Breast Cancer Res Treat (2013) 142:9–18
DOI 10.1007/s10549-013-2724-6
PRECLINICAL STUDY
Reduction of tumor angiogenesis induced by desmopressin
in a breast cancer model
Giselle V. Ripoll • Juan Garona • Marina Pifano
Hernan G. Farina • Daniel E. Gomez •
Daniel F. Alonso
•
Received: 8 August 2013 / Accepted: 3 October 2013 / Published online: 12 October 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Desmopressin (DDAVP), a synthetic peptide
analog of vasopressin, is a safe antidiuretic and hemostatic
compound that acts as a selective agonist for the vasopressin V2 membrane receptor. It is known that DDAVP
can inhibit progression of residual metastatic cells and also
improves chemotherapy effects in preclinical breast cancer
models. Here, we explored the effects of DDAVP on tumor
angiogenesis using the aggressive F3II mammary carcinoma in syngeneic Balb/c mice. Intravenous administration
of the compound (2 lg/kg) markedly decreased vascularization of growing subcutaneous tumors, as well as inhibited the early angiogenic response around intradermal
inoculation sites. In vitro studies confirmed the presence of
vasopressin V2 receptors on F3II cells and a modest antiproliferative activity of DDAVP. Interestingly, conditioned
media from F3II monolayers exposed to low doses of
DDAVP (100 nM) significantly increased angiostatin formation in the presence of purified plasminogen. Such
increase was associated with an enhancement of tumorsecreted urokinase-type plasminogen activator, suggesting
the proteolytic conversion of plasminogen to angiostatin
in vitro. Similar results were observed with the MCF-7
human breast carcinoma, a cell line known to express the
vasopressin V2 receptor. No direct effects of DDAVP
(100 nM–1 lM) were found on capillary-like tube formation by human microvascular cells HMVEC. Our studies
showed that DDAVP induces anti-angiogenic effects that
may be associated with the generation of angiostatin by
G. V. Ripoll J. Garona M. Pifano H. G. Farina
D. E. Gomez D. F. Alonso (&)
Laboratorio de Oncologı́a Molecular, Universidad Nacional de
Quilmes, R. Sáenz Peña 352, Bernal, B1876BXD Buenos Aires,
Argentina
e-mail: dfalonso@unq.edu.ar
tumor cells. Further preclinical studies with DDAVP and
other vasopressin analogs are warranted to determine their
potential in cancer management.
Keywords Tumor vascularization Vasopressin
Peptide analog Angiostatin Mammary carcinoma
Mice
Introduction
Desmopressin
(1-deamino-8-D-arginine
vasopressin,
DDAVP) is a synthetic analog of the antidiuretic hormone
vasopressin, firstly described in the late sixties [1]. In
contrast to vasopressin, which binds to the different vasopressin receptors, DDAVP is a selective agonist for the V2
cell membrane receptor [2]. This vasopressin receptor
subtype is expressed in the kidney-collecting duct, mediating the antidiuretic action, and is also present in endothelial cells, mediating most of the non-renal effects of
DDAVP, including a potent hemostatic effect [3, 4].
In the middle nineties it was postulated that vasopressin
gene-related expression is a feature of all breast cancers,
and products of this expression are attractive as potential
targets for therapy [5]. The presence of vasopressin
receptors was documented in various human cancer cell
lines [6], including breast cancer [7]. DDAVP exhibited
modest but significant antiproliferative effects on MCF-7
and Skbr3 V2 receptor-expressing human breast carcinoma
cell lines [8]. Such action was clearly mediated through
agonist V2 receptor signaling, and thus involved activation
of adenylate cyclase followed by intracellular cAMP elevation. The cytostatic effect could be blocked by the
selective V2 receptor antagonist satavaptan (SR121463)
[8]. It was also reported that the natural hormone
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vasopressin inhibited the in vitro growth of MCF-7 human
breast carcinoma cells at high concentrations [9].
In 1999, we communicated for the first time that intravenous infusion of DDAVP can inhibit the development of
metastasis in syngeneic Balb/c mice. At clinically relevant
doses, the peptide inhibited by 70 % experimental lung
colonization of aggressive F3II mammary cancer cells [10],
and dramatically decreased axillary lymph node metastasis
in a mouse model of breast tumor manipulation and surgical excision [11]. Considering the antimetastatic properties of DDAVP as well as its well-known hemostatic
effect and tolerability, we conducted a pilot veterinary
clinical trial in dogs with locally advanced mammary
cancer [12]. Perioperative administration of DDAVP at
high doses of 1 lg/kg significantly prolonged disease-free
and overall survival. An extended trial recently confirmed
these results, showing a reduced incidence of local relapses
and lung metastasis in treated animals, and a particular
survival benefit in cases with more aggressive carcinoma
[13]. It is likely that DDAVP infusion not only inhibits
perioperative metastatic events, but also combats micrometastases that occur before surgery.
We have also explored the antitumor effects of DDAVP
in combination with chemotherapeutic agents using the
F3II mammary carcinoma. Weekly cycles of intravenous
DDAVP contributed to impair aggressiveness of residual
mammary tumors during chemotherapy [14]. Although
preliminary, these preclinical results support the notion that
DDAVP complements conventional cytotoxic drugs, and
suggest the compound may affect the interaction of tumor
cells with tissue microenvironment, modulating tumorinduced angiogenesis [14, 15]. The dependence of solid
tumors on angiogenesis for sustained growth and metastasis formation is an established concept in tumor biology,
with well-documented therapeutic value [16]. Since the
pioneering work of Folkman [17], a number of antivascular
approaches have been described. The interest in angiogenesis inhibitors has been boosted by the finding of
tumor-derived angiostatic agents, such as a fragment of
plasminogen known as angiostatin [18]. Components of the
plasminogen-activator system, including the serine proteases urokinase-type (uPA) and tissue-type (tPA), are
involved in the proteolytic conversion of plasminogen to
angiostatin in vitro, although the in vivo processes are not
fully understood [19, 20]. Complete and reproducible
inhibition of angiostatin formation by cell lines secreting
uPA and/or tPA was observed with specific serine protease
inhibitors [19].
The aim of the present work was to explore the effects
of DDAVP on tumor angiogenesis using the aggressive
F3II mammary carcinoma in syngeneic mice. Intravenous
administration of the compound markedly decreased vascularization of growing subcutaneous tumors, as well as
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Breast Cancer Res Treat (2013) 142:9–18
inhibited the early angiogenic response around intradermal
tumor inoculation sites. Additional in vitro studies, also
performed in human breast carcinoma and microvascular
endothelial cell lines, indicated that the anti-angiogenic
effect of DDAVP may be associated with the generation of
angiostatin by tumor cells.
Materials and methods
Cell lines and culture conditions
The mammary carcinoma cell line F3II is a highly invasive
and metastatic variant derived from a clone of a spontaneous Balb/c mouse mammary tumor [10]. F3II cells were
maintained in Dulbecco’s modified Eagle’s medium
(DMEM, Gibco, Grand Island, New York, USA) supplemented with 5 % fetal bovine serum (FBS), 2 mM glutamine and 80 lg/ml gentamycin in monolayer culture, at
37 °C in a humidified atmosphere of 5 % CO2. The human
breast carcinoma cell line MCF-7 was routinely grown in
DMEM plus 10 % FBS. Human microvascular endothelial
cells (HMVEC) isolated from the human lung were
obtained from Lonza (Walkersville, MD, USA). The cell
line was cultured using complete endothelial cell basal
medium-2 with specific growth factors (EGM-2 MV Bullet
Kit, Lonza) and routinely cultured for up to 10 in vitro
passages.
Animals
Pathogen-free female Balb/c mice were obtained from the
School of Veterinary of La Plata National University
(UNLP, La Plata, Argentina) and kept at our animal house
facility according to an institutionally approved animal
protocol. Food and water were provided ad libitum, and
general health status of the animals was monitored daily.
Animals with an age of 8–14 weeks and an average weight
of 25 g were used.
In vivo studies
On day 0, groups of at least five mice received 2 9 105
viable F3II cells in the subcutis of the right flank. The time
of appearance of local tumors was monitored by palpation
and further confirmed by histopathology. In all cases,
tumors were diagnosed as spindle-cell carcinomas, as
expected [11]. Tumor size was measured periodically with
a caliper and tumor volume was calculated by the formula:
p/6 9 width2 9 length. Animals were sacrificed by cervical dislocation and necropsied on day 60. To examine
tumor vascularization, tumors were removed, fixed with
formalin and routinely processed for hematoxylin–eosin
Breast Cancer Res Treat (2013) 142:9–18
(H&E) staining. The presence of blood vessels was confirmed with CD31 immunohistochemistry.
Treatment with DDAVP from Ferring Pharmaceuticals
(Malmö, Sweden) was started after tumor formation at day
15. The compound was administered at a dosage within the
range that other authors have previously used and proved
enhanced antidiuretic and hemostatic effects (0.3–2 lg/kg).
These doses are clinically relevant and have the advantage
of being well characterized from a pharmacological point
of view [3]. Since DDAVP showed tachyphylaxis with
repeated daily applications [4], doses were administered on
a weekly or thrice weekly basis. Mice were administered
intravenously with weekly cycles of DDAVP at a dose of
2 lg/kg of body weight or thrice weekly at 0.3 lg/kg.
Control animals received only saline vehicle.
Complementary, we performed an intradermal angiogenesis assay, as a rapid and quantitative alternative to test
DDAVP effects on tumor-induced neovascularization.
Mice were injected intradermally with F3II cells (2 9 105
F3II cells in 0.1 ml of DMEM plus trypan blue). Each
mouse received two injections midlaterally in the thoracolumbar position of the trunk. After 4 days, animals were
sacrificed and the skin was examined under a dissecting
microscope. Quantification of vascularization was done by
measuring the vessel density around each inoculation site.
Daily intravenous doses of DDAVP (2 lg/kg) were
administered throughout the experiment.
In vitro growth assays
In vitro experiments were performed using nanomolar and
low micromolar concentrations of DDAVP, a range consistent with the in vivo dosage [10, 14]. To assess the
antiproliferative effect of DDAVP against rapidly growing
tumor cells, a range of concentrations from 100 nM to
1 lM was used with a 3-day exposure of log-phase
growing cells. F3II cells were seeded on 96-well plates
(2.5 9 103 cells/well) in DMEM plus 5 % FBS. After
24 h, the compound was added, culture was continued for
72 h and then tested by the MTT assay. Additionally,
cytostatic effects of DDAVP were examined at low cell
density by colony formation assay. F3II cells were plated at
300 cells/cm2 (6 9 102 cells/well in 24-well plates) and
grown for 7 days in complete medium in the presence of
proper concentrations of DDAVP. Cultures were then fixed
with formalin, stained with crystal violet and colonies of
[50 cells were counted. The concentration producing
50 % inhibition (IC50) was determined by plotting the
percentage of cell colonies versus drug concentration.
To evaluate direct effects of DDAVP on cultured
microvascular endothelial cells a capillary tube morphogenesis assay was conducted. HMVEC cells were seeded
on Matrigel coated wells (2.5 9 104 cells/well in 24-well
11
plates) and tube formation was assessed after 20 h in the
presence of DDAVP (100 nM, 250 nM and 1 lM). Capillary-like tube structures per field were quantified using an
inverted microscope (magnification 9100).
Antibodies
An anti-vasopressin V2 receptor antibody produced in
rabbit, recognizing a conserved human epitope of the
receptor also expressed in mice and rats, was purchased
from Santa Cruz Biotechnology Inc. (Santa Cruz, California, United States). A fluorescein (FITC)-conjugated goat
anti-rabbit IgG (Chemicon International Inc., Temecula,
CA, USA) was applied for immunofluorescence. Rabbit
polyclonal antibodies against human plasminogen (Dako,
Glostrup, Denmark), which recognize the kringle domains
of angiostatin [19], and a goat anti-rabbit peroxidase-conjugated secondary antibody were used (Bio-Rad, Hercules,
CA, USA).
Immunofluorescence detection of V2 vasopressin
receptor
Cells were seeded on glass coverslips, washed with cold
phosphate-buffered saline (PBS) pH 7.4, and then fixed
with 3 % (w/v) paraformaldehyde in PBS for 15 min. Cells
were washed with cold PBS, incubated with 50 mM
ammonium chloride for 5 min, again washed with PBS,
and incubated with 3 % FBS as blocking agent for 30 min.
Cells were then incubated with anti-V2 vasopressin
receptor primary antibody (4 lg/ml in 0.1 % FBS) for 1 h
at 37 °C. Receptor-bound antibodies were detected with a
secondary FITC-conjugated goat anti-rabbit IgG (1:400 in
0.1 % FBS) and nuclei were labeled with 40 ,6-diamidino-2phenylindole (DAPI) using the Vectashield fluorescent
mounting medium (Vector Laboratories Inc., Burlingame,
CA, USA). Samples were examined by standard fluorescence microscopy using a Nikon TE-2000 fluorescence
microscope, and pictures were processed using Nikon NISElements software (Nikon, Tokyo, Japan). Cultures of
MCF-7 human breast carcinoma cells were used as a
positive control of V2 receptor expression, as reported [7].
Negative controls consisted of omission of the primary
antibody and were consistently negative.
Digestion of plasminogen to angiostatin by cell-culture
supernatant
From semiconfluent tumor cell cultures, serum-free conditioned medium was produced by washing the cells thrice
with PBS, followed by overnight incubation with serumfree DMEM in the presence or absence of DDAVP. Conditioned medium was centrifuged to remove cell debris,
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and stored at -20 °C until further use. Purified plasminogen from Chromogenix (Mölndal, Sweden) was added to
conditioned media at a final concentration of 1 lg/ml, and
incubated at 37 °C for 24 h to allow angiostatin generation,
as described previously [19].
Zymography
For detection of plasminogen activator forms contained in
serum-free conditioned media, casein zymography was
performed using SDS–polyacrylamide gel electrophoresis
with 7.5 % separating and 4 % stacking gels. The separating gel was copolymerized with 12 lg/ml purified
plasminogen and 5 mg/ml non-fat dried milk as casein
source. After running, gels were washed with 2 % Triton
X-100 and incubated at 37 °C for 24 h in 20 mM Tris
buffer (pH 8.3) containing 15 mM EDTA. Upon staining
with Coomassie blue and destaining, the final gel had a
uniform blue background except in those regions to which
proteases had migrated and activated plasminogen to
plasmin. Molecular weights were determined by prestained standards (Bio-Rad, Hercules, CA, USA). Plasminogen-free gels were used to test plasminogen-independent protease activity. For evaluation of gelatinolytic
metalloprotease (MMP) activity, zymographic analysis was
performed using gels copolymerized with gelatin.
Breast Cancer Res Treat (2013) 142:9–18
software package. p values less than 0.05 were considered
statistically significant.
Results
Reduction of tumor volume and angiogenesis
by DDAVP administration in vivo
We first tested the effects of intravenous administration of
DDAVP in Balb/c mice-bearing subcutaneous F3II mammary tumors. F3II cells grew as highly invasive carcinoma
tumors in control animals. As shown in Fig. 1a, treatment
of mice with a single weekly dose of 2 lg/kg significantly
reduced tumor volume. A similar antitumor effect was
obtained administering DDAVP thrice weekly at 0.3 lg/
kg/dose (0.9 lg/kg/week). No improved efficacy against
mammary tumors was obtained with higher weekly doses
of 6 lg/kg (data not shown). Histopathological studies
performed on tumors treated with DDAVP showed a
marked and statistically significant decrease in tumor vascularization (Fig. 1b, c).
We further evaluated the early angiogenic response by
measuring the vessel density around intradermal tumor
inoculation sites. F3II mammary tumor cells induced a
prominent angiogenic response in control animals. Daily
intravenous administration of DDAVP at a dose of 2 lg/kg
significantly reduced tumor-induced angiogenesis (Fig. 2).
Western blot
In vitro cytostatic effect of DDAVP
For detection of plasminogen digestion and angiostatin
generation, Western blot was performed essentially as
described elsewhere [19]. Briefly, samples of conditioned
medium incubated with plasminogen were run on a 7.5 %
polyacrylamide gel. After electrophoresis, samples were
electroblotted onto polyvinylidene fluoride membranes.
Blots were blocked for 60 min in blocking solution followed
by overnight incubation with antibody diluted in blocking
solution. After washing, blots were incubated for 1 h with
peroxidase-conjugated secondary antibody. After washing,
blots were developed by chemiluminescence according to
the manufacturer’s protocol (ECL, General Electrics, Fairfield, CT, USA). Angiostatin bands were further analyzed by
densitometry using the Kodak Electrophoresis Documentation and Analysis System (EDAS 120) software (Kodak,
Rochester, NY, USA), and expressed as fold change compared with untreated control cells.
Statistical analysis
Differences in in vitro and in vivo data between control and
treated groups were evaluated with the GraphPad Prism 4.0
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We next assessed the cytostatic effect of DDAVP on logphase growing F3II mammary carcinoma cells. After a
72-h exposure, DDAVP caused a modest but significant
inhibition of cell growth, as reported previously for MCF-7
cells [8]. The higher dose of 1 lM reduced proliferation in
F3II monolayers up to 15–20 % (Fig. 3a). On the other
hand, DDAVP had a stronger effect on colony formation at
low density, with an IC50 value of 700 nM against F3II
cells (Fig. 3b).
Expression of V2 vasopressin receptors in both tumor
and endothelial cells
In order to check the expression of the V2 vasopressin
receptor for which DDAVP is the selective agonist, an
immunofluorescence assay was conducted. As shown in
Fig. 4, F3II mouse mammary tumor cells brightly expressed the V2 receptor on the cell surface in a similar pattern
than MCF-7, a human breast carcinoma cell line known to
display normal forms of all vasopressin membrane receptors plus an abnormal V2 receptor [7]. HMVEC microvascular endothelial cells were also positive for the V2
Breast Cancer Res Treat (2013) 142:9–18
b
1500
Control
DDAVP
1000
Intratumoral
Tumor Volume (mm 3 )
a
13
**
500
0
10
15
25
20
30
40
35
45
50
Time (Days)
c
*
10
Peritumoral
Vessels/HPF
20
0
Control
DDAVP
Fig. 1 Antitumor effects of DDAVP in syngeneic mice-bearing F3II
mammary tumors. Animals were injected in the subcutis with
2 9 105 F3II cells, and intravenously administered with DDAVP or
saline as a control. a Tumor growth curves of control (closed square)
and treated animals, receiving weekly DDAVP at 2 lg/kg (closed
circles) or thrice weekly DDAVP at 0.3 lg/kg (open circles). Data
represent mean ± standard error. Results are representative of three
independent experiments. **p \ 0.01 versus control, ANOVA plus
Dunnett’s test comparing the mean slopes from day 15.
b Representative micrographs of tumors from controls and mice
treated with weekly DDAVP (2 lg/kg). Areas showing intratumoral
and peritumoral vascularization are depicted. H&E staining, original
magnification 9100 (insets 9400). c Quantitative analysis of
intratumoral vascularization in controls and mice treated with weekly
DDAVP (2 lg/kg). Blood vessels per high power field (HPF, 9400)
are shown. Data represent mean ± standard error. *p \ 0.05,
unpaired t test
receptor (see also Fig. 4), as documented previously by
RT-PCR [21].
conditioned media from F3II monolayers cultured in the
presence of low, non-cytostatic doses of DDAVP (100 nM)
significantly increased angiostatin formation (Fig. 6a).
Such increase was associated with an enhancement of
tumor-secreted uPA activity (50 kDa) in conditioned media
containing purified plasminogen, as revealed by zymography (Fig. 6b). F3II monolayers also secreted a 105 kDa
protein with gelatinolytic activity, corresponding to MMP9. Although DDAVP induced a slight increase of this band,
no clear dose-dependent effects were observed (data not
shown).
Control MCF-7 human breast carcinoma cells were not
able to excise angiostatin from plasminogen under the
same experimental conditions, as previously reported [19].
However, treatment of MCF-7 monolayers with DDAVP
gave rise to a detectable band of angiostatin (Fig. 6c).
Lack of direct effects of DDAVP on endothelial cells
in vitro
Considering the anti-angiogenic effects observed in vivo, we
first investigated direct effects of DDAVP on cultured
microvascular endothelial cells. Endothelial tube formation
in HMVEC cultures on Matrigel was not significantly
affected by DDAVP at concentrations consistent with previous in vivo and in vitro studies, in a range from 100 nM to
1 lM (Table 1). Representative micrographs of capillarylike structures are depicted in Fig. 5.
Modulation of tumor-derived proteolytic activity
by DDAVP and angiostatin formation
We then explored the proteolytic production of angiostatin
by mammary tumor cells. In initial experiments, purified
plasminogen was incubated with serum-free conditioned
media from F3II monolayers for different period of times,
and reaction products were analyzed by Western blot. After
at least 20–24 h, prominent bands of about 66 and
38–44 kDa corresponding to plasmin heavy-chain and
angiostatin, respectively, were detected. Interestingly,
Discussion
DDAVP is known to inhibit development of metastasis and
contribute to impair aggressiveness of residual tumors
during chemotherapy in the aggressive F3II mouse mammary cancer model [10, 11, 14]. To the best of our
knowledge, this is the first study to examine the association
between antitumor properties of a vasopressin peptide
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a
a
Cell Proliferation
(% of control)
Control
100
***
***
75
50
100
250
500
1000
DDAVP (nM)
b
Colony formation
(% of control)
DDAVP
150
100
50
0
0
250
500
750
1000
DDAVP (nM)
b
Fig. 3 Cytostatic effect of DDAVP on F3II mammary tumor cells.
a 3-day exposure on log-phase growing cells using the MTT assay.
***p \ 0.001 versus control, ANOVA plus Dunnett’s test. b 7-day
treatment on the colony formation assay at low density. Dotted line
indicates the IC50 value (700 nM). Data represent mean ± standard
error. In both cases, results are representative of at least three
independent experiments
7
6
Vessels/mm2
5
***
4
3
2
1
0
Control
DDAVP
Fig. 2 Effect of DDAVP on the intradermal angiogenic response
induced by F3II mammary tumor cells. Mice were inoculated
intradermally with 2 9 105 F3II cells, and intravenously administered
daily with DDAVP at 2 lg/kg or saline as a control. a Representative
micrographs of tumor inoculation sites from control and DDAVPtreated mice. Original magnification 920. b Quantitative analysis of
angiogenic response. Data represent mean ± standard error. Results
are representative of three independent experiments. ***p \ 0.001,
unpaired t test
analog and its potential angiostatic effect. Intravenous
administration of DDAVP at clinically relevant doses of
0.3–2 lg/kg significantly decreased intratumor vascularization in mice bearing subcutaneous-growing F3II tumors.
Similarly, treatment inhibited the early angiogenic
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response by about 50 % in the dermis around F3II cell
inoculation sites.
Our findings suggested that DDAVP reduces tumor
angiogenesis by inducing the formation of angiostatin, a
potent naturally occurring inhibitor of angiogenesis that is
generated by cancer-mediated proteolysis of plasminogen.
A number of proteases can generate angiostatin, including
MMPs and serine proteases [18–20]. DDAVP seemed to
stimulate the secretion of plasminogen activators such as
uPA by mammary tumor cells, thus excising angiostatin
from plasminogen by controlled proteolysis. The involvement of plasminogen activators in angiostatin production
has been convincingly demonstrated more than a decade
ago by Gately et al. [22]. Plasmin generated by uPA is able
to digest either plasmin itself or intact plasminogen thus
leading to angiostatin formation. In human prostate and
bladder carcinoma cell lines, a robust correlation of
angiostatin with uPA levels, and in melanoma with tPA
levels, was documented by Westphal et al. [19]. Conversely, cells lines that were unable to produce significant
amounts of angiostatin, such as the MCF-7 human breast
carcinoma, have very low or absent uPA and tPA levels
[19].
Breast Cancer Res Treat (2013) 142:9–18
15
Table 1 Quantification of capillary-like tube structures in HMVEC
cells after treatment with DDAVP
Treatmenta
Tube formation per fieldb
Control
12.4 ± 2.9
DDAVP 100 nM
11.9 ± 3.2
DDAVP 250 nM
DDAVP 1,000 nM
9.6 ± 3.4
11.7 ± 3.1
Non-significant differences among groups (p [ 0.05, ANOVA plus
Tukey multiple comparison test)
a
HMVEC cells were seeded on Matrigel, and tube formation was
assessed after 20 h in the presence or absence of DDAVP
b
Fig. 4 Immunofluorescence detection of vasopressin receptor in
tumor and endothelial cells. Vasopressin V2 receptor expression
was detected using a specific anti-V2 antibody and a secondary
antibody labeled with FITC. a F3II mouse mammary tumor cells.
b HMVEC human microvascular endothelial cells. c MCF-7 human
breast carcinoma cells (positive control). Insets, omission of the
primary antibody (negative control). Original magnification 91,000
Data are expressed as mean ± standard deviation
DDAVP is likely to favor the production of angiostatin
by experimental tumors in vivo, with consequent antiangiogenic effect. In mice bearing experimental tumors,
angiostatin and the collagen-XVIII fragment endostatin
were able to inhibit the growth, and even reduce the volume, of established disease [23]. Moreover, these experiments showed that anti-angiogenic therapy was not
associated with acquired drug resistance [23, 24].
We cannot rule out the possibility that other underlying
mechanisms account for the antitumor activity of DDAVP.
In fact, the compound has a direct cytostatic effect on F3II
cells and human breast cancer cell lines [8]. In addition,
DDAVP induces a rapid increase in circulating von Willebrand factor (vWF) by stimulating its release mainly from
microvascular endothelial cells through a specific agonistic
action on V2 vasopressin receptors [25]. Interestingly, it was
reported that inhibition of vWF expression by short interfering RNA in endothelial cells caused an increase of in vitro
angiogenesis and vascular endothelial growth factordependent proliferation and migration [26], suggesting a
potential role for vWF in the modulation of angiogenesis.
Breast cancer is one of the most commonly diagnosed
malignancies in women and mortality due to the disease is
related to the capacity of breast tumor cells to find alternative routes to vascularization and metastasis. A multidisciplinary approach to the management of breast cancer
and the introduction of novel systemic therapies have
improved the quality of life and survival of patients. In this
regard, the potential combination of standard treatment
with novel biological agents is exciting. Perhaps the
greatest obstacle for therapy of an advanced cancer is that
the outcome of residual metastasis depends on interactions
of disseminated cells with homeostatic mechanisms which
the tumor cells usurp. DDAVP appears as a safe agent to
develop strategies for a pharmacological modulation of
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Breast Cancer Res Treat (2013) 142:9–18
Fig. 5 Lack of effects of DDAVP on endothelial tube formation by
HMVEC cells on Matrigel. Tube formation was assessed after 20 h in
the presence or absence of DDAVP. a Control; b 100 nM; c 250 nM;
No
digestion Control
a
d 1 lM. The micrographs were taken with an inverted microscope.
Original magnification 9100
DDAVP
**
Fold change of
angiostatin level
3
Plasminogen
Plasmin
Angiostatin
2
1
0
Control
b
c
No
digestion Control
DDAVP (nM)
0
100
250 1000
uPA
DDAVP
DDAVP
Plasminogen
Plasmin
Angiostatin
Fig. 6 Effect of DDAVP on angiostatin formation and tumor-derived
proteolytic activity. a Western blot analysis (left) of protein fragments
generated by digestion of purified plasminogen by serum-free
conditioned media from F3II mouse mammary tumor cells cultured
with or without DDAVP (100 nM). Bands obtained at time 0 (no
digestion of plasminogen) and after 20 h of digestion are shown.
Quantitative analysis (right) of angiostatin band (38-44 kDa) by
densitometry is also shown. Data represent mean ± standard error.
123
Results are representative of three independent experiments.
**p \ 0.01, t test. b Zymographic analysis of uPA (50 kDa) secreted
by F3II monolayers to the conditioned media after exposure to
varying concentrations of DDAVP. c Angiostatin formation by
medium conditioned by MCF-7 human breast carcinoma cells treated
with DDAVP (100 nM). Molecular weights were determined using
pre-stained standards
Breast Cancer Res Treat (2013) 142:9–18
angiostatin production and tumor-endothelial interactions
[15]. Synthetic peptides such as DDAVP are much appreciated as lead compounds for developing human therapeutics, having a great potential as therapeutic agents due
to their ease of rational design and target specificity [27].
Currently, a panel of linear and cyclic vasopressin peptide
analogs with improved antitumor effects is in development
in our laboratory. Punctual amino acid substitutions in
DDAVP generate novel synthetic oligopeptides with
enhanced cytostatic or anti-angiogenic effects [28]. However, the effects of such novel compounds on the release,
by endothelial cells, of vWF and other hemostatic factors
such as factor VIII remained to be investigated. We consider that further preclinical studies with DDAVP, as well
as with derivatized analogs, are warranted to determine
their potential utility in cancer therapy.
Acknowledgments We would like to thank Dr. Alejandra Scursoni
for expert histopathological assessment. J.G. is a research fellow and
G.V.R., H.G.F, D.E.G. and D.F.A. are members of the National
Research Council (CONICET, Argentina).
Conflict of interest This work was partially funded by the National
Agency for the Promotion of Science and Technology (ANPCYT,
Argentina) and Chemo-Romikin. The authors have no other relevant
affiliations or financial involvement with any organization or entity
with a financial interest in or financial conflict with the subject matter
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