Published OnlineFirst April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
Cancer
Prevention
Research
Research Article
Indole-3-Carbinol and 30 ,30 -Diindolylmethane Modulate
Androgen's Effect on C-C Chemokine Ligand 2 and Monocyte
Attraction to Prostate Cancer Cells
Eun-Kyung Kim1,2,3, Young S. Kim1, John A. Milner1,4, and Thomas T.Y. Wang3
Abstract
Inflammation has a role in prostate tumorigenesis. Recruitment of inflammatory monocytes to the tumor
site is mediated by C-C chemokine ligand 2 (CCL2) through binding to its receptor CCR2. We hypothesized
that androgen could modulate CCL2 expression in hormone-responsive prostate cancer cells and thereby
promote recruitment of monocytes. Given the inhibitory effect of broccoli-derived compounds indole-3carbinol (I3C) and 3,30 -diindolylmethane (DIM) on androgen-dependent pathways, we also reasoned that
I3C and DIM could modulate the effect of androgen on CCL2-mediated pathways. Dihydrotestosterone was
found to induce a time-dependent (0–72 hours) and concentration-dependent (0–1 nmol/L) increase in
CCL2 mRNA levels in androgen-responsive human prostate cancer cells (LNCaP). This increase in CCL2
mRNA corresponded with increased secretion of CCL2 protein. The effect of dihydrotestosterone was
mediated through an androgen receptor (AR)-dependent pathway as small inhibitor RNA against AR
negated the induction of CCL2. Although dihydrotestosterone also induced TWIST1 mRNA, an epithelial–
mesenchymal transition–related factor, and purported inducer of CCL2, blocking its expression with small
inhibitor RNA did not inhibit dihydrotestosterone induction of CCL2 mRNA. Moreover, conditioned media
from androgen-treated cells promoted human monocyte THP-1 cell migration and this effect was blocked by
antibody against CCL-2. Both I3C and DIM inhibited promotional effects of dihydrotestosterone on CCL2
and migration. These results show that androgen may regulate CCL2 and promote inflammatory microenvironment in prostate tumors and that this process can be blocked by broccoli-derived compounds.
Cancer Prev Res; 6(6); 519–29. 2013 AACR.
Introduction
Androgens are functionally required for the normal
growth and development of the prostate gland. In adult
males, androgens promote secretary epithelial cell survival.
However, androgens also promote prostate tumor development and progression (1, 2). Androgen deprivation is the
only clinically effective therapy for advanced prostate cancer. However, because of the relapse of castration-resistant
Authors' Affiliations: 1Nutritional Sciences Research Group, Division of
Cancer Prevention, National Cancer Institute, NIH, Bethesda; 2Department
of Nutrition and Food Science, University of Maryland, College Park; and
3
Diet, Genomics, and Immunology Laboratory, 4Beltsville Human Nutrition
Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland
Note: Current address for E.-K. Kim: Division of Food Bioscience, Department of Natural Science, Konkuk University, Chungju 380-701, Korea; and
current address for J.A. Milner: Beltsville Human Nutrition Research Center,
Agricultural Research Service, U.S. Department of Agriculture, Beltsville,
MD, USA.
Corresponding Author: Thomas T.Y. Wang, U.S. Department of Agriculture, Building 307C, Room 132, BARC-EAST, 10300 Baltimore Ave.,
Beltsville, MD 20705. Phone: 304-504-8459; Fax: 304-504-9062; E-mail:
tom.wang@ars.usda.gov
doi: 10.1158/1940-6207.CAPR-12-0419
2013 American Association for Cancer Research.
androgen-independent tumors, the long-term benefit of
androgen deprivation in patients with metastatic disease
remains controversial (3, 4). Although previous research
shows that many of the biologic effects of androgen are
likely through the regulation of androgen-responsive genes
(ARG) via an androgen receptor (AR)–mediated pathway
(5), some of the molecular effects of androgens in normal
and prostate cancer remain unresolved.
Inflammation as a causal agent has been linked to approximately 20% of human cancers (6). In prostate cancer, a
growing amount of evidence suggests a link between prostate inflammation and subsequent cancer development (712). Previous studies suggest that C-C chemokine ligand 2
(CCL2), also known as monocyte chemoattractant protein1 (MCP-1) may play pivotal role in prostate cancer tumorigenesis and invasion (13, 14). CCL2 is known to attract
monocytes to the site of inflammation and, by binding to its
receptor CCR2, directly stimulates prostate cancer cell proliferation, survival, and migration (15). Prostate cancer cells
LNCaP, C4-2B, PC-3, and VCaP produce CCL2 (16, 17).
Furthermore, recent findings suggest a role for CCL2 in
acquisition of epithelial–mesenchymal transition (EMT)
properties (18). EMT has been shown to be crucial for the
pathogenesis of tissue fibrosis and cancer (19). Prostate
tumor epithelial cells gain the ability to migrate and invade
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by differentiating through activation of biologic pathways
associated with EMT. Androgens were reported to induce
changes that are characteristic of EMT such as expression of
TWIST1, CDH1, and SNAIL; cytoskeleton reorganization;
and promotion of metastatic behavior of castration-resistant prostate cancer cells (20). TWIST1, a helix-loop-helix
transcription factor thought to be an important mediator of
EMT, is upregulated in several types of epithelial cancers
including prostate, breast, and gastric carcinomas (21–24).
Several EMT-related genes, such as CDH1, CDH2, SNAIL,
desmin, and vimentin, are putative downstream targets
regulated by TWIST1. TWIST1 also appears to be involved
in inflammatory pathways (25). TWIST1 expression in Thelper 1 (TH1) lymphocytes and bone marrow–derived
macrophages attenuates the expression of IFN-g, interleukin (IL)-2, and TNF-a, further implicating TWIST1 in the
regulation of cytokine expression (25–27). It was also
reported that CCL2 expression may be subject to regulation
by TWIST1 (28). The relationship between androgens and
these EMT-related proteins as well as the relation between
TWIST1 and CCL2 in prostate cells remains unresolved.
Cancer prevention represents an integral part of a sound
strategy in fight against cancer. Diet-derived compounds are
often the focus due to their perceived safety, indole-3carbinol (I3C) is a putative preventive compound derived
from hydrolysis of glucobrassicin from ingesting cruciferous vegetables such as broccoli, cabbage, and cauliflower
(29). I3C can be converted to the dimeric form 30 ,30 -diindolylmethane (DIM) in stomach (30, 31). Previous studies,
including our own, have shown that the I3C and DIM
inhibit prostate cancer cell growth in vitro and in vivo
(32–34). Both I3C and DIM appeared to exert their effects,
in part, through modulation of androgen-dependent pathways. In addition, these agents have also been reported to
prevent or delay the progression of cancer, through their
ability to attack cancer stem cells or EMT-type cells and
modulate inflammation in cancer cells (35, 36). The effects
of these compounds on prostate cancer–related EMT
remain unknown.
Given the potential roles of androgen in regulating prostate cancer and EMT and existent information on TWIST1
and CCL-2, we hypothesized that androgen may modulate
TWIST1 and, in turn, CCL2 expression in prostate cancer
cells, and these effect may result in promotion of monocyte
recruitment. The present study seeks to resolve these interactions. Finally, given the effects of I3C and DIM on
androgen-dependent pathways (32–34), we also sought to
determine the effects of I3C and DIM on TWIST1, CCL2,
and monocyte migration.
Materials and Methods
Chemicals and reagents
I3C, DIM, dihydrotestosterone (DHT), and dimethyl
sulfoxide (DMSO) were from Sigma Chemical. TRIzol,
AffinityScript Multiple Temperature cDNA Synthesis kit,
and TaqMan real-time PCR primers and probes were
obtained from Life Technology. On-Target plus SMART
pool siRNA targeting AR and TWIST1 were purchased from
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Cancer Prev Res; 6(6) June 2013
Thermo Fisher Scientific. HiPerFect Transfection Reagent
was purchased from Qiagen.
Cell culture
LNCaP and PC-3 human prostate cancer cells were
obtained from the American Type Culture Collection
(ATCC) and maintained in medium A [RPMI-1640 medium
with phenol red and 2 mmol/L L-glutamine (Invitrogen/Life
Technologies), 100 U/mL penicillin, and 100 mg/mL streptomycin (BioSource International) with 10% FBS (Invitrogen/Life Technologies)]. For androgen-related experiments,
cells were seeded as follows: 6-well plates at 250,000 cells/
well. After 24 hours in medium A, cells were switched to
medium B, which had the same composition as medium A
except that FBS was replaced with 10% charcoal dextran–
treated FBS (GIBCO/Life Technologies) for an additional
24 hours to minimize background androgen levels. Cells
were incubated in the presence of 5% CO2 in air at 37 C.
THP-1 cell were also purchased from ATCC and maintained
in RPMI-1640 medium with phenol red and 2 mmol/L
L-glutamine (Invitrogen/Life Technologies), 100 U/mL penicillin, and 100 mg/mL streptomycin (BioSource International) with 10% FBS (Invitrogen/Life Technologies).
RNA isolation and real-time reverse transcription PCR
(RT-PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen/
Life Technologies) and reverse transcribed to cDNA using
AffinityScript Multiple Temperature cDNA Synthesis kit
(Agilent/Life Technologies). Real-time PCR was carried out
using a TaqMan Fast Universal PCR Master Mix on a 7900HT
FAST real-time PCR System (Applied Biosystems/Life Technologies). The amplification parameters used were as follows: 95 C for 20 seconds, followed by 46 cycles of amplification at 95 C for 1 second and 60 C for 20 seconds.
Relative mRNA fold changes to control were calculated using
the comparative CT 2DDCt cycle (DCt) method following
manufacturer’s directions. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous
control for all gene expression analysis calculation.
Determination of gene expression in LNCaP cells using
RT-PCR
To examine the effects of test compounds on TWIST1,
CDH1, CDH2, SNAI1 (SNAIL1), DES (desmin), VIM
(vimentin), KLK3 (PSA), CCL2, or TNF (TNF-a) mRNA
levels, LNCaP cells were plated in 6-well plates (0.25 106
cells/well) in medium A, and after 24 hours, the media was
removed and replaced with fresh media containing vehicle
or varied concentrations of test compounds I3C at 5, 25, 50
mmol/L and DIM at 1, 5, 10 mmol/L. LNCaP cells were plated
in 6-well plates (0.25 106 cells/well) in medium A and
switched to medium B 24 hours after plating to minimize
the effect of serum hormones. The cells were then incubated
in medium B for an additional 24 hours before the treatments began. Twenty-four hours later, the medium was
replaced with fresh medium containing 1 nmol/L dihydrotestosterone with or without test compounds. For all
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I3C and DIM Inhibit Monocyte Attraction
experiments, fresh medium containing the test compounds
was changed daily and cells were harvested for total RNA
isolation using the TRIzol method (Invitrogen/Life Technologies) after 48 hours. TaqMan real-time PCR was used to
quantify expression of the mRNA.
siRNA oligonucleotide studies
siRNAs specifically block expression of AR, or TWIST1
mRNA was used to confirm the role of androgen regulation
of TWIST1, CCL2, and TNF-a mRNA expression. LNCaP
cells were seeded in 6-well plates as described above. Twenty-four hours after androgen deprivation, LNCaP cells were
treated with or without 1 nmol/L dihydrotestosterone. For
siRNA experiments, cells were transfected with 5 nmol/L of
siRNA oligonucleotides targeting AR or TWIST1 using
HiPerFect transfection reagent according to the manufacturer’s protocol. Forty-eight hours after transfection, cell
culture media were replaced with fresh media in the presence or absence of 1 nmol/L dihydrotestosterone for an
additional ours. After treatment, cells were harvested for
total RNA isolation as described above.
Transwell cell migration assay
For transwell migration assays, 2 105 THP-1 cells in 0.2
mL phenol-red free RPMI-1640 (Invitrogen/Life Technologies) were placed into the noncoated membrane top chamber (24-well insert; pore size, 8 mm; Corning Costar) and
allowed to migrate toward LNCaP cell–conditioned media
(1.2 mL) in the lower chamber. After 10 hours of incubation
with the conditioned media, cells migrated to the lower
chamber were collected by centrifugation (1,000 g for 10
minutes), resuspended in 0.1 mL PBS, and the number of
cells counted under a light microscope (Olympus CK40)
using a hemocytometer. To confirmed that CCL2 in conditioned media contributed toward migration of THP-1
cells, condition media were first treated with 0.5 mg/mL
anti-human neutralizing CCL2 antibody (R&D Systems) or
IgG isotype control (BD Pharmingen) for 1 hour and then
migration assays were conducted as above.
ELISA determination of CCL2 and TNF-a protein
LNCaP cell were treated with or without dihydrotestosterone (1 nmol/L) in presence or absence of DIM (10 mmol/
L) or I3C (50 mmol/L) for 48 hours. After incubation,
LNCaP cells conditioned media were removed, lyophilized,
and resuspended in RPMI-1640 phenol red–free media
(Invitrogen/Life Technologies); to yield 200 mg protein/mL,
50 mL was used for detection of CCL2 or TNF-a. Protein
expressions of CCL2 and TNF-a were determined by using
commercially available ELISA kits. EH2MCP1 for CCL-2,
EHTNFA for TNF-a (Thermo Scientific) were used according to the manufacturer’s instructions. The optical density
(OD) was determined at 550 or 450 nm using a multiwell
plate reader (Spectra Max Plus, Molecular Devices).
Statistical methods
Statistical analysis of data was conducted with the GraphPad Prism 4 program (GraphPad Software, Inc.). Multiple
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group data were analyzed using 1- or 2-way ANOVA followed by a Bonferroni post hoc test. An unpaired Student t
test was used to compare experiments between 2 groups.
Gene expression results are expressed as means SEM
relative to vehicle control. Data are representative of 3
independent experiments.
Results
Androgen induces TWIST1 mRNA expression through
an AR-dependent pathway
Dihydrotestosterone (0–1 nmol/L) significantly induced
TWIST1 in a dose- and time-dependent manner (Fig. 1A and
B). As shown in Fig. 1C and D, adding the AR siRNA
effectively inhibited dihydrotestosterone induction of the
well-documented androgen responsive gene PSA (Fig. 1C).
Similarly, AR siRNA also inhibited dihydrotestosterone
induction of TWIST1 mRNA (Fig. 1D).
Androgen differentially regulate putative TWIST1
downstream target genes mRNA expression
Dihydrotestosterone treatment of LNCaP cells led to
differential effects on TWIST1-responsive gene mRNA
expression. EMT-related, TWIST1-responsive genes such as,
CDH1, SNAIL, and VIM were not affected by dihydrotestosterone treatment (Fig. 2A–C), and the expression of DES
and CDH2 was below detection limits under the current
experimental condition. In contrast, CCL2 and TNF, 2
inflammation-related putative TWIST1-responsive genes,
were induced by dihydrotestosterone treatment in a timeand dose-dependent manner (Fig. 2D–G).
Androgen regulation of CCL2 and TNF-a mRNA is
through an AR but not a TWIST1-dependent pathway
Both AR and TWIST1 may both contribute to the upregulation of CCL2 and TNF mRNA levels by dihydrotestosterone treatment. siRNA against AR effectively inhibited
dihydrotestosterone (1 nmol/L)-induced increase in CCL2
and TNF-a mRNA levels (Fig. 3A and B). In contrast, siRNA
against TWIST1 did not affect the expressions of dihydrotestosterone induction of CCL2 or TNF (Fig. 3C and D).
Treatment of cells with siRNA for AR and TWIST1 lead to
about 90% and 80% inhibition of AR and TWIST expression, respectively (Fig. 3E). TWIST1, CCL2, and TNF-a
mRNA expression levels were higher in PC-3 cells than in
LNCaP cells (Fig. 4A), Furthermore, treatment of PC-3 cells
with dihydrotestosterone (1 nmol/L) did not affect the
mRNA expression levels of these genes (Fig. B and C).
Consistent with increase in CCL2 mRNA levels, dihydrotestosterone also lead to an increase in CCL2 protein levels
(Fig. 5A)
Conditioned media from dihydrotestosterone-treated
LNCaP cell increase THP-1 monocyte migration
As shown in Fig. 5B, the number of THP-1 cells migrated
toward dihydrotestosterone-treated LNCaP cells was significantly higher than in those provided the control media.
Adding dihydrotestosterone (1 nmol/L) directly into media
did not lead to increase in migration of THP-1 cells.
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6
Figure 1. Effects of DHT on TWIST1 expression in androgen-responsive LNCaP cells. LNCaP cells (0.25 10 cells/well) were plated in 6-well plates, and after
androgen deprivation, the cells were daily treated with vehicle (DMSO) or DHT and then subjected to RNA isolation and real-time PCR analysis. Real-time PCR
results are expressed as means SE of fold change relative to vehicle-treated control of 3 separate experiments with each experiment including
triplicate PCR tubes. Error bars with an asterisk are significantly different than vehicle-treated control ( , P < 0.05; , P < 0.01; , P < 0.001). A,
concentration-dependent effects of DHT on TWIST1 mRNA levels. The cells were treated daily with or without varied concentrations (0–1.0 nmol/L) of
DHT for 48 hours. B, time course of DHT effects on TWIST1 mRNA levels. LNCaP cells were daily treated with or without 1 nmol/L DHT for 0, 24, 48, or 72 hours.
C and D, effects of AR siRNA on TWIST1 and PSA mRNA levels. LNCaP cells were transfected with 5 nmol/L AR siRNA or negative control siRNA as
described in the Materials and Methods. After transfection, the cells were treated with or without DHT (1 nmol/L) for 48 hours as described in Materials
and Methods. After treatment, total RNA was isolated and mRNA for PSA and TWIST1 determined as described in Materials and Methods. Real-time
PCR results are expressed as means SE of fold change relative to vehicle-treated control of 3 separate experiments with each experiment including triplicate
PCR tubes. Error bars with an asterisk are significantly different than vehicle-treated control ( , P < 0.05; , P < 0.01; , P < 0.001). C, effect of AR siRNA on
DHT induction of TWIST1 mRNA levels. D, effect of AR siRNA on DHT induction of PSA mRNA levels. NC, negative control siRNA.
Pretreatment of conditioned media with a neutralizing
antibody against CCL2 was found to attenuate increased
migration of THP-1 cells toward dihydrotestosterone-treated conditioned media (Fig. 5C).
I3C and DIM modulate effects of androgen on CCL2 and
migration
Consistent with their effect on androgen-dependent
pathways, I3C and DIM exerted a concentration-dependent
inhibition of both TWIST1 and CCL2 mRNA expression
induced by dihydrotestosterone treatments. This response
was similar to the effects that I3C and DIM had on androgen
induction of the well-documented androgen-responsive
gene PSA mRNA (Fig. 6A–C). Furthermore, treatment of
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Cancer Prev Res; 6(6) June 2013
cells with I3C (50 mmol/L) and DIM (10 mmol/L) also
inhibited the dihydrotestosterone-induced increase in
CCL2 protein (Fig. 6D and E). Furthermore, I3C (50
mmol/L) or DIM (10 mmol/L) treatment also led to significantly reduction of dihydrotestosterone-induced migration of THP-1 cells (Fig. 6F and G).
Discussion
The molecular mechanisms underlying the etiology
and progression of prostate cancer remain unclear.
Although exposure to androgen is considered as one of the
risk factor for prostate cancer, the mechanisms are also not
completely delineated. In the present study, we reported a
novel finding that CCL2, a protein critical in attracting
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I3C and DIM Inhibit Monocyte Attraction
Figure 2. Effects of DHT on
TWIST1-related genes expression in
androgen-responsive LNCaP cells.
6
LNCaP cells (0.25 10 cells/well)
were plated in 6-well plates, and after
androgen deprivation, the cells were
daily treated with vehicle or DHT for
48 hours and then subjected to RNA
isolation and real-time PCR analysis.
Real-time PCR results are expressed
as means SE of fold change
relative to vehicle-treated control of 3
separate experiments with each
experiment including triplicate PCR
tubes. Error bars with an asterisk are
significantly different than vehicletreated control ( , P < 0.05;
, P < 0.01; , P < 0.001).
A, concentration-dependent effects
of DHT on CDH1 mRNA levels.
B, concentration-dependent effects
of DHT on SNAIL mRNA levels.
C, concentration-dependent effects
of DHT on vimentin mRNA levels.
D, concentration-dependent effects
of DHT on CCL2 mRNA levels.
E, concentration-dependent effects
of DHT on TNF-a mRNA levels.
F, time course of DHT effects on
CCL2 mRNA levels. G, time course of
DHT effects on TNF-a mRNA levels.
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Figure 3. Effects of siRNA against
AR and TWIST1 on DHT induction
of CCL2 and TNF-a mRNA
expression in LNCaP cells. LNCaP
cells were transfected with 5 nmol/
L AR siRNA, TWIST1 siRNA, or
negative control siRNA as
described in the Materials and
Methods. After transfection, the
cells were treated with or without
DHT (1 nmol/L) for 48 hours as
described in Materials and
Methods. After treatment, total
RNA was isolated and mRNA for
AR, TWIST1, CCL-2, and TNFa determined as described in
Materials and Methods. Real-time
PCR results are expressed as
means SE of fold change relative
to vehicle-treated control of 3
separate experiments with each
experiment including triplicate
PCR tubes. Error bars with an
asterisk are significantly different
than vehicle-treated control
( , P < 0.05; , P < 0.01; ,
P < 0.001). A, effects of AR siRNA
on DHT induction of CCL2 mRNA
expression. B, effects of AR siRNA
on DHT induction of TNF-a mRNA
expression. C, effects of TWIST1
siRNA on DHT induction of CCL2
mRNA expression. D, effects of
TWIST1 siRNA on DHT induction of
TNF-a mRNA expression. E,
effects of AR or TWIST1 siRNA on
AR (left) and TWIST1 (right) mRNA
expression. NC, negative control
siRNA.
monocytes (37, 38), is an androgen-responsive gene. We
show that exposure of the androgen-responsive LNCaP cell
line to dihydrotestosterone led to induction of CCL2 mRNA
as well as protein (Fig. 5A), and this effect of dihydrotestosterone on CCL2 mRNA was blocked by siRNA against AR
(Fig. 3A). This observation supports the notion that exposure to androgens can promote attraction of monocytes by
prostate cancer cells. We provide in vitro data (Fig. 5C) to
support this notion as we observed increased migration of
THP-1 toward conditioned media derived from dihydro-
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Cancer Prev Res; 6(6) June 2013
testosterone-treated cells (Fig. 5B). The involvement of
CCL2 was further strengthened because THP-1 cell migration was inhibited when conditioned media were preincubated with anti-CCL-2 antibody (Fig. 5C). These results
suggest that exposure of prostate cancer cells to androgens
may create a pro-inflammatory environment, where the
prostate cancer cell is stimulated to secrete CCL2 and attract
monocytes toward tumor site. Monocytes can produce
cytokines such as IL-6 and IL-1b (39). These cytokines
are known to stimulate prostate cancer cell growth and
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I3C and DIM Inhibit Monocyte Attraction
Figure 4. Baseline and DHTinduced mRNA levels of TWIST1,
CCL-2, and TNF-a in androgenresponsive LNCaP and
nonresponsive PC-3 cells. LNCaP
or PC-3 cells were plated in 6-well
plates, and after androgen
deprivation, the cells were daily
treated with vehicle or DHT for 48
hours and then subjected to RNA
isolation and real-time PCR analysis.
Real-time PCR results are expressed
as means SE of fold change
relative to vehicle-treated control of 3
separate experiments with each
experiment including triplicate PCR
tubes. Error bars with an asterisk are
significantly different than vehicletreated control ( , P < 0.001,
, p < 0.01). A, relative baseline
mRNA levels of TWIST1, CCL-2, and
TNF- a in LNCaP and PC-3 cells.
B, effect of DHT on genes expression
in androgen-responsive LNCaP
cells. C, effects of DHT on genes
expression in androgennonresponsive PC-3 cells.
angiogenesis (40). Therefore, upregulation of CCL2 may be
a mechanism whereby androgens promote prostate cancer
development. We also observed that the androgen-independent prostate cancer cell PC-3 expresses higher levels of
CCL2 mRNA. PC-3 cell is considered to be more advanced
than LNCaP cell in prostate cancer tumorigenesis process.
The higher expression of CCL2 in PC-3 cells than in LNCaP
cells is consistent with the more aggressive phenotype of
PC-3 and suggests that they may have a greater capacity for
monocyte recruitment. This will lead to increased inflammation in the tumor site. One may thus reason that antiinflammatory agents targeting the CCL2 pathway may delay
tumor growth in both androgen-dependent and -independent prostate tumors.
One of our initial hypotheses was that dihydrotestosterone could modulate EMT. EMT induction in cancer cells
results in the acquisition of invasive and metastatic properties. EMT-type cells, which share molecular characteristics
with cancer stem cells (CSC), are believed to play critical
roles in early cancer metastasis and drug resistance as shown
in several human malignancies including prostate cancer
(41–43). We found that LNCaP cells exposed to dihydrotestosterone significantly upregulated the EMT-related pro-
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tein TWIST1 mRNA levels. This effect of dihydrotestosterone on TWIST1 is dependent on AR as siRNA against AR
blocked the induction of TWIST1 mRNA by dihydrotestosterone (Fig. 1). TWIST1, a helix-loop-helix transcription
factor, is highly expressed in many types of human cancers
(44). Recently, TWIST1 was suggested to be an oncogene
(23, 45, 46) and confers prostate cancer cells with an
enhanced metastatic potential through promoting EMT,
and a high TWIST1 expression in human prostate cancer
is associated with an increased metastatic potential (47).
However, we did not find dihydrotestosterone to effect the
TWIST1-regulated and/or EMT-related genes such as CDH1,
SNAIL, and vimentin expression in LNCaP cells (Fig. 2A–C).
These results suggest that although androgens induced
TWIST1, they did not lead to development of an EMT
phenotype in our experimental conditions. TWIST1 was
also reported to be a regulator of CCL2 (48). We did found
that dihydrotestosterone treatment increased the mRNA of
2 purported TWIST11-responsive genes, CCL2 and TNF-a.
TNF-a is also a potent chemoattractant for several cell lines
(49). However, the effect of androgen on CCL2 and TNF-a
mRNA appeared to be AR-dependent pathway but not
TWIST1–dependent. AR siRNA and not TWIST1 siRNA
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Figure 5. Effect of DHT on CCL2 protein and LNCaP cell conditioned media–stimulated THP-1 monocyte migration. LNCaP cells were treated with or without 1
nmol/L DHT for 48 hours, conditioned media–harvested and CCL2 protein level, THP-1 migration assay conducted as described in Materials and Methods.
The results are expressed as means SE of fold change relative to vehicle-treated control of 3 separate experiments. Error bars with an asterisk are
significantly different than vehicle-treated control ( , P < 0.01; , P < 0.001. as analyzed by Bonferroni posttest). A, protein expressions of CCL2 in LNCaP
cells conditioned media. Error bars with an asterisk are significantly different than vehicle-treated control ( , P < 0.01; , P < 0.001). B, effect of conditioned
media on THP-1 cells migration. Error bars with an asterisk are significantly different than vehicle-treated control ( , P < 0.01; , P < 0.001). C, effect of CCL2blocking antibody on conditioned media–induced THP-1 migration. LNCaP cells conditioned media were treated with 0.5 mg/mL isotype control or CCL2
blocking antibody for 1 hour and then the THP-1 migration assay was conducted as described in Materials and Methods. Values with a different superscripts
(a, b, and c) are significantly different at P < 0.05.
blocked the effects of dihydrotestosterone on CCL2 and
TNF-a mRNA. These results suggest that TWIST1 is not
involved in the regulation of CCL2 or TNF-a mRNA by
dihydrotestosterone in LNCaP cells. Additional studies
are necessary to elucidate the role of dihydrotestosterone
in EMT as well as TWIST1 in the prostate cancer–EMT
relationship.
We previously reported that I3C and DIM can inhibit
LNCaP cell growth that correlated with inhibition of androgen-dependent pathways (33). Consistent with this observation, cells treated with I3C and DIM also significantly
blocked the induction of CCL2 mRNA by dihydrotestosterone (Fig. 6C). In addition, I3C and DIM treatment also
led to the inhibition of CCL2 protein expression induced by
dihydrotestosterone as well as migration of THP-1 monocytes induced by dihydrotestosterone (Fig. 6F and G). These
results indicate that exposure of cells to I3C and DIM not
only modulates the growth of androgen-responsive prostate
526
Cancer Prev Res; 6(6) June 2013
cancer cells but also indirectly influences luminal cells such
as the monocytes. We reason that I3C and DIM, through
inhibition of monocyte to the tumor site, could minimize
the exposure of tumors to cytokines such as IL-1b and IL-6.
This effect may thus attenuate the inflammatory process and
ultimately lead to delay in prostate tumor growth and
angiogenesis. Our data also support the possibility that diet
or diet-derived compounds may act through multiple
mechanisms including inflammation and angiogenesis to
prevent prostate cancer. Additional in vivo studies are warranted to validate our hypothesis. TWIST1, an androgenresponsive gene, was also modulated by I3C and DIM (Fig.
6B). The consequence of TWIST1 modulation by I3C and
DIM remains unclear and warrants further study.
In summary, we found that the androgen dihydrotestosterone promotes monocyte migration through upregulation of CCL2 mRNA and protein. The effect of dihydrotestosterone on CCL2 appeared to be independent of TWIST1
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Published OnlineFirst April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
I3C and DIM Inhibit Monocyte Attraction
Figure 6. Effect of I3C and DIM on
DHT induced gene expression in
LNCaP cells and conditioned media
stimulated THP-1 cell migration.
LNCaP cells were treated with or
without 1 nmol/L DHT for 48 hours,
cell harvested for total RNA isolation
and gene expression analysis as
described in Materials and Methods.
Conditioned media were harvested
for CCL2 protein ELISA and THP-1
migration assay as described in
Materials and Methods. The results
are expressed as means SE of fold
change relative to vehicle-treated
control of 3 separate experiments. A,
I3C and DIM inhibition of DHTinduced PSA expression. B, I3C and
DIM inhibition of DHT-induced
TWIST1 mRNA expression. C, I3C
and DIM inhibition of DHT-induced
CCL2 mRNA expression. D, I3C
repressed the protein expression of
CCL2 in DHT-treated LNCaP media.
E, DIM repressed the protein
expression of CCL2 in DHT-treated
LNCaP media. F, I3C suppressed
THP-1 migration in DHT-treated
LNCaP conditioned media. G, DIM
suppressed THP-1 migration in DHTtreated LNCaP conditioned media.
Error bars with an asterisk are
significantly different than
vehicle-treated control ( , P < 0.05;
, P < 0.01; , P < 0.001).
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Cancer Prev Res; 6(6) June 2013
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527
Published OnlineFirst April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
Kim et al.
and mediated through AR-dependent pathways. More
importantly, the diet-derived factors I3C and DIM can
modulate CCL2 and monocyte migration induced by dihydrotestosterone. These may be important mechanisms
whereby diet-derived compound delay prostate tumor
growth and development. Finally, we found that dihydrotestosterone induced EMT-related TWIST1 expression but
had no effect on other EMT-related genes downstream of
TWIST1.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): E.-K. Kim
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E.-K. Kim, J.A. Milner, T.T.Y. Wang
Writing, review, and/or revision of the manuscript: E.-K. Kim, J.A.
Milner, T.T.Y. Wang
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.-K. Kim, Y.S. Kim, J.A. Milner
Study supervision: Y.S. Kim, J.A. Milner
Grant Support
This work was supported by U.S appropriated funds to USDA project
number 1235-51530-053-00D (to T.T.Y. Wang) and the National Cancer
Institute (to E.-K. Kim, Y.S. Kim, J.A. Milner).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Authors' Contributions
Conception and design: E.-K. Kim, Y.S. Kim, J.A. Milner, T.T.Y. Wang
Development of methodology: E.-K. Kim, T.T.Y. Wang
Received October 10, 2012; revised March 4, 2013; accepted March 20,
2013; published OnlineFirst April 12, 2013.
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Published OnlineFirst April 12, 2013; DOI: 10.1158/1940-6207.CAPR-12-0419
Indole-3-Carbinol and 3′,3′-Diindolylmethane Modulate Androgen's
Effect on C-C Chemokine Ligand 2 and Monocyte Attraction to
Prostate Cancer Cells
Eun-Kyung Kim, Young S. Kim, John A. Milner, et al.
Cancer Prev Res 2013;6:519-529. Published OnlineFirst April 12, 2013.
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