Content uploaded by Hermann Rath
Author content
All content in this area was uploaded by Hermann Rath on Jan 07, 2019
Content may be subject to copyright.
JBUON 2018; 23 (Suppl 1): S53-S59
ISSN: 1107-0625, online ISSN: 2241-6293 • www.jbuon.com
E-mail: editorial_oce@jbuon.com
ORIGINAL ARTICLE
Correspondence to: Cristina Adela Iuga, PhD. Department of Proteomics and Metabolomics, MedFuture Research Center for
Advanced Medicine, “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania.
Tel: +40-722-460-298, E-mail: iugac@umfcluj.ro
Received: 21/05/2018; Accepted: 16/06/2018
Inuence of soy isoavones in breast cancer angiogenesis: a
multiplex glass ELISA approach
Alina Uifalean1,2, Hermann Rath3, Elke Hammer3, Corina Ionescu4, Cristina Adela Iuga1,5,
Michael Lalk2
1
Department of Pharmaceutical Analysis, Faculty of Pharmacy, “Iuliu Haţieganu” University of Medicine and Pharmacy,
Cluj-Napoca, Romania; 2Institute of Biochemistry, University of Greifswald, Greifswald, Germany; 3Department of Functional
Genomics, Interfaculty Institute of Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany;
4Department of Pharmaceutical Biochemistry and Clinical Laboratory, Faculty of Pharmacy, “Iuliu Haţieganu” University of
Medicine and Pharmacy, Cluj-Napoca, Romania;
5
Department of Proteomics and Metabolomics, MedFuture Research Center for
Advanced Medicine, “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania
Summary
Purpose: The aim of this study was to evaluate the anti-
angiogenic properties of soy isoavones using two breast
cancer cell lines, by measuring the concentration of 30 cy-
tokines involved in angiogenesis using a multiplex glass slide
ELISA-based array.
Methods: Estrogen-dependent MCF-7 cells and estrogen-
independent MDA-MB-231 cells were exposed to genistein
(Gen), daidzein (Dai) and a soy seed extract (Ext) for 72 hrs,
at selected concentration levels. The conditioned medium was
analyzed using a glass slide, multiplex sandwich ELISA-
based platform with uorescent detection which allowed the
identication and the quantication of 30 angiogenesis-
related cytokines.
Results: In MCF-7 cells, low, stimulatory concentrations
of test compounds determined the increase of CXCL16 and
VEGF-A level. Gen induced the greatest eect, with 1.5-fold
change compared to control. When MDA-MB-231 cells were
exposed to inhibitory concentrations, all test compounds de-
termined a reduction of CXCL16 and VEGF-A level with ap-
proximately 30%.
Conclusions: Soluble CXCL16 and VEGF-A are two pro-
moters of angiogenesis and metastasis in breast cancer.
The stimulation of these two angiogenesis-related cytokines
could represent one of the mechanisms explaining the prolif-
erative eects of low isoavone doses in estrogen-dependent
cells. In estrogen-independent cells, soy isoavones inhibited
their secretion, demonstrating promising anti-angiogenic
properties.
Key words: angiogenesis, breast cancer cells, CXCL16, ELI-
SA, isoavones, VEGF-A
Introduction
In the USA, breast, lung and colorectal cancers
account for 50% of all cancer cases expected to oc-
cur in women in 2018. Of this percentage, breast
cancer alone accounts for 30%, which embody
266,120 new diagnosed cases [1]. Breast cancer
rates are generally higher in Northern America,
Australia/New Zealand, Western Europe and low
in most of Africa and Asia [2].
Several studies have related the low incidence
rates of breast cancer in Asian countries with the lo-
cal dietary patterns, showing that soy consumption
could lower the risk of breast cancer for both pre-
and post-menopausal women in Asian countries
[3,4]. Later, these epidemiological observations
were strengthened by in vitro data, soy isoavones
and especially genistein, showing antiproliferative
This work by JBUON is licensed under a Creative Commons Attribution 4.0 International License.
Soy isoavones in breast cancer angiogenesis54
JBUON 2018; 23 (Suppl 1): S54
eects by sustaining apoptosis, antioxidant defense
and DNA repair and, not least, by inhibiting the
development of tumor angiogenesis and metastasis
[5].
Soy isoavones have been explored as prom-
ising anti-angiogenetic agents as they appear to
inhibit multiple angiogenic mechanisms, such as
regulation of vascular endothelial growth factor
(VEGF), matrix metalloproteinases (MMPs), epi-
dermal growth factor receptor (EGFR) expressions
and NF-κB, PI3-K/Akt or ERK1/2 signaling path-
ways [5,6]. However, despite the intensive research,
the anti-angiogenic potential of soy isoavones in
breast cancer remains controversial, mainly due to
their twofold eect [7].
Several in vitro assays have been developed
to assess the angiogenic properties of exogenous
agents. Most models focus on proliferation, mi-
gration, and dierentiation of endothelial cells
[8]. While these tests determine the eect or the
outcome of drugs on blood vessel formation, more
high-throughput tests have been developed in order
to identify which particular angiogenic molecules
or mechanisms are targeted by the test compounds.
Such are the glass slide ELISA-based quantitative
systems, extensively used for the rapid proling of
cytokine expression.
The main advantage of glass slides over the
single-targeted 96-well plate ELISAs or Western
blots is the possibility of performing simultane-
ous identication and quantication of multiple
cytokines, growth factors, proteases, soluble recep-
tors and other angiogenesis-associated proteins in
a single experiment. Furthermore, they are highly
specic and reproducible, require low sample vol-
umes and are well-suited for high throughput as-
says [9].
To our knowledge, no ELISA-based quantitative
array on breast cancer cells exposed to isoavones
has been performed so far. The identication and
quantication of the key molecules involved in an-
giogenesis will provide a further understanding of
isoavones’ anti-angiogenic properties. The aim of
this study was to evaluate the anti-angiogenic prop-
erties of genistein (Gen), daidzein (Dai) and a soy
seed extract (Ext) using two breast cancer cell lines,
MCF-7 and MDA-MB-231, by measuring the con-
centration of 30 cytokines involved in angiogenesis
using a multiplex glass slide ELISA-based array.
Methods
Chemical and standards
All chemicals and standards were purchased from
Sigma-Aldrich (Taufkirchen, Germany), unless otherwise
stated.
The soy extract was purchased from Hunan Gold-
liloo Pharmaceutical Co., Ltd. (Changsha, China). Accord-
ing to manufacturer’s specications, the extract was ob-
tained from soy seeds (Glycine max), using an aqueous
ethanolic solution followed by spray-drying. The extract
contains 40% isoavones, of which daidzein represents
only 1.50%, glycitein 0.12%, and genistein 0.02%. The
isoavone distribution was conrmed in our laboratory
by a validated HPLC-UV method [10].
Stock solutions of standard Gen, Dai, and Ext were pre-
pared in dimethyl sulfoxide (DMSO) and stored at -20°C.
Cell culture and culture conditions
The MCF-7 and MDA-MB-231 breast adenocarci-
noma cell lines were obtained from CLS Cell Lines Ser-
vice (Eppelheim, Germany) and routinely cultured as
previously described [11]. All cells used in experiments
were between passage number 5 and 20.
Cell treatment and sampling
The test concentrations of Gen, Dai, and Ext were
established based on a MTT test, as previously described
[11]. Briey, in MCF-7 estrogen-dependent cells, all com-
pounds induced a twofold eect, stimulating cell growth
at relatively low concentrations and causing inhibition
at higher concentrations. Therefore, we selected two con-
centration levels for each test compound: the concentra-
tions that stimulated cell proliferation by 20% compared
to control (SC
20
) and the concentrations that inhibited
cell growth by 20% compared to control (IC20). The SC20
concentrations for Gen, Dai, and Ext were 5.62 μM, 19.01
μM, and 22.59 μg/mL respectively, while the IC20 con-
centrations were 22.44 μM, 52.24 μM, and 166.34 μg/mL
respectively. For MDA-MB-231 estrogen-independent
cells, only a dose dependent inhibitory eect was ob-
served and, therefore, only the IC20 concentrations were
selected. Precisely, these IC
20
concentrations were 11.04
μM for Gen, 36.39 μM for Dai, and 26.36 μg/mL for Ext
[11].
For cell treatment, 2.4×106 MCF-7 cells or 1.2×106
MDA-MB-231 cells were seeded in 150 mm cell cul-
ture dishes (Sarstedt, Germany) in 15 mL RPMI 1640
medium supplemented with 10% heat-inactivated fetal
bovine serum, 1 mM sodium pyruvate, 1% non-essential
amino acids and 1% penicillin-streptomycin.
The dishes were shaken for 1 min to ensure the
homogeneous distribution of cells. Next, all plates were
incubated for 24 hrs to allow cell attachment. Aer 24
hrs, the medium was replaced with 28 mL fresh medium
containing the selected concentrations of Gen, Dai, Ext,
or DMSO as solvent control. In all cases, the nal con-
centration of DMSO did not exceed 0.01%. The incuba-
tion time was 72 hrs.
For sampling, 1.5 mL conditioned medium were cen-
trifuged at 2000 rpm, at 4°C for 10 min. The supernatant
was immediately frozen at -80°C until measurement.
Antibody array analysis of angiogenesis related cytokines
For the quantication of angiogenesis-associated
cytokines, we used Quantibody Human Angiogenesis
Array 3 (#QAH-ANG-3, RayBiotech, Norcross, Georgia,
Soy isoavones in breast cancer angiogenesis 55
JBUON 2018; 23 (Suppl 1): S55
USA). This is a glass slide, multiplex sandwich ELISA-
based platform which allows the identication and quan-
tication of 30 cytokines, chemokines, growth factors,
and other molecules involved in angiogenesis. The 30
spotted targets were angiogenin-1, angiostatin, C-X-C
motif chemokine ligand 16 (CXCL16), epidermal growth
factor, broblast growth factor 4, follistatin, granulo-
cyte colony-stimulating factor, granulocyte-macrophage
colony-stimulating factor, I-309, interleukin-1 beta, in-
terleukin-4, interleukin-10, interleukin-12 subunit p40,
interleukin-12 subunit p70, interferon-inducible T-cell
alpha chemoattractant, monocyte chemotactic protein 2,
monocyte chemotactic protein 3, monocyte chemotactic
protein 4, matrix metalloproteinase-1, matrix metallo-
proteinase-9, platelet endothelial cell adhesion mole-
cule-1, transforming growth factor alpha, transforming
growth factor beta-3, tyrosine-protein kinase receptor
Tie-1, tyrosine-protein kinase receptor Tie-2, urokinase
plasminogen activator surface receptor, vascular en-
dothelial growth factor-A (VEGF-A), vascular endothelial
growth factor receptor 2, vascular endothelial growth
factor receptor 3 and vascular endothelial growth factor
D. Each antibody, together with two positive controls and
a negative control, is printed in four identical spots, so
each cytokine is measured four times per sample.
The assay was conducted according to manufacturer
recommended protocol [9]. Briey, the glass slides were
rst allowed to equilibrate and dry at room temperature
for 2 hrs. In the blocking step, 100 μL sample diluent was
added into each well and the slides were incubated for
30 min at room temperature. Next, the sample diluent
was discarded and 100 μL calibration standard cytokines
or conditioned medium were added into each well. The
glass chamber was covered with adhesive lm and incu-
bated overnight, at 4°C, on a plate shaker (Titramax 101,
Heidolph Instruments, Schwabach, Germany) at 200 rpm.
On the next day, the supernatant was discarded and
each well was washed ve times with Wash Buer I
and two times with Wash Buer II. Subsequently, the
biotinylated antibody cocktail was reconstituted and
80 μL were added per well. Aer 2 hrs, the antibody
cocktail was removed, the wells were washed again with
the two washing buers and 80 μL of Cy3 Equivalent
Dye-Streptavidin were added per well. The slides were
incubated in the dark, at room temperature for 1 hr. Aer
other washing steps, the slides were carefully removed
from the gasket and allowed to dry at room temperature.
For uorescence detection, a DNA Microarray Scan-
ner (G2505C, Agilent Technologies, USA) with a scan
resolution of 10 μm was used.
Data analysis
For background subtraction and densitometry
measurement, the scanned images were analyzed using
Image Studio Lite (v.2.5.2.). For each spot, the dened
area for signal capture was a circle with a 158-micron
diameter. The median intensity of a three-pixel border
around the dened circle was used for local background
subtraction.
As our cells were cultivated in serum-containing
medium, which might contain various types of cytokines,
the median signal intensity of each cytokine of the com-
plete medium array was subtracted from the signal in-
tensity of the corresponding cytokine from each other
array.
Next, data normalization was carried out by ac-
counting for the dierences in signal intensities of
the positive control spots across all arrays. The posi-
tive control spots represent standardized amounts of
biotinylated antibody and the signal of these spots is
dependent on the amount of streptavidin-uor bound
to that antibody. This bounding capacity will propor-
tionally aect the signal intensity of every spot on the
array. Therefore, the dierences in the positive control
signals between arrays will accurately reect the dier-
ences between other spots on those arrays. The reference
array was the solvent control (medium with DMSO only)
corresponding to each cell line. The normalized values
were calculated using equation 1 [9]:
nX(Y)=X(Y)×P / P(Y)
nX(Y)= the normalized value for cytokine “X” of sample
“Y”, X(Y)= the signal density of the spots for cytokine
“X” of sample “Y”, P= the average signal density of the
positive control spots on the reference array, P(Y)= the
average signal density of the positive control spots of
sample “Y”
Statistics and visualization
The calibration curves and the statistical analysis
were executed using Prism (v.6.01, GraphPad Soware).
Each treatment was compared to the corresponding sol-
vent control according to two-way ANOVA with Sidak’s
pg/mL Fold change
CXCL16 concentration
In MCF-7 cells
Control 1140.91
Gen SC20 1775.65 1.55
Dai SC20 1691.43 1.48
Dai IC20 547.37 -2.08
In MDA-MB-231 cells
Control 1574.22
Gen IC20 1232.29 -1.28
Ext IC20 1137.21 -1.38
VEGF-A concentration
In MCF-7 cells
Control 1309.22
Gen SC20 1982.84 -1.51
Ext SC20 1808.55 -1.38
In MDA-MB-231 cells
Control 1521.43
Gen IC20 1192.17 -1.27
Dai IC20 1130.65 -1.34
Ext IC20 950.75 -1.60
Table 1. The concentration and the fold change of
signicantly altered cytokines (p<0.05, two-way ANOVA
with Sidak’s correction for multiple comparisons)
Soy isoavones in breast cancer angiogenesis56
JBUON 2018; 23 (Suppl 1): S56
correction for multiple comparisons. Dierences with
p values less than 0.05 were considered as statistically
signicant.
Results
Exposure of both breast cancer cell lines to
Gen, Dai, and Ext induced signicant changes, es-
pecially in the signal intensity of two cytokines,
CXCL16 and VEGF-A. In MCF-7 cells, SC20 of test
compounds caused an increase in CXCL16 and
VEGF-A signal intensity, while treatment of cells
with IC20 concentrations led to a reduction of
CXCL16 level. For MDA-MB-231 cells, inhibitory
concentrations of test compounds triggered a de-
crease in the VEGF-A and CXCL16 signal intensity
(Figure 1).
Next, the mean intensity of the signicantly
changed cytokines was plotted on the correspond-
ing calibration curve (Figure 2). Using these curves,
the absolute cytokine concentration was then cal-
culated (Table 1).
Discussion
Soy isoavones are known as promising anti-
angiogenic agents, acting on multiple pathways,
such as ERK1/2 signaling pathway, regulation of
Figure 1. The signal intensity of CXCL16 and VEGF-A aer MCF-7 and MDA-MB-231 cells were exposed to genistein
(Gen), daidzein (Dai), and soy extract (Ext) at test concentrations. Asterisks indicate statistically signicant dierences
(p<0.05) between solvent control and treated samples.
Figure 2. The calibration curves for CXCL16 and VEGF-A obtained by plotting their mean intensities against the pre-
determined concentrations. The curves were generated using a non-linear regression t model (R2=0.9974 for CXCL16
and R2=0.9584 for VEGF-A).
Soy isoavones in breast cancer angiogenesis 57
JBUON 2018; 23 (Suppl 1): S57
VEGF or MMPs expression [5,6]. However, there is
limited data regarding the inuence of these natu-
ral compounds on CXCL16 expression.
CXCL16, along with CXCL12 (C-X-C motif
chemokine ligand 12), belong to the superfamily of
chemotactic cytokines, which govern the immune
cell tracking between or within tissues. Through
coordinated interaction with its specic receptor,
CXCR6 (C-X-C motif chemokine receptor 6), CXCL16
also plays a crucial role in tumor growth, invasion,
angiogenesis, and metastasis in various types of
cancers such as breast adenocarcinoma [12-14],
lung cancer [15] or prostate cancer [16]. Moreover,
for prostate and breast cancers, a positive correla-
tion between CXCR6/CXCL16 expression and cancer
aggressiveness was found [17,18], higher CXCR6 ex-
pression in nest site and metastatic lymph node be-
ing responsible for breast cancer progression [12].
So far, in vitro studies have shown that soy iso-
avones can regulate other angiogenic chemokines,
such as CXCL12. In MCF-7 estrogen-dependent
cells, low doses of Gen or Dai (1–10 μM) induced a
signicant increase in CXCL12 level [19-21], trig-
gering cell proliferation and invasion. When the
same cell line was exposed to higher Gen concen-
trations (>25 μM), the CXCL12 mRNA level was
signicantly downregulated. This downregulation
resulted in a subsequent inhibition of migration
and invasion. In MDA-MB-231 cells, CXCR4 (C-X-C
motif chemokine receptor 4), the cognate receptor
of CXCL12, was downregulated by Gen in a dose-
dependent manner [19].
To our knowledge, no study has assessed the ef-
fect of soy isoavones on CXCL16 chemokine so far.
Our results show that isoavone treatment
triggers similar changes for soluble CXCL16 ex-
pression, as for CXCL12. Low doses of isoavones
(SC
20
) signicantly stimulated CXCL16 secretion
in MCF-7 cells, Gen causing the highest CXCL16
increase. When MCF-7 were exposed to higher, in-
hibitory doses of isoavones (IC20), only Dai caused
a signicant decrease. The CXCL16 decrease caused
by Dai could be due to the anti-inammatory prop-
erties of Dai, which was shown to suppress the
transcription of pro-inammatory chemokines,
such as CXCL2, by depressing PARP-1 activity [22].
However, the IC20 of Gen used in this study
(22.44 μM for MCF-7 cells) was lower than the con-
centrations used in other studies [19,23]. Therefore,
it is not excluded that higher Gen concentrations,
most likely >50 μM, could decrease the CXCL16 se-
cretion. In MDA-MB-231 cells, all test compounds
generated a decrease in the CXCL16 level.
One of the mechanisms proposed for explain-
ing the proliferative eects of CXCR6/CXCL16
breast cancer cells involves the activation of down-
stream signaling paths, such as ERK1/2 signaling
pathway [12]. Apparently, stimulation of ERK1/2
pathway activates RhoA, a member of the RhoGT-
Pase family. The eect leads to inhibition of colin
activity, responsible for the regeneration of actin
laments. In response to colin inhibition, F-actin
stability enhances, favoring breast cancer invasive-
ness and metastasis [12]. In fact, Gen can also act
as direct modulator of ERK1/2 pathway, promoting
MCF-7 cell growth through delayed and prolonged
phosphorylation of ERK1/2 [24].
An alternative explanation could rely on the
estrogenic eects of low isoavones doses. Similar
to estrogen, which upregulates CXCL12 and CXCR4
expression in breast cancer cells [25], isoavones
could upregulate CXCL16 expression and secretion
acting through the same molecular mechanisms.
Isoavone treatment also determined signi-
cant changes in the VEGF-A level. Compared to
CXCL16, VEFG-A is a much more known player in
the process of tumor angiogenesis and the most
intensively studied member of VEGF family. Ac-
tivation of the VEGF-receptor pathway triggers a
network of signaling processes that promote cell
growth, migration, and survival from pre-existing
vasculature. The concentrations of the VEGF pro-
tein and VEGF receptors in the serum of breast
cancer patients showed positive correlations with
estrogen receptor status and the clinical stage of
disease [26].
Soy isoavones, and particularly Gen, have
been intensively examined for their potential to
modulate VEGF-A secretion, especially using cul-
tured human umbilical vein endothelial cells [6,27].
In breast cancer, low concentrations (10-12-10-6 M)
of Gen have similar eects as estrogen in estrogen
receptor (ER) positive cells like MCF-7 (ER posi-
tive), MELN (derived from MCF-7 cells) and MELP
(derived from MDA-MB-231 cells and transfected
with ER) inducing VEGF-A expression signicantly.
The same eect was not observed in MDA-MB-231
cells, suggesting that ER is necessary for VEGF
stimulation [28]. On the other hand, when MDA-
MB-453 cells were exposed to high Gen concen-
tration, the VEGF mRNA expression decreased
signicantly [6] pointing to a second receptor and
signaling pathway for Gen.
Our results are in line with the existing data,
that low, stimulatory concentrations of Gen or Ext
increase VEGF-A secretion in MCF-7 cells. Appar-
ently, low isoavone doses seem to mimic again
the estrogen action, stimulating the secretion of
VEGF-A. As VEGF-A promotes cell proliferation,
upregulation of VEGF-A secretion could be one of
the mechanisms explaining the proliferative eects
of isoavones.
Soy isoavones in breast cancer angiogenesis58
JBUON 2018; 23 (Suppl 1): S58
Notably, concentrations of Gen below 5 μM
correspond to a blood plasma concentration attain-
able in a soy-rich diet [29]. As CXCL16 and VEGF-A
secretion were both stimulated at low isoavone
concentrations, special attention should be paid
to the daily phytoestrogen intake by patients with
estrogen responsive breast cancer subtype to avoid
any pro-angiogenic eects.
In estrogen-independent MDA-MB-231 cells,
IC
20
of all test compounds triggered VEGF-A de-
crease. Inhibition of VEGF-A expression is a poten-
tial strategy especially in the triple negative breast
cancer, the cancer subtype that lacks any targeted
therapy and with the worst prognosis among all
breast cancer subtypes. As isoavones are capable
of inhibiting VEGF-A secretion in MDA-MB-231
cells, they could represent promising anti-angio-
genic agents.
Conclusion
Our study investigated the potential of soy iso-
avones to modulate the main molecules involved
in angiogenesis, using a quantitative glass slide
ELISA-based array. The results showed that iso-
avones exert dose dependent eects in both cell
lines: in MCF-7 cells, low isoavone doses stimu-
lated the secretion of CXCL16 and VEGF-A, two
promoters of angiogenesis and metastasis, while
higher concentrations inhibited CXCL16 and VEGF-
A secretion in MDA-MB-231 cells. The anti-angi-
ogenic properties of isoavones could be further
exploited as an eective strategy, especially in tri-
ple negative breast cancers.
Acknowledgements
This work was supported by the “Iuliu
Haţieganu” University of Medicine and Phar-
macy Cluj-Napoca through Internal Grant No.
1491/20/28.01.2014 and the People Programme
(Marie Curie Actions) of the European Union’s Sev-
enth Framework Programme FP7/2007-2013/under
REA Grant Agreement No. 317338.
We are grateful to Philipp Westho and Ra-
mona Suharoschi for their helpful discussions and
advice.
Conict of interests
The authors declare no conict of interests.
References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018.
CA Cancer J Clin 2018;68:7-30.
2.
Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent
J, Jemal A. Global cancer statistics, 2012. CA Cancer J
Clin 2015;65:87-108.
3. Chen M, Rao Y, Zheng Y et al. Association between soy
isoavone intake and breast cancer risk for pre- and
post-menopausal women: a meta-analysis of epidemio-
logical studies. PLoS One 2014;9(2):e89288.
4.
Nagata C, Mizoue T, Tanaka K et al. Soy intake and
breast cancer risk: an evaluation based on a systematic
review of epidemiologic evidence among the Japanese
population. Jpn J Clin Oncol 2014;44:282-95.
5. Uifalean A, Schneider S, Ionescu C, Lalk M, Iuga CA.
Soy isoavones and breast cancer cell lines: molecu-
lar mechanisms and future perspectives. Molecules
2015;22:21.
6. Varinska L, Gal P, Mojzisova G, Mirossay L, Mojzis J.
Soy and breast cancer: focus on angiogenesis. Int J Mol
Sci 2015;16:11728-49.
7.
Allred CD, Allred KF, Ju YH, Virant SM, Helferich
WG. Soy diets containing varying amounts of gen-
istein stimulate growth of estrogen-dependent (MCF-
7) tumors in a dose-dependent manner. Cancer Res
2001;61:5045-50.
8. Tahergorabi Z, Khazaei M. A review on angiogenesis
and its assays. Iran J Basic Med Sci 2012;15:1110-26.
9. RayBiotech - Quantibody Human Angiogenesis Array
3 - User Manual [29.03.2018]. Available from: https://
www.raybiotech.com/quantibody-human-angiogene-
sis-array-3-1-slide/.
10.
Uifalean A, Farcas A, Ilies M, Heghes SC, Ionescu C,
Iuga CA. Assessment of isoavone aglycones variabil-
ity in soy food supplements using a validated HPLC-UV
method. Clujul Med 2015;88:373-80.
11. Uifalean A, Schneider S, Gierok P, Ionescu C, Iuga CA,
Lalk M. The impact of soy isoavones on MCF-7 and
MDA-MB-231 breast cancer cells using a global me-
tabolomic approach. Int J Mol Sci 2016;17:1443.
12.
Xiao G, Wang X, Wang J et al. CXCL16/CXCR6
chemokine signaling mediates breast cancer progres-
sion by pERK1/2-dependent mechanisms. Oncotarget
2015;6:14165-78.
13.
King J, Mir H, Singh S. Association of cytokines and
chemokines in pathogenesis of breast cancer. Prog Mol
Biol Transl Sci 2017;151:113-36.
14.
Fang Y, Henderson FC, Jr., Yi Q, Lei Q, Li Y, Chen N.
Chemokine CXCL16 expression suppresses migration
and invasiveness and induces apoptosis in breast can-
cer cells. Mediators Inamm 2014;2014:478641.
Soy isoavones in breast cancer angiogenesis 59
JBUON 2018; 23 (Suppl 1): S59
15. Liang K, Liu Y, Eer D, Liu J, Yang F, Hu K. High CXC
chemokine ligand 16 (CXCL16) expression promotes
proliferation and metastasis of lung cancer via reg-
ulating the NF-kappaB pathway. Med Sci Monit
2018;24:405-11.
16.
Richardsen E, Ness N, Melbo-Jorgensen C et al. The
prognostic signicance of CXCL16 and its receptor C-X-
C chemokine receptor 6 in prostate cancer. Am J Pathol
2015;185:2722-30.
17. Lu Y, Wang J, Xu Y et al. CXCL16 functions as a novel
chemotactic factor for prostate cancer cells in vitro.
Mol Cancer Res 2008;6:546-54.
18. Deng L, Chen N, Li Y, Zheng H, Lei Q. CXCR6/CXCL16
functions as a regulator in metastasis and progression
of cancer. Biochim Biophys Acta 2010;1806:42-9.
19.
Hsu EL, Chen N, Westbrook A et al. Modulation of
CXCR4, CXCL12, and tumor cell invasion potential in
vitro by phytochemicals. J Oncol 2009;2009:491985.
20. Habauzit D, Boudot A, Kerdivel G, Flouriot G, Pakdel F.
Development and validation of a test for environmental
estrogens: checking xeno-estrogen activity by CXCL12
secretion in breast cancer cell lines (CXCL-test). Envi-
ron Toxicol 2010;25:495-503.
21. Lecomte S, Lelong M, Bourgine G, Efstathiou T, Sali-
gaut C, Pakdel F. Assessment of the potential activity
of major dietary compounds as selective estrogen re-
ceptor modulators in two distinct cell models for pro-
liferation and dierentiation. Toxicol Appl Pharmacol
2017;325:61-70.
22.
Li HY, Pan L, Ke YS et al. Daidzein suppresses pro-
inflammatory chemokine Cxcl2 transcription in
TNF-alpha-stimulated murine lung epithelial cells
via depressing PARP-1 activity. Acta Pharmacol Sin
2014;35:496-503.
23. Lee WY, Huang SC, Tzeng CC, Chang TL, Hsu KF. Al-
terations of metastasis-related genes identied using
an oligonucleotide microarray of genistein-treated
HCC1395 breast cancer cells. Nutr Cancer 2007;58:239-
46.
24.
Liu H, Du J, Hu C et al. Delayed activation of extracellu-
lar-signal-regulated kinase 1/2 is involved in genistein-
and equol-induced cell proliferation and estrogen-re-
ceptor-alpha-mediated transcription in MCF-7 breast
cancer cells. J Nutr Biochem 2010;21:390-6.
25.
Boudot A, Kerdivel G, Habauzit D et al. Dierential
estrogen-regulation of CXCL12 chemokine recep-
tors, CXCR4 and CXCR7, contributes to the growth
eect of estrogens in breast cancer cells. PLoS One
2011;6(6):e20898.
26.
Thielemann A, Baszczuk A, Kopczynski Z, Kopczynski P,
Grodecka-Gazdecka S. Clinical usefulness of assessing
VEGF and soluble receptors sVEGFR-1 and sVEGFR-2
in women with breast cancer. Ann Agric Environ Med
2013;20:293-7.
27.
Yu X, Zhu J, Mi M, Chen W, Pan Q, Wei M. Anti-an-
giogenic genistein inhibits VEGF-induced endothelial
cell activation by decreasing PTK activity and MAPK
activation. Med Oncol 2012;29:349-57.
28. Buteau-Lozano H, Velasco G, Cristofari M, Balaguer P,
Perrot-Applanat M. Xenoestrogens modulate vascular
endothelial growth factor secretion in breast cancer
cells through an estrogen receptor-dependent mecha-
nism. J Endocrinol 2008;196:399-412.
29. Cassidy A, Brown JE, Hawdon A et al. Factors aecting
the bioavailability of soy isoavones in humans aer
ingestion of physiologically relevant levels from dif-
ferent soy foods. J Nutr 2006;136:45-51.
