Nutritional Genomics in Cancer Processes

Indole-3-Carbinol Is a Negative Regulator of Estrogen1,2

Karen J. Auborn,*y**3 Saijun Fan,*z Eliot M. Rosen,*zyy Leslie Goodwin,* Alamelu
Chandraskaren,* David E. Williams,zz# DaZhi Chen* and Timothy H. Carter*y§
*North Shore–Long Island Jewish Research Institute, Manhasset, NY 11030, Departments of
y
Otolaryngology and zRadiology, Long Island Jewish Medical Center, Long Island Campus of
Albert Einstein College of Medicine, New Hyde Park, NY 11040, Departments of **Microbiology and
Immunology and yyDevelopmental and Molecular Biology, Albert Einstein College of Medicine,
Bronx, NY 10461, zzDepartment of Environmental and Molecular Toxicology and # Linus Pauling Institute,

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Oregon State University, Corvallis, OR 97331 and §Department of Biological Sciences,
St. Johns University, Jamaica, NY 11530

ABSTRACT Studies increasingly indicate that dietary indole-3-carbinol (I3C) prevents the development of
estrogen-enhanced cancers including breast, endometrial and cervical cancers. Epidemiological, laboratory, animal
and translational studies support the efficacy of I3C. Whereas estrogen increases the growth and survival of tumors,
I3C causes growth arrest and increased apoptosis and ameliorates the effects of estrogen. Our long-range goal is to
best use I3C together with other nutrients to achieve maximum benefits for cancer prevention. This study examines
the possibility that induction of growth arrest in response to DNA damage (GADD) in genes by diindolylmethane
(DIM), which is the acid-catalyzed condensation product of I3C, promotes metabolically stressed cancer cells to
undergo apoptosis. We evaluated whether genistein, which is the major isoflavonoid in soy, would alter the ability of
I3C/DIM to cause apoptosis and decrease expression driven by the estrogen receptor (ER)-a. Expression of GADD
was evaluated by real-time reverse transcription–polymerase chain reaction. Proliferation and apoptosis were mea-
sured by a mitochondrial function assay and by fluorescence-activated cell sorting analysis. The luciferase reporter
assay was used to specifically evaluate expression driven by ER-a. The estrogen-sensitive MCF-7 breast cancer cell
line was used for these studies. We show a synergistic effect of I3C and genistein for induction of GADD expression,
thus increasing apoptosis, and for decrease of expression driven by ER-a. Because of the synergistic effect of I3C
and genistein, the potential exists for prophylactic or therapeutic efficacy of lower concentrations of each phyto-
chemical when used in combination. J. Nutr. 133: 2470S–2475S, 2003.

KEY WORDS:  indole-3-carbinol  diindolylmethane  genistein  estradiol

Indole-3-carbinol (I3C)4 and its biologically active dimer
diindolylmethane (DIM), which are obtained from the dietary
consumption of cruciferous vegetables (Brassicas), are promis-
ing agents for the prevention of estrogen-enhanced cancers. A
1
Published in a supplement to The Journal of Nutrition. Presented at the
‘‘Nutritional Genomics and Proteomics in Cancer Prevention Conference’’ held
September 5–6, 2002, in Bethesda, MD. This meeting was sponsored by the
Center for Cancer Research, National Cancer Institute; Division of Cancer Preven-
tion, National Cancer Institute; National Center for Complementary and Alternative
Medicine, National Institutes of Health; Office of Dietary Supplements, National
Institutes of Health; Office of Rare Diseases, National Institutes of Health; and the
American Society for Nutritional Sciences. Guest editors for the supplement were
Young S. Kim and John A. Milner, Nutritional Science Research Group, Division of
Cancer Prevention, National Cancer Institute, Bethesda, MD.
2
This work is supported by National Institutes of Health grants 2R01-
CA733850 (to K. J. Auborn) and R01-CA82599 (to E. M. Rosen). The contents are
solely the responsibility of the authors.
3
To whom correspondence should be addressed. E-mail: kauborn@nshs.edu.
4
Abbreviations used: BRCA-1, breast cancer 1; DIM, diindolylmethane;
DMSO, dimethyl sulfoxide; ER, estrogen receptor; ERE, estrogen receptor ele-
ment; GADD, growth arrest in response to DNA damage; I3C, indole-3-carbinol;
PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcriptase–poly-
merase chain reaction; UV, ultraviolet.
combination of epidemiological and experimental data provides
suggestive evidence that a high intake of cruciferous vegetables
protects against some cancers at various sites (1). In a nation-
wide study of postmenopausal women in Sweden, consumption
of cruciferous vegetables was inversely associated with breast
cancer risk (2). Although cruciferous vegetables have a number
of cancer-preventing compounds, I3C alone shows efficacy for
the prevention of breast (3), endometrial (4) and cervical can-
cers (5) in animal models. Importantly, I3C shows efficacy for
treatment of precancerous lesions of the cervix in translational
human studies (6).
In estrogen-sensitive cells, I3C/DIM and estrogen have op-
posing activities on cells. Estrogen promotes tumor growth,
whereas I3C suppresses it. For example, the K14-HPV16
mouse, which has transgenes for the oncogenes from human
papillomavirus type 16, only develops cervical cancer when
estrogen is given chronically (7). However, dietary I3C pre-
vents cervical cancer in these estrogen-treated mice (5). This is
consistent with many in vitro studies that show that estrogen
increases cell proliferation (8,9) and I3C causes growth arrest
(10). Immunohistochemistry studies determined (5) that

0022-3166/03 $3.00 Ó 2003 American Society for Nutritional Sciences.

2470S
 INDOLE-3-CARBINOL AND ESTROGEN
PCNA (a component of DNA-d polymerase) is robustly
expressed in the cervical epithelium of estrogen-treated mice
(both transgenic and normal mice), and that this increase is
reduced by dietary intake of I3C. Studies in breast cancer cells
show that estrogen inhibits the effects of a variety of
proapoptotic agents (11). In cervical cells, estrogen inhibits
apoptosis that is induced by cisplatin, taxol and ultraviolet
(UV) radiation, and this inhibition by estradiol is dose
dependent (our unpublished data). On the other hand, I3C
and DIM induce apoptosis of both breast cancer and cervical
cells in vitro (12,13) and induce apoptosis in the cervical
epithelium of mice given estrogen (13). When estradiol and
I3C are together, the amount of apoptosis depends on the
relative concentrations of each (our unpublished data).
A number of mechanisms exist (that are not mutually
exclusive) whereby I3C (or DIM) can diminish the effects of
estrogen on tumor growth. First, I3C and DIM induce enzymes
such as CYP1A1, which converts estrone to 2-hydroxyestrone
(14) and ultimately results in metabolites that are antipro-
liferative and proapoptotic (15,16). Alternative metabolism
(16a-hydroxylation) of estradiol results in compounds that in-
crease proliferation and anchorage independent growth (9,17).
Second, in the case of genes driven by the estrogen receptor
(ER)-a, I3C acts as a negative regulator (18). The tumor sup-
pressor breast cancer 1 (BRCA-1), whose expression is up-
regulated by I3C/DIM (19), also inhibits the expression of
genes driven by ER-a (20). Moreover, I3C and BRCA-1 work
together to abrogate ER-a–driven expression (19). Using sub-
tractive hybridization, Chen et al. (21) determined that ex-
pression of a battery of genes driven by estrogen was abrogated
by DIM. Speculation is that I3C/DIM and estradiol modulate
the ER and the aryl hydrocarbon receptor (21). Thus, estrogen
could modulate the activity of I3C/DIM as well. Finally, in the
absence of estrogen, I3C and DIM induce many genes that
have the potential to induce growth arrest and apoptosis and
therefore might counteract the effects of estradiol. For example,
Cover et al. (10) determined that cyclin-dependent kinase
6 was induced by DIM, which should cause growth arrest of
breast cancer cells. In our own studies, we determined that
expression of > 100 genes was changed by a short (4–6 h)
treatment with DIM (22). Many genes that encode for trans-
cription factors and are involved in the endoplasmic reticulum
stress response were upregulated, whereas a number of genes
involved in proliferation were downregulated.
In this study, we investigated some of the genes [e.g., the
growth-arrest genes collectively named for growth arrest in re-
sponse to DNA damage (GADD)] that were robustly upregu-
lated by DIM (22), because these should provide insight into
mechanisms whereby I3C/DIM can overcome the growth and
survival effects of estrogen on tumor growth. We hypothesized
a mechanism whereby I3C/DIM can specifically target tumor
cells, and we have determined that genistein, another phyto-
chemical that is the major isoflavonoid in soy, can act sy-
nergistically with I3C/DIM to kill these cells.
MATERIALS AND METHODS
Reagents
17b-Estradiol, I3C, genistein and propidium iodide were purchased
from Sigma (St. Louis, MO). DIM was a gift from Dr. M. Zeligs
(Bioresponse, Boulder, CO). All the vectors used in this study were
described previously (18,19) including the ER-a expression plasmid
driven by the cytomegalovirus promoter and the ER element–
thymidine kinase–luciferase (ERE-TK-LUC) reporter, which is comp-
osed of the vitellogenin A2 estrogen-responsive elements that control
the thymidine kinase (TK) promoter and luciferase in plasmid pGL2.
2471S
Cell lines and cell culture
The breast cancer cell line MCF-7 was purchased from the
American Type Culture Collection (Manassas, VA). All cells were
maintained as monolayer cultures at 378C in 7% CO2 and were grown
in Dulbecco’s modified Eagle’s medium (DMEM) that contained 4.5 g
of glucose and bicarbonate/L (GIBCO-BRL, Gaithersburg, MD) sup-
plemented with 110 mg of sodium pyruvate/L, 200 mmol glutamine/L,
100 mL of fetal bovine serum/L and 100,000 U each of penicillin and
streptomycin/L.
Mitochondrial function assay for cell viability
Assays were preformed as described previously (13). Cells were
trypsinized, seeded at 10,000 cells/well in 96-well plates that contained
100 mL of medium/well and incubated for 16 h. The medium was
changed to 200 mL that contained either dimethyl sulfoxide (DMSO)
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as a solvent control, DIM and/or genistein with 6 replicate wells/
condition. Viability was determined after 72 h by reduction of 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)
-2H-tetrazolium (MTS) using the Cell Titer Aqueous One kit
(Promega, Madison, WI) according to the manufacturer’s instructions.
Absorbance at 595 nm of the solutions was determined with
a multiwell plate reader, and protein concentration was measured
with the MicroBCA kit (Pierce, Rockford, IL).
Fluorescence-activated cell sorting analysis
Subconfluent monolayers were treated with DMSO solvent control
or genistein and/or DIM for 48 h, trypsinized, washed in phosphate
buffered saline, fixed in 70% ethanol and incubated with 500 U of
RNase/mL. The DNA was stained using 50 mg of propidium iodide/mL
and sorted by fluorescence using a Becton Dickinson FACScan with
CellQuest software (Palo Alto, CA).
Real-time reverse transcription–polymerase chain
reaction analysis
Total RNA from cells was prepared using reagents from Qiagen
(Valencia, CA) followed by cDNA synthesis using a T-7–linked
oligo(dT) primer (reagents were from GIBCO-BRL, Grand Island,
NY). The cDNA were quantified by real-time polymerase chain re-
action (PCR) analysis using the TaqMan PCR core reagent kit and the
ABI Prism 7700 sequence-detector system (PE Biosystems). In brief,
the PCR reaction contained 0.5 mmol primers/L, 0.1 mmol TaqMan
probe carboxyfluorescein (FAM)/L, 3.5 mmol MgCl2/L, 0.2 mmol
dNTP/L, 0.25 U of uracil DNA glycosylase (AmpErase UNG), 0.625 U
of Taq polymerase (AmpliTaq Gold) and the cDNA in TaqMan buffer.
PCR conditions were 508C for 2 min, 958C for 10 min and 45 cycles of
958C for 30 s and 608C for 1 min. Values were calculated using the
software provided with the ABI Prism 7700 system. Each sample was
run in triplicate, and mean values were used for data analysis. Results
were normalized to b-actin expression and were expressed as a fold
change with respect to DMSO-treated values. For GADD-34, the
forward primer was cga ctg caa agg cgg c, the reverse primer was cag
gaa atg gac agt gac ctt ct and the TaqMan probe was 59-tetrachloro-
fluorescein phosphoramidite (TET)-caa gcg ccc aga aac ccc tac tca tg-
6-carboxy-tetramethylrhodamine (TAMRA). For GADD-153, the
forward primer was ctg aat ctg cac caa gca tga, the reverse primer was
aag gtg ggt agt gtg gcc c and the TaqMan probe was TET-caa ttg gga
gca tca gtc ccc cac t-TAMRA. For Gadd45-a, the forward primer
was aag tgc tca gca aag ccc tg, the reverse primer was gct tgg ccg ctt cgt
aca and the TaqMan probe was TET-tca gcg cac gat cac tgt cgg
g-TAMRA. For b-actin, the forward primer was cct ggc acc cag cac
aat, the reverse primer was gcc gat cca cac gga gta ct and the TaqMan
probe was TET-atc aag atc att gct cct cct gag cgc-TAMRA.
Reporter gene assay for 17b-estradiol–activated
ER-a–mediated transcriptional activity
As described previously (18,20), subconfluent cells plated in 24-
well culture dishes were cotransfected with the luciferase reporter
2472S SUPPLEMENT

plasmid that contains ERE (ERE-TK-LUC, 0.5 mg/L) and the ER-a
expression vector (0.5 mg/L) using the lipofection reagent Lipofectin
(GIBCO-BRL, Gaithersburg, MD) according to the manufacturer’s
instructions. Cells were cultured an additional 24 h in medium with or
without 17b-estradiol, I3C and/or genistein. After lysis with luciferase
lysis buffer (Promega), lysates were analyzed for luciferase activity
using a liquid scintillation counter (model LS60001C, Beckman,
Fullerton, CA), and the data was normalized by protein concentration.

RESULTS
We hypothesized that certain genes may be instrumental in
determining how I3C/DIM ameliorates estrogen induction of
tumor growth and eventually causes growth arrest and apop-

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tosis of these cells (Fig. 1). We considered the involvement of
GADD because of our recent information that their expression
is robustly upregulated by DIM (22). GADD are a group of
proteins (GADD-153, GADD-45a, -b and -g and GADD-34)
that induce growth arrest and apoptosis by different pathways.
Moreover, BRCA-1, which is induced by I3C/DIM, not only
induces expression to GADD-45 (23) but also inhibits estrogen
signaling that is dependent on ER-a (20). Finally, as shown
below, genistein also can induce expression of GADD and can
affect ER signaling.

DIM and genistein synergistically induce GADD

Genistein, an isoflavonoid from soy that is considered to be
an anticancer phytochemical (24), inhibits glucose-regulated
protein (25). This activity would counteract the protective
response to ER stress, and raises the possibility that genistein
could be an adjunct to I3C/DIM by modulation of the endo-
plasmic reticulum stress response in the direction of increased
growth arrest and apoptosis. We first asked how genistein might
affect the expression of GADD. We evaluated effects of DIM
and genistein separately and together on the expression of
GADD and used real-time RT-PCR to measure the response in
estrogen-sensitive MCF-7 cells. As shown in Figure 2A, a short-
time (6-h) treatment with either DIM (100 or 50 mmol/L) or FIGURE 2 DIM and genistein synergistically induce growth arrest in
genistein (5 or 25 mmol/L) increases expression of GADD-34. response to DNA damage (GADD). MCF-7 cells were treated with
dimethyl sulfoxide (DMSO, a solvent control), genistein, DIM or both DIM
Very little increase is detected using 25 mmol DIM/L. However and genistein for 16 h. The cDNA made from mRNA was used as
the combination of 5 mmol genistein/L and 25 mmol DIM/L a template to amplify GADD-34 (A), GADD-153 (B), GADD-45a (C) and
results in a synergistic increase in the expression of this GADD. b-actin by real-time reverse transcription–polymerase chain reaction (RT-
Results also are shown for GADD-153 (Fig. 2B) and GADD- PCR) as described in Methods and Materials. Treatment of cells was
45a (Fig. 2C). Similar results occur with the other isoforms of DMSO (1), 100 mmol DIM/L (2), 50 mmol DIM/L (3), 25 mmol DIM/L (4), 25
GADD-45 and are virtually identical in C33A cells (un- mmol genistein/L (5), 5 mmol genistein/L (6) and the combination of 25
published data). mmol DIM/L 1 5 mmol genistein/L (7). The amount of estrogen in media
(10% fetal calf serum) was 10ÿ15 mol/L.

DIM and genistein synergistically increase apoptosis

FIGURE 1 Model for a subset of genes induced by indole-3-
carbinol/diindolylmethane (I3C/DIM) that may counteract estrogen-
enhanced tumors.
If DIM and genistein synergistically induce GADD, then
growth arrest and apoptosis should be a consequence of this
induction. We used two methods [a mitochondrial function
assay and fluorescence-activated cell sorting (FACS) analysis]
to evaluate growth arrest and apoptosis. In the mitochondrial
function assay (Fig. 3), DIM and genistein work together to
decrease cell viability. When increasing concentrations of DIM
are used together with 5 mmol genistein/L (a concentration of
genistein that enhances growth in MCF-7 cells when used
alone), increased killing of cells occurs at concentrations of
DIM as low as 20 mmol/L (Fig. 3A) compared to the require-
 INDOLE-3-CARBINOL AND ESTROGEN
ment for DIM concentrations to be . 50–60 mmol/L when cells
are exposed to DIM alone. DIM (50 mmol/L) counteracts the
proliferative effect of genistein, and genistein (used at increas-
ing concentrations) potentiates cell killing by DIM (Fig. 3B).
Using FACS analysis (Fig. 4A), the profiles of subdiploid
(putative apoptotic cells, M1), G1 (M2), S (M3) and G2 (M4)
2473S
FIGURE 3 DIM and genistein work together to decrease
viability. MCF-7 cells were treated for 72 h with DMSO (solvent
controls), or 5 mmol genistein/L and increasing amounts of DIM
from 0 to 100 mmol/L (A) or 50 mmol DIM/L and increasing
amounts of genistein from 0 to 50 mmol/L (B). A minimum of six
replicate wells was employed per condition. Viability was
determined by a mitochondrial function assay [3-(4,5-dime-
thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS)]. The amount of estrogen in media (10%
fetal calf serum) was 10ÿ15 mol/L.
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are identical for cells treated for 24 h with DMSO (solvent
control), 25 mmol DIM/L or 5 mmol genistein/L. However, the
fraction of putatively apoptotic cells is dramatically increased
when cells are treated with the combination of both DIM and
genistein (Fig. 4, A and B). Results with C33A cells (both
assays) are identical to those of MCF-7 cells.
FIGURE 4 DIM and genistein synergistically increase
apoptotic cells. MCF-7 cells were treated with DMSO, 25 mmol
DIM/L, 5 mmol genistein/L or 25 mmol DIM/L 1 5 mmol genis-
tein/L. Cells were treated for 48 h, stained with propidium
iodide and sorted by fluorescence (abscissa). (top and middle
panels) M1, subN2 cells; M2, G1; M3, S; M4, G2/M. (bottom
panel) The gated percent values of M1 cells for the four
conditions were 6.5, 4.8, 4.4 and 42.3%.
2474S
FIGURE 5 DIM and genistein synergistically inhibit estrogen
signaling. MCF-7 monolayers were transfected with a plasmid that
expresses estrogen receptor-a and a plasmid for luciferase expression
driven by the estrogen response element (ERE). The cells were then
treated with 61 mmol estradiol/L, 625 mmol/L genistein and 650 mmol
I3C/L and were assayed for luciferase activity 24 h later as described in
Methods and Materials. The final bar is not experimental data but the
theoretical value if the effects of genistein and I3C had been additive.
DIM and genistein synergistically decrease estrogen
signaling driven by ER-a
Because genistein is a weak estrogen (26) but able to com-
pete with estradiol for the ER (27), we wanted to know what
the effect of I3C and genistein together have on the expression
of genes driven by the ER-a (Fig. 5). Using MCF-7 cells treated
with I3C (50 mmol/L), genistein (25 mmol/L) or the combi-
nation of I3C and genistein, we evaluated the amount of
luciferase that genes produce when driven by an estrogen-
responsive enhancer as described previously (18,20). I3C de-
creases estrogen-driven luciferase activity. Treatment with
genistein results in an even greater decrease. Treatment with
the two phytochemicals reduces expression significantly more
than would have been predicted if the effect of the two phyto-
chemicals were additive, which indicates a clearly synergistic
effect.
DISCUSSION
We provide insight into additional mechanisms whereby
I3C/DIM can counteract the growth and survival of tumors in
estrogen-sensitive cells. We performed additional analysis that
confirms that not only does DIM induce GADD, but also
that DIM and genistein synergistically induce expression of
GADD. Consistent with their effects on the induction of
GADD proteins, genistein and DIM work better together than
alone to increase apoptosis. Another way by which I3C/DIM
can lead to the growth arrest of estrogen-sensitive cancer cells is
by interfering with estrogen signaling. Here, too, genistein and
DIM are synergistic in inhibiting estrogen signaling by ER-a.
Our discovery that DIM induces GADD and other proteins
involved in the endoplasmic reticulum stress response (22) not
only supports the possibility that GADD contribute to the
growth arrest and apoptosis associated with I3C/DIM, but also
may answer (at least in part) why I3C/DIM seems to specifically
target tumor cells opposed to normal cells. The importance of
the tumor microenvironment in malignant progression has re-
ceived much less attention in the literature than the cellular
events that trigger oncogenesis. Tumor cells protect themselves
SUPPLEMENT
from changes in the microenvironment such as decreased
availability of oxygen and nutrients by engaging a biochemical
pathway called the metabolic stress response. In vivo cancer
cells are likely to be chronically stressed. The cellular response
to hypoxia, hypoglycemia and nutrient starvation includes the
synthesis of protective proteins and cell cycle arrest, which can
lead to apoptosis or survival and also can involve induction of
genes that promote angiogenesis and tissue remodeling. In
other words, the fate of the stressed cell is survival by adap-
tation to the stressful conditions or elimination by programmed
cell death. Obviously, the desired outcome for cancer preven-
tion is growth arrest and apoptosis.
The fact that genistein works synergistically with I3C/DIM
has a number of implications. Importantly (at least for induc-
tion of GADD, apopotosis and inhibition of estrogen-increased
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gene expression), the concentrations of these phytochemicals
used in vitro to achieve these activities are more in line with
concentrations that people acquire from eating the relevant
foods. Additionally, people are exposed to combinations of
foods and their bioactive constituents. Although sorting out
how diets ultimately may affect a cell necessarily involves eval-
uating individual nutrients, the study of interactions between
nutrients (especially well-studied nutrients) is the next step.
Clearly, the Asian diet, which is considered protective against
breast and some other cancers, must involve many bioactive
compounds and their interactions.
ACKNOWLEDGMENTS
We would like to thank Dr. Kai Liu and Kathy Ripali for their
technical assistance.
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