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Cancer Letters 318 (2012) 221–225
Contents lists available at SciVerse ScienceDirect
Cancer Letters
journal homepage: www.elsevier.com/locate/canlet
Blockade of VEGF-A suppresses tumor growth via inhibition of autocrine signaling
through FAK and AKT
Jungwhoi Lee a, Taeyun Ku a,b, Hana Yu a, Kyuha Chong a,b, Seung-Wook Ryu a,c, Kyungsun Choi a,c,
Chulhee Choi a,b,c,⇑
a
b
c
Cell Signaling and Bio Imaging Laboratory, Department of Bio and Brain Engineering, KAIST, Daejeon 305-701, Republic of Korea
Graduate School of Medical Science and Engineering, KAIST, Daejeon 305-701, Republic of Korea
KAIST Institute for the BioCentury, KAIST, Daejeon 305-701, Republic of Korea
a r t i c l e
i n f o
Article history:
Received 24 September 2011
Received in revised form 17 November 2011
Accepted 9 December 2011
Keywords:
VEGF
FAK
AKT
Apoptosis
Autocrine signaling
a b s t r a c t
Blockade of VEGF signaling using RNA interferences, a neutralizing antibody, an antagonizing soluble
VEGF receptor, and a receptor tyrosine kinase inhibitor induced anti-tumor effects in human astrocytoma
U251-MG and fibrosarcoma HT-1080 in vitro in a dose-dependent manner. Furthermore, blockade of
VEGF-A using the doxycycline-inducible VEGF-A RNA interference system showed a significant antitumor effect in a murine HT-1080-xenograft model. Anti-tumor effect through the blockade of VEGF signaling was mediated by FAK and AKT pathway in vitro and in vivo. These results collectively indicate that
VEGF-A and its receptors can act as key inducer of tumor growth as well as angiogenesis.
Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Even though targeting the VEGF and VEGFRs pathway has been
proven effective in the treatment of human cancers [1,2], the multitude of biological actions of VEGF blockade such as vessel disruption
and normalization prompts us to exploit the alternative role of VEGF
inhibition and its therapeutic potential [3]. Traditional anti-angiogenic strategies attempt to inhibit new vessel formation and/or to
destroy existing vessels to starve the tumor from its nutrients [4];
however, it is now becoming increasingly clear that normalizing,
and not only pruning, the tumor vessels can be beneficial [3].
Moreover, the heterogeneity of clinical responsiveness and adverse
effects has motivated new attraction in re-searching biological
meanings of VEGF and its suppression in the complex tumor
microenvironment [5,6].
We have previously shown that blockade of VEGF signaling induced a significant anti-tumor effects in various human cancer cell
types in vitro, especially human malignant glioblastoma and fibrosarcoma cells [7]. On the contrary, we had also shown that blockade of VEGF improved the perfusion of tumor-associated blood
vessels as well as vascular permeability [8]. In the present study,
we tried to verify the effects of anti-VEGF treatment on the angiogenesis or tumor-growth, and investigate alternative mechanisms
responsible for anti-tumor effects of VEGF blockade other than
ablating tumor-associated vessels using in vitro and in vivo models.
Our reports are the first to suggest that pertinent inhibition of
autocrine and paracrine VEGF signaling function may be effective
to inhibit tumor growth in consort with anti-angiogenesis for
VEGF-blockade dependent cancers.
2. Materials and methods
2.1. Cell culture and reagents
Abbreviations: FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase; HIF-1a, hypoxic inducible factor 1a; siRNA, small interfering RNA;
shRNA, small hairpin RNA; VEGF, vascular endothelial growth factor; VEGFR,
vascular endothelial growth factor receptor; Z-VAD, carbobenzoxy-valyl-alanyl
-aspartyl-[O-methyl]-fluoromethylketone.
⇑ Corresponding author at: Department of Brain and Bioengineering, KAIST, 291
Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Tel.: +82 42 350 4321;
fax: +82 42 350 4380.
E-mail address: cchoi@kaist.ac.kr (C. Choi).
URL: http://csbi.kaist.ac.kr (C. Choi).
0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2011.12.014
HT-1080 (a human fibrosarcoma cell line) and U251-MG (a human glioblastoma
cell line) were obtained from the American Type Culture Collection (ATCC, Manassa,
VA). The cells were grown in DMEM (Gibco BRL, Gaithersburg, MD, USA) medium
supplemented with 10% Fetal Bovine Serum (Gibco) and 1 Â 105 unit/L Penicilin100 mg/L Streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified
atmosphere containing 5% CO2. AKT inhibitor (LY294002) and FAK inhibitor
(SC203950) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Antibodies for FLK-1 and FLT-1 were obtained from Santa Cruz Biotechnology
and for phospho-FAK (Y397 and Y576/577), FAK, phospho-AKT (Ser473), AKT,
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J. Lee et al. / Cancer Letters 318 (2012) 221–225
phospho-ERK (Thy202/204), ERK, NRP-1, caspase-3, cleaved caspase-3, CD31, PCNA,
HIF-1a, and GAPDH were obtained from Cell Signaling Technology (Beverly, MA,
USA). Recombinant vascular endothelial growth factor-A was obtained from R&D
Systems (Minneapolis, MN, USA).
2.2. siRNA transfection
siRNA transfection was performed using an effectene (Qiagen, Hilden, Germany), according to the manufacturer’s protocol as described previously [7]. The
siRNA oligonucleotides that encode VEGF-A, FLT-1, FLK-1, NRP-1, and scrambled
control were obtained from Bioneer (Daejeon, Korea). The sequences of the siRNAs
were as follows: VEGF-A, 50 -AAAUGUGAAUGCAGACCAA-30 -dTdT, FLT-1: 50 -GACUC
UCUUCUGGCUCCUA-30 -dTdT, FLK-1: 50 -CUCCUAAUGAGAGUUCCUU-30 -dTdT, NRP1: 50 -GUCCGAAUCAAGCCUGCAA-30 -dTdT and scrambled control, 50 -CCUACGCCAAUUUCGU-30 -dTdT.
2.3. Measurement of cell death
To assess cell death, LDH (lactate dehydrogenase) assay (Promega, Madison, WI,
USA) was carried out according to the manufacturer’s protocol. After 30 min incubation at room temperature, the absorbance was measured at 490 nm by using a
microplate reader (Bio-Rad, Richmond, CA, USA). To evaluate cell viability, WST-1
reagent (Nalgene, Rochester, NY) was used as previously [7].
2.4. Western blot analysis and ELISA
Nuclear/cytosol Fractionation Kit (Bio Vision, Mountain View, CA, USA) was performed according to the manufacturer’s instruction using the tumor samples extracted from mice. Extracted proteins were separated by SDS–PAGE as previously
described [9]. The cytoplasmic fractions were probed with GAPDH, and nuclear fractions were probed with PARP (poly ADP-ribose polymerase). Human VEGF DuosetÒ
ELISA Development System (R&D system, Minneapolis, USA) was performed according to the manufacturer’s instruction using the blood samples extracted from mice.
2.5. Generation of VEGF-specific shRNA stable cell lines
For stable suppression of VEGF-A expression by short hairpin (sh)-activated gene
silencing vector system, plasmids expressing shRNAs were constructed by synthesizing cDNA oligonucleotides bearing the target sequence, Xhol1 and HindIII linker, and
then ligated into XhoI and HindIII sites of pSingle-tTs-shRNA vector (tTs-VEGF shRNA,
Clontech). The target sequences for VEGF, corresponding to region at nucleotides
379–397 of human VEGF mRNA (GeneBank accession No. GI6631028), were 50 AAATGTGAATGCAGACCAA-30 . One day after transfection with the tTs-VEGF-A shRNA
constructs, HT-1080 cells were grown in Dulbecco’s complete medium containing
1 g/LG418 for 3 days followed by an additional 6 days of culture in Dulbecco’s complete medium containing 400 mg/L G418 for the selection of stable transfectants.
2.6. Xenograft tumor model
Balb/c nude mice were obtained from Orient (Seongnam, Korea) at 5–6 weeks of
age. All mice were housed and handled in accordance with the Animal Research
Committee’s Guidelines at KAIST (Daedeok Science Town, Daejeon, Republic of Korea). When tumors reached an average size approximately 3 cm3, water containing
200 mg/L doxycycline was fed to mice. Tumor volume was calculated using the following formula: V = 0.523 LW2 (L = length, W = width). Animals were anesthetized
by ketamine and xylazine, perfused with PBS, and followed by fixation with formalin (Sigma–Aldrich, St. Louis, MO, USA). Specimens were excised, immersed in formalin, and transferred to 30% sucrose (Sigma) solution. Immunohistochemistry was
performed using the VECTASTAINÒ ABC-kit (Vector Laboratories, Inc., Burlingame,
CA, USA) as manufacturer’s recommendation.
2.7. Statistical analysis
Data are presented as the mean ± standard deviation (SD). Levels of significance
for comparisons between two independent samples were determined using the
Student’s t-test. Groups were compared by one-way analysis of variance (ANOVA)
with Tukey’s post hoc test applied to significant main effects (SPSS 12.0 K for Windows; SPSS Inc., Chicago, IL, USA).
3. Results
3.1. Blockade of VEGF-A induced apoptotic cell death via inhibition of
AKT and FAK
Previously, we have demonstrated that VEGF blockade induced
a significant cell death in human glioblastoma and fibrosarcoma
cell lines [7]. We further confirmed the functional dependency of
HT-1080 and U251-MG cells on VEGF-A by showing the dosedependent induction of cell death by inhibition of VEGF signaling
using anti-VEGF-A monoclonal antibody, soluble VEGF receptor
and receptor tyrosine kinase inhibitor (Fig. 1).
To elucidate the anti-cancer mechanism of VEGF-A blockade,
we examined the signal transduction pathways that are specifically activated by VEGF stimulation (Fig. 2A). Treatment with
VEGF-A induced a time-dependent increase in FAK and AKT phosphorylation. Inhibition of VEGF-A-induced activation of AKT and
FAK by pharmacological inhibitors induced a significant cell death
in a dose-dependent manner, which was significantly suppressed
by pretreatment of a pan-caspase inhibitor, z-VAD (Fig. 2B). The
involvement of caspases was confirmed by immunoblot analysis
specific for active caspase-3 (Fig. 2C). Treatment with pharmacological inhibitors of AKT and FAK induced a significant cell death
even in the absence of exogenous VEGF-A (data not shown). The
involvement of autocrine FAK and AKT activation in HT-1080 cells
was confirmed by immunoblot analysis (Fig. 2D). Similar results
were obtained using U251-MG cells (data not shown). Interestingly, inhibition of FAK also suppressed phosphorylation of AKT;
while specific inhibition of AKT had little effect on FAK phosphorylation, suggesting that FAK might be an upstream signaling event
in this setting. To confirm that, we further inhibited FAK expression
using siRNAs to demonstrate that knockdown of FAK suppressed
both FAK and AKT phosphorylation in HT-1080 cells (Fig. 2E). Similar results were also obtained in U251-MG cells (data not shown).
3.2. Inhibition of tumor-derived VEGF-A suppressed tumor growth
independently from anti-angiogenic activities
We constructed a doxycycline-inducible sh-VEGF HT1080 cell
line (supplementary figure), then investigated the effect of specific
targeting of tumor-derived VEGF-A in vitro and in vivo. First, we
checked the knock-down efficacy of VEGF-A in these cells in vitro
(Fig. 3A). Treatment with tetracycline induced a significantly
reduction of VEGF-A accompanied by inhibition of AKT and FAK
phosphorylation, confirming that AKT and FAK are downstream
signals responsible for VEGF-dependency. Secretion of VEGF-A
was also significantly suppressed by inducible expression of shRNA
specific for VEGF-A (data not shown). Cell viability also decreased
significantly upon induction of VEGF knockdown in vitro (Fig. 3B).
Such an anti-proliferative effect of VEGF knockdown was also observed in HT-1080 xenograft model transplanted with the doxycycline-inducible sh-VEGF HT1080 cells (Fig. 3C). The tumor-laden
mice were fed with doxycycline when the tumors reached to an
average size approximately 3 cm3. Control tumors grew to an average size of 6.24 ± 1.9 cm3 by 46 days after transplantation; while
the shRNA-inducible tumors remained to an average size of
2.94 ± 0.9 cm3 by 46 days after 6 days of doxycycline treatment.
The serum level of human-specific VEGF-A was significantly lower
in shRNA-inducible tumors compared to the control (Fig. 3D).
We further investigated the possible therapeutic mechanisms of
inhibition of tumor-derived VEGF-A by histochemical analysis. As
expected, the vessel areas and number of CD31-positive endothelial cells were significantly reduced in VEGF-suppressed tumors
(Fig. 4A and B), which were consistent with previous results of ours
and others [3,8]. To verify the effect of tumor vessel reduction, we
next examined the level of HIF-1a protein, a hypoxia-sensitive protein, in the cytosol and nuclear fractions extracted from the tumors
(Fig. 4C). In VEGF-suppressed tumors, the level of HIF-1a protein
was substantially lower compared to the control tumors, indicating
that the tumor vessel reduction was not associated with vessel disruption leading to tissue hypoxia but rather tumor vessel normalization. To confirm the hypothesis, we analyzed the involvement of
tissue hypoxia with tumor cell apoptosis in the core and peripheral
regions of the tumors (Fig. 4D). In the central necrotic regions, both
223
J. Lee et al. / Cancer Letters 318 (2012) 221–225
A
P <0.01
P <0.01
P <0.01
B
P <0.01
*
95
90
*
*
*
85
*
*
*
*
80
75
20
*
*
*
HT1080
U251
**
**
*
10
0
0 2 5 10
0 2 5 10
- - - -
- - - -
VEGF-Trap - - - -
- - - -
0 10 50 100
0 10 50 100 (mg/L)
α-VEGF
*
P =0.046
Cell Death (%)
Viability (%)
P <0.01
HT1080
U251
100
(mg/L)
SU1498
0
5
10 20 50
0
5 10 20 50 (μmol/L)
Fig. 1. Dose-dependent anti-cancer effect of VEGF blockade. (A) HT-1080 and U251-MG cells were treated with varying doses of an anti-VEGF-A neutralizing antibody and
VEGF-Trap for 72 h. Viability was measured by WST-1 assay. (B) Cells were incubated with varying doses of SU1498 for 20 h, and the cell death was measured by LDH assay.
(n = 3; Tukey’s post hoc test was applied to significant group effects in ANOVA, ÃP < 0.05, ÃÃP < 0.01).
A
B
VEGF-A
0
( / L)
10
30
60
120
P <0.001
P <0.001
(min)
*
70
Cell Death (%)
p-FAK
FAK
p-AKT
AKT
P =0.02
60
**
50
**
40
30
*
*
P =0.01
*
20
10
GAPDH
0
SC203950
LY294002
VEGF
Z-VAD
C
(kDa)
Caspase-3
32
Cleaved
Caspase-3
19
GAPDH
SC203950
LY294002
VEGF
Z-VAD
+
-
+
+
-
+
+
-
+
+
+
+
+
+
0 5 10 20 50 50
- - - - - + + + + + +
- - - - - +
D
- - - - - - (μmol/L)
0 5 10 20 50 50 (μmol/L)
+ + + + + +
- - - - - +
E
p-FAK
(Y397)
p-FAK
(Y576/577)
p-FAK
(Y397)
p-FAK
(Y576/577)
FAK
FAK
p-AKT
p-AKT
AKT
AKT
GAPDH
SC203950
LY294002
Wortmannin
GAPDH
-
-
-
+
-
+
scrambled
si-RNA
si-FAK
+
-
-
+
Fig. 2. FAK and AKT are downstream signals of VEGF. (A) HT-1080 cells were incubated with VEGF-A (50 lg/L) for varying time periods, and the cell lysates were subjected to
western blot analysis using antibodies specific for phosphor-FAK (Y397), total FAK, phosphor-AKT (S473), total AKT and GAPDH. (B) Cells were treated with varying doses of
SC203950 (FAK inhibitor) and LY29400 (AKT inhibitor) in the presence of exogenous VEGF-A (50 lg/L) for 20 h. Cytotoxicity was measured by LDH assay. (n = 3; Tukey’s post
hoc test was applied to significant group effects in ANOVA, ÃP < 0.05, ÃÃP < 0.01). (C) Cells were incubated in the absence or presence of VEGF-A (50 lg/L) or Z-VAD (10 lmol/L)
for 1 h, and further treated with SC203950 or LY294002 for 20 h. Cell lysates were subjected to immunoblot analysis using antibodies against total caspase-3, cleaved
caspase-3 and GAPDH. (D) Cells were incubated in the absence or presence of varying doses of pharmacological inhibitors of AKT and FAK for 20 h, and the cell lysates were
examined for expression of phosphor-FAK (Y397 and Y576/577), FAK, phospho-AKT (S473), AKT and GAPDH. (E) Cells were transfected with siRNA specific for FAK, and then
examined for expression of various proteins by immunoblot analysis.
VEGF-suppressed and control tumors exhibited high levels of HIF1a and active caspase-3. The number of HIF-1a positive cells was
lower in VEGF-suppressed tumors even though the difference is
not statistically significant. On the contrary, in the peripheral regions where the blood perfusion is higher than the core regions
[8], the level of HIF-1a protein was quite lower in both VEGF-suppressed and control tumors; however, the level of active caspase-3
was significantly higher in VEGF-suppressed tumors. These results
suggest that the reduction of tumor size in VEGF-suppressed tumors was not associated with tissue hypoxia. The in vivo involvement of downstream kinases for VEGF dependency was also
confirmed by immunoblot analysis using tumor extracts (Fig. 4E).
4. Discussion
In the present study, we demonstrated that selective inhibition
of tumor-derived VEGF-A can suppress tumor growth in vivo by
inhibition of autocrine tumor growth signals. We further showed
that FAK and AKT are the downstream signal transduction pathways responsible for VEGF-A dependency in human glioblastoma
and fibrosarcoma cells; while the FAK signaling seems to be upstream to the AKT pathway. This is the first report to our knowledge in that anti-VEGF therapy might exert its anti-cancer effects
in some VEGF-dependent malignancies independently from its
anti-angiogenic effect whether it is disruptive or normalizing,
224
J. Lee et al. / Cancer Letters 318 (2012) 221–225
A
B
sh-Vector sh-VEGF-A
VEGF-A
sh-Vector
p-FAK
sh-VEGF-A
Viability (%)
100
FAK
p-AKT
90
*
80
70
AKT
-
Dox
-
+
+
GAPDH
C
-
+
-
D
+
Dox - (n=3)
9
8
7
6
5
4
3
2
1
0
Dox + (n=4)
Serum VEGF-A
(rel. secretion)
Tumor volume (cm3)
Dox
*
*
Dox (-/+)
1.75
Dox -
1.50
Dox +
*
*
1.25
1.00
*
0.75
0.50
40
41
42
43
44
45
46
0
Days after cancer cells implantation
3
6
Days after doxycyclin treatment
Fig. 3. In vivo effect of VEGF silencing in tumor xenograft model. (A) and (B) HT-1080 cells stably expressing control or shVEGF vectors were incubated in the absence or
presence of 2 mg/L doxycycline, and the cell lysates were examined for expression of VEGF-A, FAK and AKT. Cell viability was measured using WST-1 assay (a representative
of more than three independent experiments. n = 5, ÃP < 0.05). (C) The effect of VEGF-A silencing on tumor growth in vivo (ÃP < 0.05). (D) Human-specific VEGF expression was
determined by ELISA in the plasma of the mice. VEGF level is expressed as the quantity of relatively secreted human VEGF (n = 3; Tukey’s post hoc test was applied to
significant group effects in ANOVA, ÃP < 0.05; y scale 1.00 indicates 13500 ng/L of VEGF-A in the plasma).
60
50
**
40
C
# of CD31+ vessel/
microscopic fields
B
Vessel areas/
microscopic fields
A
30
20
10
0
Dox
C
90
+
N
C
+
N
GAPDH
70
PARP
60
Dox
-
HIF-1α
*
80
50
-
sh-VEGF-A
Dox
E
-
Dox
+
sh-VEGF-A
+
VEGF-A
p-FLK-1
D
central region
500
# of positive
stained cells
peripheral region
P =0.19
n.s
P =0.01
400
FLK-1
HIF-1α
Cleaved
Caspase-3
p-FAK
FAK
300
p-AKT
200
100
AKT
0
PCNA
Dox
-
+
-
+
GAPDH
Fig. 4. Anti-cancer effect of VEGF silencing is not associated with anti-angiogenic action. (A) Blood vessel areas (pixels) and the number of CD31 positive vessels (B) were
assessed by immunohistochemical staining in shVEGF expressing HT-1080 tumors. Magnification, Â200. (C) Fractionated lysates (C, cytosol fraction; N, nuclear fraction) from
tumor samples was subjected to the immunoblot analysis for HIF-1a expression. GAPDH and PARP were measured to test the efficacy of cell fractionation. (D) The number of
HIF-1a and cleaved caspase-3 positive cells was analyzed in tumors. Peripheral region means the outside of three quarters in the section of tumor samples. Magnification,
Â200. (E) The lysates from tumor samples were tested for VEGF-A, p-FLK-1, FLK-1, p-FAK, FAK, p-AKT, AKT, PCNA and GAPDH by immunoblot analysis.
J. Lee et al. / Cancer Letters 318 (2012) 221–225
and that AKT and FAK signaling pathways can be potential therapeutic targets in VEGF-sensitive tumors.
Multiple pro-angiogenic functions are attributed to VEGFs,
including the activation of endothelial cell invasion, migration,
and proliferation [10]. Tight balance between pro-angiogenic and
anti-angiogenic signals in the tumor microenvironment that governs the response of endothelial cells is critical. Accordingly, VEGF,
one of the most effective pro-angiogenic factor, can critically shift
this balance to angiogenesis process [11]. In agreement with this,
vessel areas and CD31-positive vessels were significantly
decreased when tumor-derived VEGF-A was specifically targeted.
The reduction of tumor vessels seems to be the result of tumor
vessel normalization rather than disruptive changes of tumorassociated vessels since there were less hypoxic markers in
VEGF-suppressed tumors.
Given that inhibition of VEGF decreases tumor growth in vivo,
our results are consistent with previous studies [12,13]. However,
the reduction of tumor sizes seems to be independent from both
tumor vessel normalization and disruption, which are usually regarded the leading mechanisms responsible for anti-cancer effect
of VEGF blockade [2,3,14]. In concordance with our initial hypothesis, we could conclude that the reduction of tumor growth was
closely related to the autocrine or paracrine VEGF signaling in
the cancer cells based on several findings. Firstly, the immunereactivity to active caspase-3, a marker for apoptotic cell death,
was even higher in properly perfused tumor periphery of VEGFsuppressed tumors, indicating that increased cell death was not related to vessel disruption. Secondly, the tissue level of HIF-1a, a
specific marker of hypoxia, was significantly lower in VEGF-suppressed tumors, where the tumor growth was markedly inhibited.
Lastly, the downstream signals responsible for tumor growth
in vitro were also remarkably reduced in vivo in VEGF-suppressed
tumors. Even though these results suggest that the reduction of tumor size in VEGF-suppressed tumors might not be associated with
tissue hypoxia, the reduction of tissue oxygen demand by substantial cell death might also result in relative non-hypoxic condition
in VEGF-suppressed tumors. In summary, blockade of VEGF evoked
prominent anti-tumor effect via inhibition of autocrine and/or paracrine VEGF/VEGFRs signaling independently from angiogenesis
through the FAK and AKT pathway. This mechanism may contribute to the adoption of patient-specific therapeutic strategy targeting VEGF/VEGFRs signaling.
225
Acknowledgments
This research was supported by the Bio & Medical Technology
Development Program of the National Research Foundation (NRF)
funded by the Korean Government (MEST) (No. 2010-0030001).
We authors thank prof. G.Y. Koh (Department of Biological Sciences, KAIST) for providing the VEGF-Trap.
References
[1] N. Ferrara, K. Alitalo, Clinical applications of angiogenic growth factors and
their inhibitors, Nat Med 5 (1999) 1359–1364.
[2] P. Carmeliet, R.K. Jain, Angiogenesis in cancer and other diseases, Nature 407
(2000) 249–257.
[3] P. Carmeliet, R.K. Jain, Principles and mechanisms of vessel normalization for
cancer and other angiogenic diseases, Nat Rev Drug Discov 10 (2011) 417–427.
[4] J. Folkman, Tumor angiogenesis: therapeutic implications, New Engl J Med 285
(1971) 1182–1186.
[5] T.T. Batchelor, A.G. Sorensen, E. di Tomaso, W.T. Zhang, D.G. Duda, K.S. Cohen,
K.R. Kozak, D.P. Cahill, P.J. Chen, M. Zhu, M. Ancukiewicz, M.M. Mrugala, S.
Plotkin, J. Drappatz, D.N. Louis, P. Ivy, D.T. Scadden, T. Benner, J.S. Loeffler, P.Y.
Wen, R.K. Jain, AZD2171 a pan-VEGF receptor tyrosine kinase inhibitor
normalizes tumor vasculature and alleviates edema in glioblastoma patients,
Cancer Cell 11 (2007) 83–95.
[6] H.X. Chen, J.N. Cleck, Adverse effects of anticancer agents that target the VEGF
pathway, Nat Rev Clin Oncol 6 (2009) 465–477.
[7] J. Lee, H. Yu, K. Choi, C. Choi, Differential dependency of human cancer cells on
vascular endothelial growth factor-mediated autocrine growth and survival,
Cancer Lett 309 (2011) 145–150.
[8] M. Choi, K. Choi, S.W. Ryu, J. Lee, C. Choi, Dynamic fluorescence imaging for
multiparametric measurement of tumor vasculature, J Biomed Opt 16 (2011)
046008.
[9] J. Jeon, J. Lee, C. Kim, Y. An, C. Choi, Aqueous extract of the medicinal plant
Patrinia villosa Juss. Induces angiogenesis via activation of focal adhesion
kinase, Microvasc Res 80 (2010) 303–309.
[10] N. Ferrara, W.J. Henzel, Pituitary follicular cells secrete a novel heparinbinding growth factor specific for vascular endothelial cells, Biochem Biophys
Res Commun 161 (1989) 851–858.
[11] G. Bergers, L.E. Benjamin, Tumorigenesis and the angiogenic switch, Nat Rev
Cancer 3 (2003) 401–410.
[12] J.Y. Yoo, J.H. Kim, Y.G. Kwon, E.C. Kim, N.K. Kim, H.J. Choi, C.O. Yun, VEGFspecific short hairpin RNA-expressing oncolytic adenovirus elicits potent
inhibition of angiogenesis and tumor growth, Mol Ther 15 (2007) 295–302.
[13] A. Hanyu, K. Kojima, K. Hatake, K. Nomura, H. Murayama, Y. Ishikawa, S. Miyata,
M. Ushijima, M. Matsuura, E. Ogata, K. Miyazawa, T. Imamura, Functional in vivo
optical imaging of tumor angiogenesis growth and metastasis prevented by
administration of anti-human VEGF antibody in xenograft model of human
fibrosarcoma HT1080 cells, Cancer Sci 100 (2009) 2085–2092.
[14] H. Choi, M. Choi, K. Choi, C. Choi, Blockade of vascular endothelial growth
factor sensitizes tumor-associated vasculatures to angiolytic therapy with a
high-frequency ultrashort pulsed laser, Microvasc Res 82 (2011) 141–146.
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