Grape seed proanthocyanidins inhibit angiogenesis via the

downregulation of both vascular endothelial growth factor and

angiopoietin signaling
Shuangsheng Huang
a,

, 1
, Ninggang Yang
b, 1
, Yuanyuan Liu
c
, Lamei Hu
d
, Jin Zhao
a
,
Jing Gao
a
, Yongquan Li
a
, Caili Li
a
, Xiaosu Zhang
a
, Tao Huang
a
a
Medical College of Northwest University for Nationalities, Lanzhou 730030, China
b
Department of Urology, Lanzhou First People's Hospital, Lanzhou, 730030, China
c
Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China
d
Institute of Pharmacology, School of Basic Medical Science, Lanzhou University, Lanzhou 730000, China
A R T I C L E I N F O A B S T R A C T
Article history:
Received 2 February 2012
Revised 27 May 2012
Accepted 29 May 2012
Vascular endothelial growth factor (VEGF)/VEGF receptor 2 and angiopoietin 1/tyrosine
kinase with immunoglobulin and epidermal growth factor homology domains 2 signaling
pathways regulate different, but complementary, aspects of blood vessel growth in
tumors. Simultaneous inhibition of both pathways not only exhibits additive
antiangiogenic effects but also overcomes the resistance to anti-VEGF therapy. Grape
seed proanthocyanidins (GSPs) are widely consumed dietary supplements with
antiangiogenic activity. However, the molecular mechanisms underlying their
antiangiogenic action have not been fully understood. We hypothesized that GSPs
modulate multiple signaling pathways to exhibit antiangiogenic effects. In the present
study, we aimed to test this hypothesis by examining the effects of GSPs on human
microvascular endothelial cell1 and chick chorioallantoic membrane. Our results showed
that GSPs inhibited the migration, matrix metalloproteinase2 and 9 secretion, and tube
formation of human microvascular endothelial cell1 in vitro in a dose-dependent
manner. In addition, chick chorioallantoic membrane angiogenesis assay showed that
GSPs inhibited neovascularization in a dose-dependent manner. Furthermore, we
demonstrated that GSPs inhibited the phosphorylation of VEGF receptor 2 and tyrosine
kinase with immunoglobulin and epidermal growth factor homology domains 2 as well as
downstream signaling component extracellular signalregulated kinase 1/2. In summary,
these data suggest that GSPs inhibit both VEGF and angiopoietin 1 signaling to execute
the antiangiogenic effects and indicate that GSPs could be developed as a pharmacologically
safe chemopreventive agent against cancer.
2012 Elsevier Inc. All rights reserved.
Keywords:
Grape seed proanthocyanidins
Angiogenesis
VEGFR2
Tie2
HMEC-1 endothelial cells
Chick chorioallantoic membrane
N U T R I T I O N R E S E A R C H 3 2 ( 2 0 1 2 ) 5 3 0 5 3 6
Abbreviations: Ang, angiopoietin; CAM, chorioallantoic membrane; ERK, extracellular signalregulated kinase; FBS, fetal bovine serum;
GSPs, grape seed proanthocyanidins; HMEC-1, human microvascular endothelial cell-1; MAPK, mitogen-activated protein kinase; MMP,
matrix metalloproteinase; SRB, sulforhodamine B; Tie2, tyrosine kinase with immunoglobulin and epidermal growth factor homology
domains 2; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.
Corresponding author. Medical College of Northwest University for Nationalities, Lanzhou, 730030, China. Tel.: +86 931 2928013.
E-mail address: huangshsh02@qq.com (S. Huang).
1
The first two authors contributed equally to the work.
0271-5317/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.nutres.2012.05.012
Avai l abl e onl i ne at www. sci encedi r ect . com
www. nr j our nal . com
1. Introduction
Angiogenesis is involved in many physiological and patho-
logical conditions, such as wound healing, embryonic devel-
opment, atherosclerosis, arthritis, and tumor. During tumor
development, newly generated vessels not only provide
oxygen and nutrients for tumor growth but also promote
tumor metastasis [1]. The process of angiogenesis is regulated
in malignant tissues by the balance of pro- and antiangiogenic
factors. Vascular endothelial growth factor (VEGF) is the most
important proangiogenic factor [2]. Vascular endothelial
growth factor signaling involved in angiogenesis is mainly
mediated by VEGF receptor 2 (VEGFR2) expressed on the
surface of endothelial cells. The binding of VEGF to VEGFR2
facilitates autophosphorylation of VEGFR2 and then activates
diverse intracellular signaling molecules, including phosphoi-
nositide 3-kinase/AKT kinase, phospholipase C, protein
kinase C, and mitogen-activated protein kinase (MAPK)/
extracellular signalrelated kinase (ERK), which ultimately
leads to endothelial cell activation, proliferation, migration,
and survival [3,4]. Many antiangiogenic agents targeting VEGF
or VEGFR2 have been approved for the treatment of metastatic
colorectal cancer, renal cell cancer, and hepatocellular carci-
noma [5]. However, tumor could circumvent the anti-VEGF
therapy via the activation and/or upregulation of other
proangiogenic signaling pathways, including fibroblast
growth factor, ephrin, and angiopoietin (Ang) families that
drive angiogenesis and tumor progression [6]. In addition,
interference with VEGF-mediated signaling events is only
effective in preventing the early growth of neovessels. Mature
vessels covered with pericytes from more established tumors
are largely resistant to these inhibitors [7].
Angiopoietin 1 (Ang1) is another important proangiogenic
factor. Angiopoietin 1 exerts its biological effects by binding to
tyrosine kinase withIg and epidermal growth factor homology
domains 2 (Tie2) on endothelial cells [8]. It is generally
accepted that VEGF/VEGFR2 signaling is essential for drawing
endothelial cells from preexisting blood vessels and stimulat-
ing their growth, whereas Ang1/Tie2 signaling is important for
sustaining the interaction between endothelial and mural
cells and stabilizing the vasculature [9]. Accumulating evi-
dence has suggested that blocking both VEGFR2 and Tie2
signaling pathways could overcome the resistance to anti-
VEGF therapy [1012].
Grapes are one of the most widely consumed fruits in the
world and are rich in polyphenols of whichabout 60%to 70%is
found in grape seeds as dimers, trimers, and other oligomers
of flavan-3-ols and known commonly as proanthocyanidins.
Grape seed proanthocyanidins (GSPs) have demonstrated
chemopreventive and/or chemotherapeutic effects in various
cancer cell cultures and animal models [13,14]. Recent studies
further showed that GSPs exhibited antiangiogenic effects in
human prostate and breast cancer via the inhibition of VEGF
signaling [15,16]. However, GSPs could regulate multiple
signaling pathways such as nuclear factorB, MAPK, and
phosphoinositide 3-kinase/Akt in tumor cells [17]. Therefore,
we hypothesized that GSPs alter multiple signaling pathways
that contribute to its antiangiogenic actions. Thus, in the
present investigation, we tested our hypothesis by examining
the effects of GSPs on human microvascular endothelial cells
(HMEC-1) and chick chorioallantoic membrane (CAM) as
models for understanding potential signaling mechanisms
influenced by these compounds.
2. Methods and materials
2.1. Materials and reagents
Grape seed proanthocyanidins, consisting of at least 95%
proanthocyanidins, 1.8% proanthocyanidins B2, and 60%
oligomers, were purchased from Jianfeng Company (Tianjin,
China). Recombinant human VEGF and Ang1 were purchased
from Peprotech (Rocky Hill, New Jersey). Growth factor
reduced Matrigel was purchased from Becton Dickinson
(Bedford, Massachusetts). MCDB131 medium, epithelial
growth factor, hydrocortisone, sulforhodamine B (SRB), and
gelatin from porcine skin were purchased from Sigma (St
Louis, Missouri). Fetal bovine serum(FBS) was purchased from
Lanzhou HyClone (Lanzhou, China). Millicell cell culture
inserts were purchased from Millipore (Bedford, Massachu-
setts). Primary antibodies to VEGFR2, Tie2, phospho-ERK1/2,
and actin were purchased from Santa Cruz Biotechnology
(Santa Cruz, California). Primary antibodies to phospho-
VEGFR2 (Tyr1175), phospho-Tie2 (Tyr992), and ERK1/2 were
purchased from Cell Signaling (Beverly, Massachusetts).
2.2. Cell culture
Human microvascular endothelial cells were cultured in
MCDB131 medium supplemented with 1.18 mg/mL NaHCO
3
,
20% inactivated FBS, 10 ng/mL epithelial growth factor, and
1 g/mL hydrocortisone in a humidified atmosphere of 5%CO
2
at 37C.
2.3. Cell viability assay
To examine the effect of GSPs on the viability of HMEC-1 cells,
we performed cell viability assay using SRB method as
described previously [18]. Briefly, exponentially growing cells
were seeded in 96-well plates with the final volume 100 L per
well. After 24 hours, cells were treated with various concen-
trations of GSPs for indicated time. The cultures were then
fixed at 4C for 1 hour with ice-cold 50% trichloroacetic acid to
give a final concentration of 10%. Fixed cells were rinsed 5
times with deionized water and stained for 10 minutes with
0.4% SRB dissolved in 0.1% acetic acid. The wells were then
washed 5 times with 0.1% acetic acid and left to dry overnight.
The absorbed SRB was dissolved in 150 L 1% Tris base (pH
10.5). The absorbance of extracted SRB was measured on a
microplate reader at 515 nm.
2.4. Cell migration assay
To examine the effect of GSPs on the migration ability of
HMEC-1 cells, we performed cell migration assay as described
previously [19]. Briefly, 2 10
5
cells were suspended in 0.4 mL
serum-free MCDB131 medium supplemented with different
concentrations of GSPs or vehicle and added to upper
531 N U T R I T I O N R E S E A R C H 3 2 ( 2 0 1 2 ) 5 3 0 5 3 6
compartment of cell culture inserts. The lower compartment
contained 0.6 mL MCDB131 medium supplemented with 20%
FBS. After incubation for 24 hours at 37C, the nonmigrated
cells on the upper face of the membrane were removed with a
cotton swab. The migrated cells on the bottom surface of the
membrane were fixed with methanol and stained with 0.1%
crystal violet. Images from 5 randomly selected microscopic
fields were obtained under light microscopy. The number of
migrated cells was counted with Image-plus software. Each
sample was repeated 3 times.
2.5. Gelatin zymography
To examine the effect of GSPs on the enzymatic activities of
matrix metalloproteinase (MMP)2 and MMP-9 in HMEC-1
cells, we performed gelatin zymography as described previ-
ously [19]. Briefly, subconfluent HMEC-1 cells were incubated
for 24 hours in the absence or presence of different concen-
trations of GSPs in serum-free MCDB131 medium. The
conditioned medium was collected. After incubation with
sample buffer for 0.5 hours at 37C, the sample was separated
by electrophoresis on a 7.5% sodium dodecyl sulfate poly-
acrylamide gel containing 1% gelatin under nonreduced
conditions. The gel was washed twice with washing buffer
(50 mmol/L Tris-HCl [pH 7.5], 100 mmol/L NaCl, and 2.5%
Triton X-100) for 1 hour and then incubated in incubation
buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, and 10
mmol/L CaCl
2
) at 37C for 36 hours. Next, the gel was stained
with 0.25% Coomassie Brilliant Blue R250 and then destained
with 25% methanol and 7.5% acetic acid. Gelatinase activity
was detected as clear bands against a dark blue background.
2.6. Tube formation assay
To examine the effect of GSPs on the tube formation of HMEC-
1 cells, we performed tube formation assay as described
previously [20]. Briefly, Matrigel was distributed in 96-well
plates with 60 L per well and allowed to solidify at 37C for at
least 1 hour. The HMEC-1 cells were seeded at a density of 7.5
10
4
per well in 100-L culture medium containing different
concentrations of GSPs or vehicle control. Plates were then
incubated at 37C and 5% CO
2
for 8 hours until forming an
intact network in the control group. Images were captured
under inverted microscope (Olympus, IX41, Tokyo, Japan).
2.7. Chick CAM angiogenesis assay
To examine the in vivo antiangiogenic effect of GSPs, we
performed chick CAM assay as described previously [21].
Briefly, fertilized white Leghorn chicken eggs were incubated
under conditions of constant humidity at 37C. On day 8 of
incubation, a small hole was punched over the air sac to
detach the CAM from the eggshell; and then a square window
was opened on the broad side of the egg to expose CAM. Sterile
cellulose disks adsorbed with 40-L solution containing
various concentrations of GSPs or vehicle were placed onto
an avascular area of CAM. The eggs were returned to incubator
and incubated for 3 days. During this period, GSPs were added
every day. Three days later, the neovascular zones were
photographed and calculated.
2.8. Western blot analysis
To determine the effects of GSPs on VEGF- and Ang1-
dependent signaling cascades, HMEC-1 cells cultured in
serum-free MCDB131 medium were pretreated with or with-
out various concentrations of GSPs for 4 hours and then
stimulated with 50 ng/mL VEGF for 10 minutes or 200 ng/mL
Ang1 for 30 minutes. The whole-cell extracts were prepared in
lysis buffer supplemented with proteinase inhibitors (50
mmol/L NaF, 0.2 mmol/L Na
3
VO
4
, 1 mmol/L phenylmethyl-
sulfonyl fluoride, 2 g/mL aprotinin, and 2 g/mL leupeptin).
Equal amounts of protein from each sample were resolved by
6% to 10% sodium dodecyl sulfate polyacrylamide gels,
transferred onto the polyvinylidene fluoride membrane, and
probed with specific antibodies, followed by exposure to a
horseradish peroxidaseconjugated secondary antibody. Im-
munoreactivity was visualized by exposure to x-ray filmusing
enhanced chemiluminescence detection.
2.9. Statistical analyses
Results were expressed as means standard error (SE).
Statistical analyses were performed by Student t test using the
SPSS version 12 statistical analysis package (SPSS Inc, Chicago,
Illinois). A P value less than .05 was considered significant.
3. Results and discussion
3.1. Effect of GSPs on the cell viability of HMEC-1 cells
Grape seed proanthocyanidin treatment for 72 hours signif-
icantly inhibited cell viability of HMEC-1 cells in a dose-
dependent manner, with an IC
50
value of 99.32 g/mL.
However, treatment with GSPs (25-200 g/mL) for 24 hours
did not change cell viability of HMEC-1 cells (Fig. 1). Therefore,
0
20
40
60
80
100
120
C
e
l
l

v
i
a
b
i
l
i
t
y

(
%

o
f

C
o
n
t
r
o
l
)
24 h 72 h
***
**
***
Control 25 50 100 200
GSP (ug/ml)
Fig. 1 Effect of GSPs on the cell viability of HMEC-1 cells.
After treatment with GSPs for indicated time, the viability
was measured with SRB assay. Results were representative
of 3 independent experiments and are expressed as means
SE of 6 cultures with the vehicle control as 100%. **P < .01,
***P < .001 vs vehicle control.
532 N U T R I T I O N R E S E A R C H 3 2 ( 2 0 1 2 ) 5 3 0 5 3 6
all the following experiments were performed with GSP
treatment not exceeding 24 hours.
3.2. GSPs inhibit cell migration of HMEC-1 cells
Cell migration is necessary for endothelial cells to form
blood vessels during angiogenesis. After stimulating HMEC-
1 cells with 20% serum for 24 hours, a large number of
cells migrated to the lower side of the filter. Grape seed
proanthocyanidins significantly inhibited serum-induced
migration of HMEC-1 cells in a dose-dependent manner.
The inhibition of HMEC-1 cell migration by GSPs at 25, 50,
100, and 200 g/mL was 12.7%, 26.3%, 51.5%, and 87.9%,
respectively (Fig. 2A).
3.3. GSPs inhibit the secretion of MMP-2 and MMP-9 by
HMEC-1 cells
The secretion of MMPs is crucial for extracellular matrix
degradation, which is required for angiogenesis. After HMEC-1
cells were treated with various concentrations of GSPs for 24
hours in serum-free medium, the conditioned medium was
collected and assayed for MMP activity. Gelatin zymography
showed that MMP-2 and MMP-9 activities in the conditioned
mediumwere markedly reduced by GSPs in a dose-dependent
manner (Fig. 2B).
3.4. GSPs suppress the tube formation of HMEC-1 cells
Maturation of migrated endothelial cells into a tubelike
structure is a critical step for the formation of functional
vessels. As shown in Fig. 2C, HMEC-1 cells seeded on the
growth factorreduced Matrigel formed elongated and
robust tubelike structures within 8 hours. However, GSPs
significantly suppressed tube formation of HMEC-1 cells in
a dose-dependent manner. In particular, when treated
with 200 g/mL GSPs, most of the cells appeared as
unorganized cell aggregates; and the tube formation was
completely disrupted.
0
20
40
60
80
100
120
M
i
g
r
a
t
e
d

c
e
l
l
s

(
%

o
f

C
o
n
t
r
o
l
)
***
***
***
Control 25 50 100 200
A
Control GSP (200 ug/ml)
Control 25 50 100 200
MMP-9
MMP-2
B
Control GSP (25 ug/ml)
GSP (50 ug/ml) GSP (100 ug/ml)
GSP (200 ug/ml)
C
GSP (ug/ml)
GSP (ug/ml)
Fig. 2 Grape seed proanthocyanidins inhibit the cell
migration, MMP-2 and -9 secretion, and tube formation of
HMEC-1 cells. A, The migration of HMEC-1 cells was assessed
by cell migration assay as described in Methods and
materials. Representative photomicrographs were shown.
Values were representative of 3 independent experiments
and are expressed as means SE of 5 fields with the vehicle
control as 100%. *** P < .001 vs vehicle control. B, The
secretion of MMP-2 and -9 by HMEC-1 cells was detected by
gelatin zymography. The clear zone of gelatin digestion
indicated the presence of MMP-2 and -9. The photographs
were the representative of 3 independent experiments. C,
The HMEC-1 cells were seeded on the Matrigel-coated
96-well plates at the density of 7.5 10
4
per well in the
presence of different concentrations of GSPs or vehicle
control. After 8 hours, photographs of endothelial tube
formation were taken. Results were representative of 3
independent experiments.
533 N U T R I T I O N R E S E A R C H 3 2 ( 2 0 1 2 ) 5 3 0 5 3 6
3.5. GSPs inhibit angiogenesis in vivo
To further confirm the antiangiogenic effect of GSPs, we
examined the antiangiogenic activity of GSPs in vivo. The
results showed that CAM in the control group showed well-
developed zones of neovascularization around the sterile
cellulose disks after 3 days of incubation. In contrast, CAM
neovascularization, especially the small vessels, was signifi-
cantly suppressed by GSPs in a dose-dependent manner
(Fig. 3). However, GSP treatment did not affect the preexisting
vessels. These results indicate that GSPs dramatically inhib-
ited angiogenesis in vivo and that the antiangiogenic effect
was not due to their cytotoxic effect.
3.6. GSPs inhibit VEGF-induced phosphorylation of
VEGFR2 and ERK1/2 in HMEC-1 cells
Vascular endothelial growth factor/VEGFR2 signaling is a key
pathway that regulates endothelial cell proliferation, migra-
tion, and differentiation [22]. As shown in Fig. 4A and C,
stimulation of HMEC-1 cells with exogenous VEGF (50 ng/mL)
for 10 minutes significantly enhanced the phosphorylation of
VEGFR2. Pretreatment with GSPs significantly blocked VEGF-
induced phosphorylation of VEGFR2 without affecting total
VEGFR2 expression level. To identify the downstream signal-
ing pathway targeted by GSP treatment, we examined the
expression and phosphorylation of ERK1/2, one of the key
signaling pathway components supporting endothelial cell
survival, migration, and differentiation. We found that GSPs
inhibited the phosphorylation of ERK1/2 in HMEC-1 cells, but
did not affect total ERK1/2 level. Our results are consistent
with previous reports that grape seed extracts inhibited
angiogenesis via the suppression of VEGFR/MAPK signaling
pathway [16].
3.7. GSPs inhibit Ang1-induced phosphorylation of Tie2
and ERK1/2 in HMEC-1 cells
Activation of Tie2 by Ang1 has been linked to the promotion of
endothelial cell survival, and the induction of migration and
sprouting [23]. Therefore, we examined the activation of Tie2
in HMEC-1 cells. Following the stimulation with Ang1 (200 ng/
mL) for 30 minutes, Tie2 was strongly tyrosine phosphorylat-
ed in HMEC-1 cells. However, preincubation of HMEC-1 cells
with various concentrations of GSPs led to dramatic suppres-
sion of Ang1-stimulated Tie2 tyrosine phosphorylation.
Furthermore, we found that Ang1 markedly increased the
phosphorylation of ERK-1/2 in HMEC-1 cells, but that pre-
treatment with GSPs significantly inhibited Ang1-stimulated
ERK-1/2 activation (Fig. 4B and D).
Recent studies have shown that proanthocyanidins ex-
hibit a range of beneficial effects such as reducing hepatic
lipid accumulation [24], inhibiting autoimmunity [25], and
providing cardioprotection [26]. In the present study, we
Control GSP (5 ug/egg) GSP (20 ug/egg)
C B A
0
20
40
60
80
100
120
V
a
s
c
u
l
a
r

d
e
n
s
i
t
y

(
%

o
f

C
o
n
t
r
o
l
)
***
***
D
Control 5 20
GSP (ug/egg)
Fig. 3 Grape seed proanthocyanidins inhibit in vivo angiogenesis in CAM. Fertilized chicken eggs were incubated for 7 days,
and then cellulose disks saturated with different concentrations of GSPs were placed on the CAMs every day. Three days later,
patterns of angiogenesis on the CAM were photographed. Vehicle control (A), 5 g GSPs per egg (B), or 20 g GSPs per egg (C). D,
Vascular densities around cellulose disk were quantified. Values were expressed as means SE of 4 to 5 separate eggs with the
vehicle control as 100%. ***P < .001 vs vehicle control.
534 N U T R I T I O N R E S E A R C H 3 2 ( 2 0 1 2 ) 5 3 0 5 3 6
provide several lines of evidence to show that GSPs exhibit
antiangiogenic effects both in vitro and in vivo and that this
is attributed to the inhibition of both VEGF and Ang1
signaling. Vascular endothelial growth factor/VEGFR2 and
Ang1/Tie2 signaling pathways are known to regulate differ-
ent but complementary aspects of blood vessel growth in
tumors. Vascular endothelial growth factor/VEGFR2 signaling
plays important role in new blood vessels formation,
whereas Ang1/Tie2 signaling contributes to new blood
vessels' maturation and stabilization. Simultaneous inhibi-
tion of both pathways not only exhibits additive inhibitory
effects on angiogenesis but also overcomes the resistance to
anti-VEGF therapy. Our new findings provide support for our
hypothesis that GSPs inhibit multiple angiogenic pathways
such as VEGF/VEGFR2 and Ang1/Tie2 signaling to execute the
antiangiogenic actions.
However, it is important to note that GSPs are a complex
mixture of various polyphenol compounds including procya-
nidins B1 to B5, procyanidin C1, and procyanidin B5-3-gallate.
Further studies will be needed to determine whether the
inhibition of VEGFR2 and Tie2 phosphorylation is due to one
kind of active component alone or the combinative effects of
different components. In addition, the effective concentration
of GSPs in this study is relatively higher than those in previous
reports. This may be related to the fact that the methods for
preparing GSPs are different, resulting in the variation of the
concentration of active components in GSPs.
In summary, our results showed that GSPs inhibited the
migration, MMP-2 and -9 secretion, and tube formation of
HMEC-1 cells in vitro in a dose-dependent manner. In
addition, chick CAM assay showed that GSPs inhibited
neovascularization in a dose-dependent manner. Further-
more, we demonstrated that GSPs inhibited the phosphory-
lation of VEGFR2 and Tie2 as well as downstream signaling
component ERK1/2. The inhibition of both VEGF and Ang1
signaling by GSPs may explain the strong antiangiogenic
VEGF
GSP (ug/ml) 0 0 50 100 200
A
P-VEGFR2
VEGFR2
P-ERK1/2
ERK1/2
Actin
Ang1
GSP (ug/ml) 0 0 50 100 200
B
P-Tie2
Tie2
P-ERK1/2
ERK1/2
Actin
0
0.2
0.4
0.6
0.8
1
1.2
p
-
V
E
G
F
R
2
/
V
E
G
F
R
2

r
a
t
i
o
GSP (ug/ml) 0 0 50 100 200
VEGF
**
*
C
0
0.2
0.4
0.6
0.8
1
1.2
p
-
T
i
e
/
T
i
e

r
a
t
i
o
GSP (ug/ml) 0 0 50 100 200
Ang1
***
***
*
*
D
Fig. 4 Grape seed proanthocyanidins inhibit VEGF-induced VEGFR2 phosphorylation, Ang1-induced Tie 2 phosphorylation,
and the activation of downstream effector ERK1/2. The HMEC-1 cells were pretreated with various concentrations of GSPs for 4
hours and stimulated with 50 ng/mL VEGF for 10 minutes (A) or 200 ng/mL Ang1 for 30 minutes (B). Western blotting analysis
was performed using anti-p-VEGFR2, VEGFR2, anti-p-Tie2, Tie2, p-ERK, and ERK antibodies. Actin served as loading control.
Densitometric analysis (values are means SE) revealed that GSPs reduced the ratios of phosphorylated VEGFR2/VEGFR2 (C)
and phosphorylated Tie/Tie (D).The significance was assessed with a Student t test. *P < .05, **P < .01, and ***P < .001 compared
VEGF or Ang1 alone.
535 N U T R I T I O N R E S E A R C H 3 2 ( 2 0 1 2 ) 5 3 0 5 3 6
effects of GSPs, which could inhibit both sprouting angiogen-
esis and the maturation of blood vessels. These data indicate
that GSPs could be developed as a pharmacologically safe
chemopreventive agent against cancer.
Acknowledgment
This work was supported by the grant from National Natural
Science Foundation of China (no. 30700142 to Shuangsheng
Huang) and Scientific Research InnovationTeamof Northwest
University for Nationalities (no. XBMUCXTD-2011-1).
The authors declare no conflicts of interest.
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