Indole-3-Carbinol Is a Negative Regulator of Estrogen12
- Karen J. Auborn*,†,**,3,
- Saijun Fan*,‡,
- Eliot M. Rosen*,ࠠ,
- Leslie Goodwin*,
- Alamelu Chandraskaren*,
- David E. Williams‡‡#,
- DaZhi Chen*, and
- Timothy H. Carter*,†§
+ Author Affiliations
- ↵3To whom correspondence should be addressed. E-mail: kauborn@nshs.edu.
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)-α.
Expression of GADD was evaluated by real-time reverse
transcription–polymerase chain reaction. Proliferation and apoptosis
were measured 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-α. 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-α. Because of the synergistic effect of I3C and genistein, the potential exists for prophylactic or therapeutic efficacy of
lower concentrations of each phytochemical when used in combination.
Indole-3-carbinol (I3C)4 and its biologically active dimer diindolylmethane (DIM), which are obtained from the dietary consumption of cruciferous
vegetables (Brassicas), are promising agents
for the prevention of estrogen-enhanced cancers. A 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 nationwide 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 cancers ( 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 opposing 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 prevents 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 PCNA (a component of DNA-δ
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 antiproliferative and proapoptotic ( 15, 16). Alternative metabolism (16α-hydroxylation) of estradiol results in compounds that increase proliferation and anchorage independent growth ( 9, 17). Second, in the case of genes driven by the estrogen receptor (ER)-α, I3C acts as a negative regulator ( 18). The tumor suppressor breast cancer 1 (BRCA-1), whose expression is upregulated by I3C/DIM ( 19), also inhibits the expression of genes driven by ER-α ( 20). Moreover, I3C and BRCA-1 work together to abrogate ER-α–driven expression ( 19). Using subtractive hybridization, Chen et al. ( 21) determined that expression 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 transcription 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 response
to DNA damage (GADD)] that were robustly upregulated 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 phytochemical that is the major
isoflavonoid in soy, can act synergistically with I3C/DIM to kill these
cells.
MATERIALS AND METHODS
Reagents
17β-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-α
expression plasmid driven by the cytomegalovirus promoter and the ER
element–thymidine kinase–luciferase (ERE-TK-LUC) reporter,
which is composed of the vitellogenin A2
estrogen-responsive elements that control the thymidine kinase (TK)
promoter and
luciferase in plasmid pGL2.
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 37°C 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) supplemented 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 μL of medium/well and incubated for 16 h. The medium was changed to 200 μL
that contained either dimethyl sulfoxide (DMSO) 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 μg 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
reaction (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 μmol primers/L, 0.1 μmol 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 50°C for 2 min, 95°C for 10 min and 45 cycles
of 95°C for 30 s and 60°C 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 β-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
5′-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-α, 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 β-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 17β-estradiol–activated ER-α–mediated transcriptional activity
As described previously ( 18, 20), subconfluent cells plated in 24-well culture dishes were cotransfected with the luciferase reporter plasmid that contains
ERE (ERE-TK-LUC, 0.5 μg/L) and the ER-α expression vector (0.5 μg/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 17β-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 apoptosis 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-45α, -β and -γ 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-α ( 20). Finally, as shown below, genistein also can induce expression of GADD and can affect ER signaling.
FIGURE 1
Model for a subset of genes induced by indole-3-carbinol/diindolylmethane (I3C/DIM) that may counteract estrogen-enhanced
tumors.
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
endoplasmic 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 μmol/L) or genistein (5 or 25 μmol/L) increases expression of GADD-34. Very little increase is detected using 25 μmol DIM/L. However the combination of 5 μmol genistein/L and 25 μmol DIM/L results in a synergistic increase in the expression of this GADD. Results also are shown for GADD-153 ( Fig. 2B) and GADD-45α ( Fig. 2C). Similar results occur with the other isoforms of GADD-45 and are virtually identical in C33A cells (unpublished data).
FIGURE 2
DIM and
genistein synergistically induce growth arrest in response to DNA damage
(GADD). MCF-7 cells were treated with dimethyl
sulfoxide (DMSO, a solvent control),
genistein, DIM or both DIM and genistein for 16 h. The cDNA made from
mRNA was used as
a template to amplify GADD-34 (A), GADD-153 (B), GADD-45α (C) and β-actin by real-time reverse transcription–polymerase chain reaction (RT-PCR) as described in Methods and Materials. Treatment
of cells was DMSO (1), 100 μmol DIM/L (2), 50 μmol DIM/L (3), 25 μmol DIM/L (4), 25 μmol genistein/L (5), 5 μmol genistein/L (6) and the combination of 25 μmol DIM/L + 5 μmol genistein/L (7). The amount of estrogen in media (10% fetal calf serum) was 10−15 mol/L.
DIM and genistein synergistically increase apoptosis
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 μmol 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 μmol/L ( Fig. 3A) compared to the requirement for DIM concentrations to be >50–60 μmol/L when cells are exposed to DIM alone. DIM (50 μmol/L) counteracts the proliferative effect of genistein, and genistein (used at increasing 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) are identical for cells treated
for 24 h with DMSO (solvent control), 25 μmol DIM/L or 5 μmol 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 3
DIM and genistein work together to decrease viability. MCF-7 cells were treated for 72 h with DMSO (solvent controls), or
5 μmol genistein/L and increasing amounts of DIM from 0 to 100 μmol/L (A) or 50 μmol DIM/L and increasing amounts of genistein from 0 to 50 μmol/L (B).
A minimum of six replicate wells was employed per condition. Viability
was determined by a mitochondrial function assay
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS)]. The amount of estrogen in
media (10% fetal calf serum) was 10−15 mol/L.
FIGURE 4
DIM and genistein synergistically increase apoptotic cells. MCF-7 cells were treated with DMSO, 25 μmol DIM/L, 5 μmol genistein/L or 25 μmol DIM/L + 5 μmol genistein/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%.
DIM and genistein synergistically decrease estrogen signaling driven by ER-α
Because genistein is a weak estrogen ( 26) but able to compete 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-α ( Fig. 5). Using MCF-7 cells treated with I3C (50 μmol/L), genistein (25 μmol/L) or the combination 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 decreases 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
phytochemicals were additive, which indicates a
clearly synergistic effect.
FIGURE 5
DIM and genistein synergistically inhibit estrogen signaling. MCF-7 monolayers were transfected with a plasmid that expresses
estrogen receptor-α and a plasmid for luciferase expression driven by the estrogen response element (ERE). The cells were then treated with ±1
μmol estradiol/L, ±25 μmol/L genistein and ±50 μmol 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.
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-α.
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 received much less attention in the literature than the
cellular
events that trigger oncogenesis. Tumor cells
protect themselves 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 adaptation to
the stressful conditions or elimination by
programmed cell death. Obviously, the desired outcome for cancer
prevention is
growth arrest and apoptosis.
The fact that genistein works
synergistically with I3C/DIM has a number of implications. Importantly
(at least for induction
of GADD, apopotosis and inhibition of
estrogen-increased 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
evaluating 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.
Footnotes
-
↵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 Prevention, 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.
ليست هناك تعليقات:
إرسال تعليق