# Modulation of CXCR4, CXCL12, and Tumor Cell Invasion Potential In Vitro by Phytochemicals

Authors: Erin L. Hsu; Natalie Chen; Aya Westbrook; Feng Wang; Ruixue Zhang; Robert T. Taylor; Oliver Hankinson
Journal: Journal of Oncology
 (2009)

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## Abstract

CXCR4 is a chemokine receptor frequently overexpressed on primary tumor cells. Organs to which these cancers metastasize secrete CXCL12, the unique ligand for CXCR4, which stimulates invasion and metastasis to these sites. Similar to our previous work with the chemoprotective phytochemical,  3,3′-diindolylmethane (DIM), we show here that genistein also downregulates CXCR4 and CXCL12 and subsequently lowers the migratory and invasive potentials of breast and ovarian cancer cells. Moreover, genistein and DIM elicit a significantly greater cumulative effect in lowering CXCR4 and CXCL12 levels than either compound alone. Our data suggest a novel mechanism for the protective effects of phytochemicals against cancer progression and indicate that in combination, these compounds may prove even more efficacious.


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## Body

## 1. Introduction

Phytochemicals
have long been implicated in a protective effect for cancer.  Soybean- and cruciferous vegetable-enriched
diets seem to be particularly protective, but the mechanisms for this
anticarcinogenicity are not fully understood. Genistein, a dietary
phytoestrogen belonging to the isoflavone class of flavonoids, is thought to
have anticarcinogenic activities, particularly for breast and prostate cancer
[1, 2].  Dietary soy has been shown in
mice to inhibit prostate tumor growth through inhibition of cell proliferation,
increased apoptosis, and reduced microvessel density [3].  Epidemiology studies of Asian women indicate
that consumption of a traditional diet high in soy confers significant
protection against breast cancer [4].3,3′-diindolylmethane
(DIM), a breakdown product of glucobrassicins, which are found in cruciferous
vegetables, has been shown in vivo to have protective effects for breast
cancer [5].  We have shown previously
that DIM lowers the levels of CXCR4 and CXCL12, a chemokine receptor and its
unique ligand required for the metastasis of breast cancer [6, 7]. In
addition to mediating the directional homing of primary breast cancer cells to
secondary organ sites, CXCR4 and CXCL12 are important in other aspects of
cancer progression, such as adhesion, proliferation, and angiogenesis.  These effects are not limited to breast
cancer, as the interaction between CXCR4 and CXCL12 is implicated in the
progression of many different types of cancer.The
biological effects of genistein are extensive and include antioxidant activity,
weak estrogenic/antiestrogenic activity, upregulation of apoptosis, inhibition
of angiogenesis, inhibition of DNA topoisomerase II, and inhibition of protein
tyrosine kinases (PTKs).  Genistein has
been shown to regulate specific sex steroid receptors, inhibit NFkB,
downregulate TGF-β, and inhibit EFG-stimulated growth.  Furthermore, genistein inhibits
2,3,7,8-tetrachlorodibenzo-p-dioxin
(dioxin)-induced CYP1A1 activity, and isoflavones can prevent the
CYP1A1-mediated binding of benzo[a]pyrene (B[a]P) metabolites to DNA [8].Our
previous observations that DIM downregulates CXCR4 and CXCL12 in breast and
ovarian cancer cells represent a novel mechanism for the chemoprotective
effects of this phytochemical.  Here, we
demonstrate that these effects are not unique to DIM, but can also be seen with
genistein.  Interestingly, we see that
the combined effect of DIM and genistein elicits a greater downregulation of
CXCR4 and CXCL12 than either compound alone, indicating that the phytochemicals
used in combination may be even more potent in their chemoprotective
properties. We also demonstrate that like DIM, genistein specifically inhibits
chemotaxis and chemoinvasion of breast and ovarian cancer cells toward CXCL12 in vitro.


## 2. Materials and Methods

### 2.1. Chemical Reagents and Cell Culture

Genistein was purchased from Sigma (St. Louis, Mo, USA), DIM was
purchased from LKT Laboratories (St. Paul, Minn, USA), and the remaining
phytochemicals were a kind gift from Dr. David Heber (University of California,
Los Angeles, Calif, USA).  BG-1 cells were generously
provided by Dr. Kenneth Korach (National Institute of Environmental Health
Sciences, NC, USA).  MCF-7 and MDA-MB-231
cells were purchased from the American Type Culture Collection (ATCC, Manassas,
Va, USA).  MCF-7 and BG-1 cells were maintained
in Minimal Essential Medium.  MDA-MB-231
cells were maintained in Dulbecco's
Modified Eagle's Medium containing 4 mM L-glutamine. All media were purchased from Invitrogen
(Carlsbad, Calif, USA) and supplemented with 10% fetal bovine serum (FBS; Omega,
Tarzana, Calif, USA), 100 U/mL penicillin/100μg/mL
streptomycin solution
(Gemini Bio-Products, West Sacramento, Calif,
USA), and 0.25 U/mL Amphotericin B (Omega).
Cells were maintained at 37°C under 5% CO2.  For all experiments, chemicals were dissolved in DMSO and administered to cells with a
final concentration of DMSO at 0.1% in the medium.


### 2.2. RNA Extraction, cDNA Synthesis, and Real-Time PCR

RNA isolation, cDNA
synthesis, and taqman multiplex real-time
PCR were performed as previously described [6].
CXCR4 and CXCL12 cDNAs were amplified using Assays on Demand (Affinity Bioreagents, Golden, Colo, USA; product
numbers Hs00607978_s1 and Hs00171022_m1, resp.; sequences proprietary).  Quantities were normalized to those for the 36B4
ribosomal housekeeping gene. The forward and reverse primers used for 36B4
quantification were 5′-CCACGGTGCTGAACATGCT-3′ and 5′-TCGAACACCTGCTGGATGAC-3′,
respectively.  The 36B4 probe sequence
was 5′-Texas Red-ACCATCTCCCCCTTCTCCTTTGGGCT-Iowa Black-3′. All primers and
probes were purchased from Integrated DNA Technologies (Coralville, Iowa, USA).  Real-time PCR was carried out using the
ICycler IQ (BioRad, Hercules, Calif, USA) or 7500 Fast (Applied Biosystems,
Foster City, Calif, USA) under standard protocols.  Data were analyzed using the ICycler or ABI
software and Microsoft Excel, and significance was evaluated using Student’s t-test.


### 2.3. Flow Cytometry

For surface staining of CXCR4 and
intracellular staining of CXCL12, cells were grown to 70% confluence and
treated with genistein for 24 or 48 hours.
In the case of CXCL12 quantification, the cells were also cotreated with
1μg/mL Brefeldin A (GlogiPlug; BD
PharMingen, Franklin Lakes, NJ, USA) for the final six hours of incubation to
inhibit protein secretion.  Cells were
harvested and stained as previously described [6, 9] with either primary CXCL12
antibody (R&D Cat. no. MAB350; Minneapolis, Minn, USA), primary CXCR4
antibody (Affinity Bioreagents Cat. no. OPA1-01101; Ill, USA) or the
appropriate primary IgG isotype control antibody, followed by staining with
either goat antimouse or antirabbit IgG-FITC secondary antibodies
(BD-PharMingen and Caltag, Carlsbad, Calif, USA, resp.).  Fluorescence was quantified using a FACScan analytic
flow cytometer (Becton Dickinson, UCLA Flow Cytometry Core Facility).  Data were analyzed using FCS Express3 Lite
Software (DeNovo, Inc., Thornhill, ON, Canada).


### 2.4. Chemotaxis and Chemoinvasion Assays

Cells
were pretreated with genistein, DIM, or the combination, followed by chemotaxis
and invasion assays, performed as we have described previously [6].  After migration/invasion, MTS assays
(Promega, Madison, Wis) were performed in each individual transwell to control
for small variations in cell number.
After detection of the formazan product at 490 nm, cells on the inserts
were washed with PBS and those on the upper layer were gently removed with a
prewet Q-tip.  Cells on the lower layer
were fixed in 100% methanol and stained with crystal violet.  Membranes were manually excised from the
inserts, mounted on microscope slides, and divided into 8 equal sections.  Cells in one random viewing field from each
section were counted at 40x magnification, and the average was calculated.  Average counts from each insert were normalized
to the equivalent MTS values.  Data are
expressed as chemotaxis or chemoinvasion indices, which were defined as the
normalized number of cells in the experimental group relative to the control
group.  Statistical analyses were
performed using a two-tailed Student’s t-test.


## 2.1. Chemical Reagents and Cell Culture

Genistein was purchased from Sigma (St. Louis, Mo, USA), DIM was
purchased from LKT Laboratories (St. Paul, Minn, USA), and the remaining
phytochemicals were a kind gift from Dr. David Heber (University of California,
Los Angeles, Calif, USA).  BG-1 cells were generously
provided by Dr. Kenneth Korach (National Institute of Environmental Health
Sciences, NC, USA).  MCF-7 and MDA-MB-231
cells were purchased from the American Type Culture Collection (ATCC, Manassas,
Va, USA).  MCF-7 and BG-1 cells were maintained
in Minimal Essential Medium.  MDA-MB-231
cells were maintained in Dulbecco's
Modified Eagle's Medium containing 4 mM L-glutamine. All media were purchased from Invitrogen
(Carlsbad, Calif, USA) and supplemented with 10% fetal bovine serum (FBS; Omega,
Tarzana, Calif, USA), 100 U/mL penicillin/100μg/mL
streptomycin solution
(Gemini Bio-Products, West Sacramento, Calif,
USA), and 0.25 U/mL Amphotericin B (Omega).
Cells were maintained at 37°C under 5% CO2.  For all experiments, chemicals were dissolved in DMSO and administered to cells with a
final concentration of DMSO at 0.1% in the medium.


## 2.2. RNA Extraction, cDNA Synthesis, and Real-Time PCR

RNA isolation, cDNA
synthesis, and taqman multiplex real-time
PCR were performed as previously described [6].
CXCR4 and CXCL12 cDNAs were amplified using Assays on Demand (Affinity Bioreagents, Golden, Colo, USA; product
numbers Hs00607978_s1 and Hs00171022_m1, resp.; sequences proprietary).  Quantities were normalized to those for the 36B4
ribosomal housekeeping gene. The forward and reverse primers used for 36B4
quantification were 5′-CCACGGTGCTGAACATGCT-3′ and 5′-TCGAACACCTGCTGGATGAC-3′,
respectively.  The 36B4 probe sequence
was 5′-Texas Red-ACCATCTCCCCCTTCTCCTTTGGGCT-Iowa Black-3′. All primers and
probes were purchased from Integrated DNA Technologies (Coralville, Iowa, USA).  Real-time PCR was carried out using the
ICycler IQ (BioRad, Hercules, Calif, USA) or 7500 Fast (Applied Biosystems,
Foster City, Calif, USA) under standard protocols.  Data were analyzed using the ICycler or ABI
software and Microsoft Excel, and significance was evaluated using Student’s t-test.


## 2.3. Flow Cytometry

For surface staining of CXCR4 and
intracellular staining of CXCL12, cells were grown to 70% confluence and
treated with genistein for 24 or 48 hours.
In the case of CXCL12 quantification, the cells were also cotreated with
1μg/mL Brefeldin A (GlogiPlug; BD
PharMingen, Franklin Lakes, NJ, USA) for the final six hours of incubation to
inhibit protein secretion.  Cells were
harvested and stained as previously described [6, 9] with either primary CXCL12
antibody (R&D Cat. no. MAB350; Minneapolis, Minn, USA), primary CXCR4
antibody (Affinity Bioreagents Cat. no. OPA1-01101; Ill, USA) or the
appropriate primary IgG isotype control antibody, followed by staining with
either goat antimouse or antirabbit IgG-FITC secondary antibodies
(BD-PharMingen and Caltag, Carlsbad, Calif, USA, resp.).  Fluorescence was quantified using a FACScan analytic
flow cytometer (Becton Dickinson, UCLA Flow Cytometry Core Facility).  Data were analyzed using FCS Express3 Lite
Software (DeNovo, Inc., Thornhill, ON, Canada).


## 2.4. Chemotaxis and Chemoinvasion Assays

Cells
were pretreated with genistein, DIM, or the combination, followed by chemotaxis
and invasion assays, performed as we have described previously [6].  After migration/invasion, MTS assays
(Promega, Madison, Wis) were performed in each individual transwell to control
for small variations in cell number.
After detection of the formazan product at 490 nm, cells on the inserts
were washed with PBS and those on the upper layer were gently removed with a
prewet Q-tip.  Cells on the lower layer
were fixed in 100% methanol and stained with crystal violet.  Membranes were manually excised from the
inserts, mounted on microscope slides, and divided into 8 equal sections.  Cells in one random viewing field from each
section were counted at 40x magnification, and the average was calculated.  Average counts from each insert were normalized
to the equivalent MTS values.  Data are
expressed as chemotaxis or chemoinvasion indices, which were defined as the
normalized number of cells in the experimental group relative to the control
group.  Statistical analyses were
performed using a two-tailed Student’s t-test.


## 3. Results

### 3.1. Genistein Downregulates CXCR4 and CXCL12 in Breast and Ovarian Cancer Cells

MCF-7 and MDA-MB-231 breast cancer cells and
BG-1 ovarian cancer cells were treated with concentrations of genistein ranging
from 1–100μM (at a
constant final DMSO concentration) for 24 hours, after which time CXCR4 and
CXCL12 mRNAs were quantified by real-time PCR.
CXCR4 mRNA levels were significantly decreased at 1 μM genistein in all
three cell lines (Figures 1(a)–1(c)).  Maximal downregulation was seen at 100 μM, 50 μM, and 30 μM genistein in each cell line, respectively. Significant
downregulation of CXCL12 mRNA occurred at a concentration of 50 μM genistein in
MCF-7 cells and 10 μM genistein in BG-1 cells (Figures 1(a)–1(c)). Interestingly,
low doses of genistein (1–10 μM)
significantly increased CXCL12 mRNA
levels in MCF-7 cells, but had the opposite effect in BG-1 cells at these
doses.  MDA-MB-231 cells do not express
detectable levels of CXCL12.Dose-response curves for CXCR4 and
CXCL12 mRNAs after treatment of breast and ovarian cancer cells with 1–10μM genistein. (a) CXCR4 and CXCL12 mRNA quantification after treatment of MCF-7 cells with
increasing concentrations of genistein for 24 hours. (b) CXCR4 quantification after
treatment of MDA-MB-231 cells with genistein for 24 hours. MDA-MB-231 cells do
not express CXCL12 as determined by real-time PCR. (c) CXCR4 and CXCL12
quantification after treatment of BG-1 cells with increasing doses of genistein
for 24 hours. All data were normalized to corresponding mRNA quantities for the
36B4 housekeeping gene.  P*<.05, P**<.01, P***<.001; relative to 0 μM genistein
control.
(a)
MCF-7(b)
MDA-MB-231(c)
BG-1Flow
cytometric analysis was used to quantify surface CXCR4 and intracellular CXCL12
expression.  Cells were treated with 30,
70, or 100μM genistein for 24 hours, harvested, stained, and analyzed for
surface expression levels of CXCR4.
Relative to DMSO-treated controls, CXCR4 was found to be downregulated
by genistein in MCF-7, MDA-MB-231, and BG-1 cells at all three doses (Figures 2(a)–2(c)).  Analysis of CXCL12 intracellular expression
showed that relative to DMSO-treated control cells, CXCL12 levels were reduced
by all three doses of genistein in both MCF-7 and BG-1 cells (Figures 2(a), 2(c)).Genistein downregulates CXCR4 surface expression and intracellular
CXCL12 expression. Downregulation of
CXCR4 protein expression in MCF-7, MDA-MB-231, and BG-1 cells at concentrations
of genistein ranging from 0–100 μM for a 24-hour
period (a)–(c).  Downregulation of intracellular CXCL12
protein expression in (a) MCF-7 and (c)
BG-1 cells at 30–100 μM genistein.
Fluorescence distribution plots depict one representative experiment.  Isotype controls are represented by filled
histograms. Mean fluorescence intensities (MFI) and their corresponding
standard deviations are derived from three independent experiments.
(a)
MCF-7(b)
MDA-MB-231(c)
BG-1


### 3.2. Downregulation of CXCR4 and CXCL12 Is Greater after Treatment with Both Genistein and DIM than with Either Compound Alone

We previously found that DIM downregulates CXCR4 and CXCL12 mRNAs and proteins in invasive breast and ovarian
cancer cells [6].  We therefore tested
whether the downregulation elicited by cotreatment with both DIM and genistein
was additive or synergistic in these cell lines.  Using real-time PCR, we quantified gene
expression after treatment of MDA-MB-231 (CXCR4 only) or BG-1 (both CXCR4 and
CXCL12) with 20 μM DIM, 100 μM genistein, or the combination. We found that in
both cell lines, the degree of downregulation of CXCR4 was greater after
treatment with both phytochemicals in combination (Figures 3(a)-3(b)).  In MDA-MB-231 cells, DIM reduced levels of
CXCR4 by 57%, genistein by 51%, and the combination by 85% (Figure 3(a)).  In BG-1 cells, DIM downregulated CXCR4 by
49%, genistein by 79%, and DIM + genistein by 90%.  Similarly, DIM lowered the levels of CXCL12
by 50%, genistein lowered levels by 84%, and genistein by 91% (Figure 3(b)).  These effects cannot be attributed to
cytotoxicity, since neither DIM, genistein, nor the combination was cytotoxic
at these doses for the 24 hour time period (data not shown).In combination, genistein and DIM are more efficacious in the
downregulation of CXCR4 and CXCL12 mRNAs than either compound alone. The effect of downregulation of CXCR4 in (a) MDA-MB-231 and (b) BG-1 cells by 20 μM DIM and 100 μM genistein in combination is
greater than their individual effects. The same is seen in (b) BG-1 cells after CXCL12 quantification.  Ctl = DMSO alone; P**<.01, P***<.001.
(a)
MDA-MB-231(b)
BG-1


### 3.3. Genistein Specifically Inhibits Chemotaxis and Chemoinvasion of Breast and Ovarian Cancer Cells toward CXCL12

Since
CXCR4 and CXCL12 mediate directional migration, and since we saw previously
that DIM inhibited chemotaxis and chemoinvasion of these cells, we performed
assays to determine whether genistein could inhibit migration through
fibronectin and/or invasion through matrigel. We found that pretreatment with
genistein inhibited the directional migration of MCF-7 cells, with a resulting
migration rate similar to the background rate (DMSO-treated cells exposed to no
CXCL12 gradient, Figure4(a)).  However,
we did not see a further reduction in migration after cotreatment with
genistein and DIM, presumably because both chemicals used independently at
these doses fully reduced migration to background levels. This result indicates
that the degree to which genistein and DIM downregulate surface CXCR4 levels at
these doses may be sufficient to significantly impact the homing of breast
cancer cells to areas of high CXCL12 expression.  As we have seen previously with DIM, the
inhibition of chemotaxis by genistein specifically affects migration toward
CXCL12, since an inhibition of MCF-7 cell chemotaxis toward FBS was not
observed.DIM
specifically inhibits chemotaxis and chemoinvasion of breast and ovarian cancer
cells toward CXCL12. (a) Quantification of chemotaxis of MCF-7
cells toward CXCL12 or FBS after treatment with DMSO alone (Ctl), 20 μM DIM,
70 μM genistein, or the combination of DIM + genistein for 24 hours.  (b)-(c) Chemotaxis and chemoinvasion of
MDA-MB-231 cells toward CXCL12 or IL-6 were quantified after pretreatment with
DIM and/or genistein as in (a). (d) Quantification of chemoinvasion
toward CXCL12 or FBS after treatment of BG-1 cells with DIM and/or
genistein.  P*<.05, P**<.01, P***<.001; compared with control + chemoattractant.
(a)
Chemotaxis of MCF-7 cells(b)
Chemotaxis of MDA-MB-231 cells(c)
Chemoinvasion of MDA-MB-231 cells(d)
Chemoinvasion of BG-1 cellsMCF-7
cells do not invade through matrigel (a synthetic extracellular matrix), and we were therefore unable to
evaluate invasive potential in these cells.
However, MDA-MB-231 breast cancer cells are invasive and we were able to
quantify both chemotactic and chemoinvasive potential in this cell line.  We found that genistein, DIM, and the
combination significantly inhibited chemotaxis and chemoinvasion of these cells
toward CXCL12, but not toward IL-6, a known in vitro chemoattractant for MDA-MB-231 cells (Figures4(b)-4(c)).  This result indicates that the inhibitory
effects of DIM and genistein are specific for CXCL12-induced
chemoattraction/chemoinvasion and is not merely a general effect on migration
or invasion. A similar result was seen with BG-1 ovarian cancer cells, which
are also invasive in vitro
(Figure 4(d)). These cells do not migrate through matrigel toward IL-6 (data not shown), but did exhibit moderate chemoinvasion toward FBS.


## 3.1. Genistein Downregulates CXCR4 and CXCL12 in Breast and Ovarian Cancer Cells

MCF-7 and MDA-MB-231 breast cancer cells and
BG-1 ovarian cancer cells were treated with concentrations of genistein ranging
from 1–100μM (at a
constant final DMSO concentration) for 24 hours, after which time CXCR4 and
CXCL12 mRNAs were quantified by real-time PCR.
CXCR4 mRNA levels were significantly decreased at 1 μM genistein in all
three cell lines (Figures 1(a)–1(c)).  Maximal downregulation was seen at 100 μM, 50 μM, and 30 μM genistein in each cell line, respectively. Significant
downregulation of CXCL12 mRNA occurred at a concentration of 50 μM genistein in
MCF-7 cells and 10 μM genistein in BG-1 cells (Figures 1(a)–1(c)). Interestingly,
low doses of genistein (1–10 μM)
significantly increased CXCL12 mRNA
levels in MCF-7 cells, but had the opposite effect in BG-1 cells at these
doses.  MDA-MB-231 cells do not express
detectable levels of CXCL12.Dose-response curves for CXCR4 and
CXCL12 mRNAs after treatment of breast and ovarian cancer cells with 1–10μM genistein. (a) CXCR4 and CXCL12 mRNA quantification after treatment of MCF-7 cells with
increasing concentrations of genistein for 24 hours. (b) CXCR4 quantification after
treatment of MDA-MB-231 cells with genistein for 24 hours. MDA-MB-231 cells do
not express CXCL12 as determined by real-time PCR. (c) CXCR4 and CXCL12
quantification after treatment of BG-1 cells with increasing doses of genistein
for 24 hours. All data were normalized to corresponding mRNA quantities for the
36B4 housekeeping gene.  P*<.05, P**<.01, P***<.001; relative to 0 μM genistein
control.
(a)
MCF-7(b)
MDA-MB-231(c)
BG-1Flow
cytometric analysis was used to quantify surface CXCR4 and intracellular CXCL12
expression.  Cells were treated with 30,
70, or 100μM genistein for 24 hours, harvested, stained, and analyzed for
surface expression levels of CXCR4.
Relative to DMSO-treated controls, CXCR4 was found to be downregulated
by genistein in MCF-7, MDA-MB-231, and BG-1 cells at all three doses (Figures 2(a)–2(c)).  Analysis of CXCL12 intracellular expression
showed that relative to DMSO-treated control cells, CXCL12 levels were reduced
by all three doses of genistein in both MCF-7 and BG-1 cells (Figures 2(a), 2(c)).Genistein downregulates CXCR4 surface expression and intracellular
CXCL12 expression. Downregulation of
CXCR4 protein expression in MCF-7, MDA-MB-231, and BG-1 cells at concentrations
of genistein ranging from 0–100 μM for a 24-hour
period (a)–(c).  Downregulation of intracellular CXCL12
protein expression in (a) MCF-7 and (c)
BG-1 cells at 30–100 μM genistein.
Fluorescence distribution plots depict one representative experiment.  Isotype controls are represented by filled
histograms. Mean fluorescence intensities (MFI) and their corresponding
standard deviations are derived from three independent experiments.
(a)
MCF-7(b)
MDA-MB-231(c)
BG-1


## 3.2. Downregulation of CXCR4 and CXCL12 Is Greater after Treatment with Both Genistein and DIM than with Either Compound Alone

We previously found that DIM downregulates CXCR4 and CXCL12 mRNAs and proteins in invasive breast and ovarian
cancer cells [6].  We therefore tested
whether the downregulation elicited by cotreatment with both DIM and genistein
was additive or synergistic in these cell lines.  Using real-time PCR, we quantified gene
expression after treatment of MDA-MB-231 (CXCR4 only) or BG-1 (both CXCR4 and
CXCL12) with 20 μM DIM, 100 μM genistein, or the combination. We found that in
both cell lines, the degree of downregulation of CXCR4 was greater after
treatment with both phytochemicals in combination (Figures 3(a)-3(b)).  In MDA-MB-231 cells, DIM reduced levels of
CXCR4 by 57%, genistein by 51%, and the combination by 85% (Figure 3(a)).  In BG-1 cells, DIM downregulated CXCR4 by
49%, genistein by 79%, and DIM + genistein by 90%.  Similarly, DIM lowered the levels of CXCL12
by 50%, genistein lowered levels by 84%, and genistein by 91% (Figure 3(b)).  These effects cannot be attributed to
cytotoxicity, since neither DIM, genistein, nor the combination was cytotoxic
at these doses for the 24 hour time period (data not shown).In combination, genistein and DIM are more efficacious in the
downregulation of CXCR4 and CXCL12 mRNAs than either compound alone. The effect of downregulation of CXCR4 in (a) MDA-MB-231 and (b) BG-1 cells by 20 μM DIM and 100 μM genistein in combination is
greater than their individual effects. The same is seen in (b) BG-1 cells after CXCL12 quantification.  Ctl = DMSO alone; P**<.01, P***<.001.
(a)
MDA-MB-231(b)
BG-1


## 3.3. Genistein Specifically Inhibits Chemotaxis and Chemoinvasion of Breast and Ovarian Cancer Cells toward CXCL12

Since
CXCR4 and CXCL12 mediate directional migration, and since we saw previously
that DIM inhibited chemotaxis and chemoinvasion of these cells, we performed
assays to determine whether genistein could inhibit migration through
fibronectin and/or invasion through matrigel. We found that pretreatment with
genistein inhibited the directional migration of MCF-7 cells, with a resulting
migration rate similar to the background rate (DMSO-treated cells exposed to no
CXCL12 gradient, Figure4(a)).  However,
we did not see a further reduction in migration after cotreatment with
genistein and DIM, presumably because both chemicals used independently at
these doses fully reduced migration to background levels. This result indicates
that the degree to which genistein and DIM downregulate surface CXCR4 levels at
these doses may be sufficient to significantly impact the homing of breast
cancer cells to areas of high CXCL12 expression.  As we have seen previously with DIM, the
inhibition of chemotaxis by genistein specifically affects migration toward
CXCL12, since an inhibition of MCF-7 cell chemotaxis toward FBS was not
observed.DIM
specifically inhibits chemotaxis and chemoinvasion of breast and ovarian cancer
cells toward CXCL12. (a) Quantification of chemotaxis of MCF-7
cells toward CXCL12 or FBS after treatment with DMSO alone (Ctl), 20 μM DIM,
70 μM genistein, or the combination of DIM + genistein for 24 hours.  (b)-(c) Chemotaxis and chemoinvasion of
MDA-MB-231 cells toward CXCL12 or IL-6 were quantified after pretreatment with
DIM and/or genistein as in (a). (d) Quantification of chemoinvasion
toward CXCL12 or FBS after treatment of BG-1 cells with DIM and/or
genistein.  P*<.05, P**<.01, P***<.001; compared with control + chemoattractant.
(a)
Chemotaxis of MCF-7 cells(b)
Chemotaxis of MDA-MB-231 cells(c)
Chemoinvasion of MDA-MB-231 cells(d)
Chemoinvasion of BG-1 cellsMCF-7
cells do not invade through matrigel (a synthetic extracellular matrix), and we were therefore unable to
evaluate invasive potential in these cells.
However, MDA-MB-231 breast cancer cells are invasive and we were able to
quantify both chemotactic and chemoinvasive potential in this cell line.  We found that genistein, DIM, and the
combination significantly inhibited chemotaxis and chemoinvasion of these cells
toward CXCL12, but not toward IL-6, a known in vitro chemoattractant for MDA-MB-231 cells (Figures4(b)-4(c)).  This result indicates that the inhibitory
effects of DIM and genistein are specific for CXCL12-induced
chemoattraction/chemoinvasion and is not merely a general effect on migration
or invasion. A similar result was seen with BG-1 ovarian cancer cells, which
are also invasive in vitro
(Figure 4(d)). These cells do not migrate through matrigel toward IL-6 (data not shown), but did exhibit moderate chemoinvasion toward FBS.


## 4. Discussion

The
antiestrogenic activity of genistein may mediate, in
part, the protective effects of soy for breast and other cancers [10].  Although other nonestrogenic mechanisms of
action such as PTK and topoisomerase II inhibition likely play a role in these
protective effects, we describe here an additional novel mechanistic pathway in
which genistein and possibly other phytochemicals may be protective. We have
found that genistein downregulates CXCR4 in the ER-positive (ER+) breast cancer
cell line, MCF-7, the ER-negative (ER−) breast cancer cell line, MDA-MB-231,
and the ER+ ovarian cancer cell line, BG-1. Furthermore, CXCL12, the unique
ligand for CXCR4, is downregulated by genistein in both MCF-7 and BG-1
cells.  We show that this downregulation
results in a subsequent inhibition of migration and invasion of these cells
toward CXCL12 in vitro.We
found previously that DIM downregulates CXCR4 and CXCL12 in MCF-7, MDA-MB-231,
and BG-1 cells [6]. We show here that when used in combination, the effects of
DIM and genistein on CXCR4 and CXCL12 mRNA levels are greater than with either
compound alone, suggesting that phytochemicals used in combination may increase
the efficacy of their protective effects.
At 20 μM DIM and 70 μM genistein, we did not find the combination to
further inhibit chemotaxis or chemoinvasion of breast or ovarian cancer cells
since these concentrations of the phytochemicals fully inhibit these processes.
However, at the slightly lower doses of 10 μM DIM and 50 μM genistein, the
combination does appear to be more effective at inhibiting chemotaxis of
MDA-MB-231 cells. At these latter doses, the individual phytochemicals did not
fully reduce chemotaxis of these cells to background levels. These results
therefore suggest that lower doses of the phytochemicals used in combination
may be equally or more effective in chemoprotection than a higher dose of a
single phytochemical.The
mechanisms of action of DIM and genistein in the downregulation of CXCR4 and
CXCL12 remain to be determined. DIM and genistein both act as weak
agonists/antagonists of the estrogen receptor, and it is therefore possible
that the compounds can act through the same mechanism. Importantly however, we
see downregulation of CXCR4 and a subsequent inhibition of chemotaxis and
chemoinvasion in MDA-MB-231 cells, which do not express the estrogen receptor.
On the other hand, DIM is a ligand for the AHR, whereas genistein has not been
shown to bind the AHR in the cell lines we used. Genistein is a specific PTK
inhibitor as well as an inhibitor of topoisomerase II, and may modulate TGF-β
signal transduction [11, 12].
Interestingly, both genistein and DIM are known to inhibit NFkB in
breast, prostate, and pancreatic cancer cells [13–15].  Since CXCR4 is regulated at the
transcriptional level by NFkB, it is plausible that the mechanism of CXCR4
and/or CXCL12 downregulation by DIM and/or genistein could be through NFkB
inactivation or downregulation [16, 17].Condoning
the general use of genistein as a supplement is at this point
controversial.  Soy products are
extensively consumed in Asian populations without apparent adverse effects, but
experimental data have led to concerns about the safety of genistein and other
constituents of soy.  Although it was
well tolerated, high doses of genistein in chronic studies caused an increase in
the weights of the kidney, spleen, adrenal, and testes in male rats and an
increase in liver, kidney, spleen, ovary, and uterus weights in female rats
[18].  In the same study, histological
changes were seen in the reproductive organs of both male and female rats.
These findings were attributed to the estrogenic properties of genistein.  Genistein can behave as both an estrogen and
an antiestrogen, and the net estrogenic effect of the chemical has proven
difficult to quantify [11].  A chronic
exposure study very recently carried out by the National Toxicology Program
showed a significant increase in the incidence of mammary gland adenoma and
adenocarcinoma (combined) [19].  Importantly,
the time of administration appears to significantly impact whether genistein
elicits a protective, adverse, or no effect [20].  For example, dimethylbenzanthracene
(DMBA)-induced mammary cancer was reduced after prepubertal and combined
prepubertal and adult administration of genistein, but not after prenatal-only
or adult-only treatments [2].Dose also plays a critical role in the biological effects of
genistein; although doses greater than 10μM over an extended period of time
inhibit the growth of both ER+ and ER− breast cancer cells, low doses of
genistein (<1 μM) appear to stimulate the growth of ER+ breast cancer cells [21, 22].
Interestingly, CXCL12 has been shown to mediate the proliferative
effects of estradiol in breast cancer cells [23].  We noted a significant increase in CXCL12
mRNA after treatment with low doses (1–10 μM) of genistein
in MCF-7 cells, although this effect was not seen in MDA-MB-231 or BG-1 cells.
An upregulation of CXCL12 by low doses of genistein remains to be confirmed in
tissues that serve as common sites of metastasis such as the lung and bone.  However, in light of what is known about the
toxic and potentially carcinogenic effects of genistein, this observation underscores the importance of thorough
safety analyses prior to condoning the use of phytochemicals as dietary
supplements.  In particular, it highlights the importance of
dose, especially at the tissue level, when assessing the impact of genistein on
the development and progression of breast and other cancers.The doses of
genistein and DIM described here are likely achievable in humans upon
supplementation, especially at the tissue level.  Total genistein plasma concentrations of up
to 20μM were obtained after feeding human volunteers a genistein supplement
[24–26].  Furthermore, genistein was
found to accumulate in certain organs to considerably higher concentrations
[27], and this is likely to be the case for fatty tissues, such as the breast,
in particular.  When mice were
administered a single oral dose of DIM, a serum concentration of approximately
5 μM was achieved.  Much higher
concentrations accumulated in certain organs [28].  Thus, the concentrations of genistein and DIM
that we have used in our studies are likely to reflect attainable doses in the
human.Since CXCR4 and CXCL12 are implicated in the
progression of many different cancers, it is of interest to determine whether
DIM and/or genistein downregulate these proteins in cancer cell lines of other
origins. It will also be important to investigate whether CXCL12 is
downregulated by phytochemicals in tissues that serve as preferred sites of metastasis
for these cancers, such as the lung and bone. Furthermore, since many other phytochemicals
have been implicated in cancer protection, it will be prudent to determine
whether phytochemicals other than DIM and genistein exert similar effects on CXCR4
and CXCL12 levels. Finally, phytochemicals in combination should be tested in vivo to determine whether a
potentiated effect can be achieved.


## 5. Conclusion

DIM
has been suggested as a potential chemotherapeutic for ER+ breast cancers.  Surprisingly however, we have found that DIM
and genistein downregulate CXCR4 and CXCL12 in both ER+ and ER− cell lines,
indicating that these phytochemicals may be effective in the treatment of both
early- and late-stage breast cancers. Effective therapies for advanced disease
are lacking, and the potential use of compounds as innocuous as phytochemicals
for treatment of either early- or late-stage cancers is an attractive
alternative.  Furthermore, the increased
response of CXCR4 and CXCL12 downregulation by DIM and genistein in combination
may prove useful in eliciting a potentiated effect in vivo and perhaps
allow for optimization of the associated biological responses.



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