because the
minimum concentration of genistein required to affect apoptosis is not
known, the dosage range used in this study may not be physiologically
appropriate. Nevertheless, the utilization of genistein as a
chemopreventive agent requires further assessment because the range of
dosages affecting cell proliferation and death in vivo has not been
established for hormone-dependent cancers. Furthermore, the
interaction or combination effects of genistein with the other
phytoestrogens found in food have not been resolved.
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FOOTNOTES |
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3 Abbreviations used: AI, apoptotic index; ER,
estrogen receptor; ERE, estrogen response element; FBS, fetal bovine
serum; IC50, 50% inhibitory concentration; JNK, c-jun
N-terminus kinase; RT-PCR, reverse transcription-polymerase
chain reaction; SAPK, stress-activated protein kinase; SRB,
sulforhodamine B.
Manuscript received June 1, 2000. Initial review completed June 30, 2000. Revision accepted August 22, 2000.
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