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Fiorio Pla et al. BMC Cancer 2014, 14:939
http://www.biomedcentral.com/1471-2407/14/939
RESEARCH ARTICLE
Open Access
Differential sensitivity of prostate tumor derived
endothelial cells to sorafenib and sunitinib
Alessandra Fiorio Pla2,3,4,5†, Alessia Brossa1†, Michela Bernardini2,3†, Tullio Genova2, Guillaume Grolez4,5,
Arnaud Villers6, Xavier Leroy7, Natalia Prevarskaya4,5, Dimitra Gkika4,5† and Benedetta Bussolati1*†
Abstract
Background: Prostate cancer is the second leading cause of male cancer death in developed countries. Although
the role of angiogenesis in its progression is well established, the efficacy of anti-angiogenic therapy is not clearly
proved. Whether this could depend on differential responses between tumor and normal endothelial cells has not
been tested.
Methods: We isolated and characterized three lines of endothelial cells from prostate cancer and we tested the
effect of Sunitinib and Sorafenib, and the combined treatment with the anti-androgen Casodex, on their angiogenic
functions.
Results: Endothelial cells isolated from prostate tumors showed angiogenic properties and expression of androgen
and vascular endothelial cell growth factor receptors. Sunitinib affected their proliferation, survival and motility while
Sorafenib only showed a minor effect. At variance, Sunitinib and Sorafenib showed similar cytotoxic and anti-angiogenic
effects on normal endothelial cells. Sorafenib and Sunitinib inhibited vascular endothelial cell growth factor receptor2
phosphorylation of prostate cancer endothelial cells, while they differentially modulated Akt phosphorylation as no
inhibitory effect of Sorafenib was observed on Akt activation. The combined treatment of Casodex reverted the
observed resistance to Sorafenib both on cell viability and on Akt activation, whereas it did not modify the response
to Sunitinib.
Conclusions: Our study demonstrates a resistant behavior of endothelial cells isolated from prostate cancer to
Sorafenib, but not Sunitinib. Moreover, it shows the benefit of a multi-target therapy combining anti-angiogenic and
anti-hormonal drugs to overcome resistance.
Keywords: Anti-angiogenic therapy, VEGF receptor, Androgen receptor, Prostate cancer, Drug resistance
Background
Prostate cancer is one of the most common malignancies
and remains the second leading cause of cancer death in
men [1]. The improved understanding of prostate cancer
biology in recent years led to the development of drugs directed against precise tumorigenesis-associated molecular
pathways [2]. Angiogenesis, the development of new blood
vessels, is recognized as one of the hallmarks of malignancy
and prostate vasculature has been shown to play an important role in regulating the size and function of prostate
malignancies [3,4]. Accordingly, several anti-angiogenic
* Correspondence: benedetta.bussolati@unito.it
†
Equal contributors
1
Department of Molecular Biotechnology and Health Sciences, Molecular
Biotechnology Centre, University of Torino, via Nizza 52, 10126 Torino, Italy
Full list of author information is available at the end of the article
drugs have been tested in phase II and III trials in
prostate cancer patients [5], including the oral nonselective tyrosine kinase inhibitors Sunitinib and Sorafenib. These two drugs share their activity on vascular
endothelial growth factor-receptors (VEGFRs), platelet
derived growth factor-receptor beta (PDGFRβ), cKIT
and RET, expressed on the cell membrane. In addition,
Sorafenib is also able to directly act on the RAF intracellular pathway [6].
A bulk of evidence indicates that tumor blood vessels
differ significantly from normal vessels for the structural
organization and for the properties of endothelium
[7-12]. This suggests that tumor vascularization depends
on mechanisms alternative to the simple recruitment
from adjacent tissue of pre-existing blood vessels [13].
© 2014 Fiorio Pla et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Fiorio Pla et al. BMC Cancer 2014, 14:939
http://www.biomedcentral.com/1471-2407/14/939
The most remarkable abnormality reported for tumorderived endothelial cells (TEC) is the chromosomal instability [9]. In addition, serial analysis of gene expression showed that TEC express genes not shared by
blood vessels that reside in normal tissues [11]. Embryonic genes are expressed also by the endothelial cells derived from tumors [10,14,15]. Finally, TEC present
functional alterations linked to increased survival, proliferation and angiogenic properties [13], as well as resistance to chemotherapeutics [16]. All these molecular and
functional alterations in TEC may result in altered sensitivity to the anti-angiogenic therapy. However, information on the phenotype of TEC derived from prostate
tumor and on their sensitivity to anti-angiogenic drugs
is limited [17].
In the present study, we isolated and characterized
three lines of TEC from three different prostate cancer
human samples (PTEC). Moreover, we evaluated the effect of two anti-angiogenic drugs, Sunitinib and Sorafenib, on typical aspects of the angiogenic process such as
the ability to form functional blood vessels in vivo,
in vitro proliferation, survival, tubulogenesis and motility. Finally, as androgen receptor (AR) stimulation was
reported to promote endothelial cell proliferation, we explored the possible effect of a combined treatment with
anti-androgen and anti-angiogenic drugs.
Methods
Cancer tissue sampling
Prostate tissue samples (prostate adenocarcinoma) were
obtained by Prof. Arnauld Villers and Prof Xavier Leroy
(Dept. of Urology, Regional University Hospital of Lille,
France) from 3 patients with a mean age of 58 years
(ranging from 57 to 59) who underwent radical prostatectomy. Immediately after prostate removal (delay <
10 min), small pieces of tissue (at least 6 tissue samples
from 0.5 to 1 cc) were grossly dissected by the pathologist from the left area, the right peripheral zone and the
transitional area. To ensure tissue was malignant and to
confirm the Gleason score, histological analysis of sections was performed on each sample by the same pathologist (Table 1). Patient verbal and written information
and signed consent form required by the tissue collection unit by law was performed and obtained for all patients. This study was in accordance with the ethical
requirements of the tissue collection unit of the Centre
Page 2 of 13
Hospitalier Régional Universitaire de Lille, University
Lille Nord de France.
Drugs
Sunitinib malate (Sigma-Aldrich, St Louis, MO, USA),
was resuspended in DMSO to a final concentration of
10 mM and stored at +4°C, according to the manufacturer’s instructions. Sorafenib (Bayer Pharmaceuticals,
Leverkusen, Germany) was resuspended in DMSO to a
stock concentration of 10 mM and stored at −20°C.
Bicalutamide (Casodex) (Sigma-Aldrich, St Louis, MO,
USA), was resuspended in DMSO to a stock concentration of 10 mM, according to the manufacturer’s instructions. Drugs were diluted into the culture medium
shortly before performing the assays.
Isolation of PTEC and other cell types
Prostate tumor endothelial cells (PTEC) were isolated
on the basis of endothelial-specific culture conditions.
For the isolation of PTEC, specimens were finely minced
and digested in RPMI (Lonza, Basel, Switzerland) containing Collagenase IV (Sigma-Aldrich, St Louis, MO,
USA) for 30 minutes at 37°C. After washings in medium
plus 10% fetal calf serum (FCS, Seromed, Poly-Labo),
the cell suspension was forced through a graded series
of meshes to separate the cell components from stroma
and aggregates. Cells (2×104/cm2) where then plated in
ECAF (Endothelial Cells Attachment Factor, SigmaAldrich, St Louis, MO, USA)-coated plates in EndoGRO
MV-VEGF medium (Merck-Millipore, Billerica, Massachusetts, USA) containing 5% FCS, and maintained in
culture for at least 6 passages. To avoid a possible fibroblast contamination, cells were cultured at passage one
for three days with D-valine-substituted DMEM (SigmaAldrich, St Louis, MO, USA). Breast tumor endothelial
cells (BTEC) were isolated and characterized as previously described [18]. Human umbilical vein endothelial
cells (HUVEC) and microvascular endothelial cells
(HMEC) were obtained from the umbilical vein or from
derma, respectively, as previously described [19]. All
endothelial cells were maintained in culture in EndoGRO MV-VEGF medium containing 5% FCS.
Human Embryonic Kidney (HEK) 293 and Lymph
Node Carcinoma of Prostate (LNCaP) C4- 2 cells were
grown in DMEM and RPMI 1684 (Invitrogen), respectively, supplemented with 10% FCS, L-glutamine (5 mM)
(Sigma-Aldrich, St Louis, MO, USA) and kanamycin
Table 1 Characteristics of the patients used for the isolation of PTEC
Patient
Age
Gleason score
Stage
PSA (ng/ml)
Androgen ablation
01
57
9 (4 + 5)
pT3b N0 M0
7.88
NO
02
59
9 (4 + 5)
pT3a N0 M0
11.46
NO
03
58
7 (3 + 4)
pT3 N0 M0
8.03
NO
Fiorio Pla et al. BMC Cancer 2014, 14:939
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(100 mg/ml) (Sigma-Aldrich, St Louis, MO, USA). Cells
were transfected with 2 μg of pcDNA4-AR construct
using FuGENE HD reagent (Roche Diagnostics, France),
as described [20].
Flow cytofluorimetric and immunofluorescence analysis
For cytofluorimetric analysis, PTEC lines were detached
from plates with a non-enzymatic cell dissociation solution (Sigma-Aldrich, St Louis, MO, USA), washed and
stained (30 min at 4°C) with the following fluorescein
isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophtcocyanin (APC)-conjugated antibodies: PDGFRβ, CD31
(all from BD Bioscience, Franklin Lakes, NJ, USA) CD105,
VEGR2 (all from MiltenyiBiotec, Bergisch Gladbach,
Germany), c-KIT (Dako, Glostrup, Denmark), TIE2,
VEGFR1, VEGFR3 (all from R&D Systems, Minneapolis,
MN, USA). Isotypes (all from MiltenyiBiotec, Bergisch
Gladbach, Germany) were used as negative controls. Cells
were subjected to cytofluorimetric analysis (FACScan
Becton Dickinson, Franklin Lakes, NJ, USA) at each
culture passage. Indirect immunofluorescence was performed on cells cultured on chamber slides (Nunc, Roskilde,
Denmark). Cells were fixed in 3.5% paraformaldehyde
containing 2% sucrose and permeabilized with Hepes-Triton
X-100 0.1% for 10 minutes at 4°C. The anti-pan-cytokeratin
polyclonal Ab (Biomeda, Foster City, California, USA)
was used. Texas Red goat anti-rabbit IgG (Molecular
Probes, Eugene, OR, USA) was used as secondary
antibody. Hoechst 33258 dye (Sigma-Aldrich, St Louis,
MO, USA) was added for nuclear staining. Confocal
microscopy analysis was performed using a Zeiss LSM 5
Pascal model confocal microscope (Carl Zeiss, Oberkochen,
Germany).
In vitro tubule formation
In vitro formation of capillary-like structures was studied
on growth factor-reduced Matrigel (BD Bioscience,
Franklin Lakes, NJ, USA) in 24-well plates. PTEC,
HUVEC and HMEC (3,5 × 104 cells/well) were seeded
onto Matrigel-coating in EndoGRO MV-VEGF medium
containing 5% FCS and treatments performed in duplicate. Cell organization onto Matrigel was periodically
imaged with a Nikon Eclipse Ti inverted microscope
using a Nikon Plan 10X/0,10 objective and cells were
kept on a stage incubator at 37°C and 5% CO2 during
the experiment (OKOLab, Italy). Images were acquired
at 2 h time intervals using MetaMorph software.
Image analysis was performed with ImageJ software:
images at 18 hours of treatment were analyzed: number
of nodes (intersections formed by at least three detectable cells) and total tubule length (aligned cells connecting nodes) were measured for each field. Number of
nodes and tubule length were normalized to maximum
values and their sum for each condition was used to
Page 3 of 13
express the grade of organization in “capillary-like”
structures in terms of arbitrary units (A.U.). At least ten
fields for each condition were analyzed in each independent experiment. Graphs show mean values of three
independent experiments, error bars show standard
error. Values are expressed as mean ± S.E.M.
In vivo tubule formation
To evaluate the tubule formation in vivo, 2 × 106 PTEC
were implanted subcutaneously into SCID mice (Charles
River, Wilmington, Massachusetts) within growth factor–reduced Matrigel (BD Biosciences, Franklin Lakes,
NJ, USA) as previously described [10]. Briefly, cells were
harvested and resuspended in 150 μl DMEM plus 250 μl
of Matrigel, chilled on ice and injected subcutaneously
into the left back of SCID mice (n = 4). After 7 days,
mice were sacrificed, and endothelial plugs recovered
and processed for histology. Typically, the overlying skin
was removed, and gels were cut out by retaining the
peritoneal lining for support, fixed in 10% buffered formalin, and embedded in paraffin. Sections (3 μm) were
cut and stained with hematoxylin and eosin and were
examined under a light microscope system.
Proliferation assay and MTT
For the cytotoxicity or the proliferation assay, cells were
plated in the growth medium at a concentration of 3000
cells/well in a 96-multiwell plate and left in adhesion
overnight. The day after the culture medium was removed and cells were incubated with Sunitinib or Sorafenib in RPMI 2% FCS. After 48 h, DNA synthesis was
detected after as incorporation of 5-bromo-2-deoxyuridine (BrdU) using an enzyme-linked immunosorbent
assay kit (Roche, Penzberg, Germany). Cytotoxicity was
evaluated by MTT (3-(4,5-dimetiltiazol-2-yl)-2,5-difenil
tetrasodium bromide) (Merck-Millipore, Billerica, Massachusetts, USA), according to manufacturer’s instructions. Data are expressed as the mean ± S.E.M of the
media of absorbance of at least three different experiments performed with all the three lines in the study in
triplicate, normalized to the positive control (vehicle
alone). To evaluate the IC50 of both Sunitinib and Sorafenib on HUVEC, MTT data were analysed using Calcusyn software. Results are expressed as mean ± S.E.M. of
five different experiments.
Cell migration
Migration was assessed using silicone culture inserts
(ibidi GmbH, Munich, Germany) in 12-well culture
plates. Inserts had two 70 μl wells, both of which were
used to plate cells. PTEC, HUVEC or BTEC (1 ×
105cells/ml) were plated on 1% gelatin coating in EndoGRO MV-VEGF medium containing 5% FCS. Cells were
maintained in incubator until confluence was reached.
Fiorio Pla et al. BMC Cancer 2014, 14:939
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Cell monolayers were starved 12 hours in DMEM 0%
FCS before removing the inserts and thus generating the
“wound area”. Floating cells were removed by wash in
PBS solution, and monolayers were treated with test
conditions (in duplicate). EndoGRO MV-VEGF medium
5% FCS was used as positive control, whereas DMEM
0% FCS served as negative control. Cell migration was
imaged with a Nikon Eclipse Ti inverted microscope
using a Nikon Plan 4X/0, objective and cells were kept
on a stage incubator at 37°C and 5% CO2 during the experiment (OKOLab, Italy). Images were acquired at 2 h
time intervals using MetaMorph software.
MetaMorph software was used to calculate migration
rate (%) by measuring the distance covered by cells between two subsequent time points (4 fields measurements for each image). Measurements were made for
each time point and at least 10 fields for each condition
were analyzed in each independent experiment. Graphs
show mean ± S.E.M of three independent experiments.
Western blot analysis
PTEC and HUVEC were grown in ENDOGRO MVVEGF medium containing 5 and 10% FCS respectively,
until cells reached confluence. Cells were then incubated
10 or 30’ with vehicle, Sunitinib or Sorafenib (1 and
2.5 μM) and lysed at 4°C for 30 min in RIPA buffer (20
nMTrisHCl, 150 nMNaCl, 1% deoxycholate, 0.1% SDS,
1% Triton X-100, pH 7.8) supplemented with protease
inhibitor cocktail, PMSF (all from Sigma-Aldrich, St
Louis, MO, USA) and PhosStop (Roche, Penzberg,
Germany). Conditions for Western blotting were as
described previously [21]. Polyvinylidene fluoride membranes were blocked and incubated overnight with goat
polyclonal anti VEGFR2 (R&D Systems, Minneapolis,
MN, USA) antibody (1:2000); or rabbit polyclonal anti
Phospho-VEGFR2-Tyr951 (p-VEGFR2; sc-101821, Santa
Cruz Biotechnology, Santa Cruz, CA, USA) antibody
(1:100); rabbit polyclonal anti p44/42MAPK (ERK1/2)
(9102, Cell Signalling, Danvers, MA, USA) antibody
(1:1000); rabbit polyclonal anti Phospho-p44/42 MAPK
(pERK1/2) (Thr202/Tyr204) (4370, Cell Signalling,
Danvers, MA, USA) antibody (1:1000); rabbit polyclonal
anti Akt (9272, Cell Signalling) antibody (1:3000); rabbit
monoclonal IgG Phospho-Akt (pAkt; Ser473) (4508, Cell
Signalling) antibody (1:2000); rabbit polyclonal anti AR
(N-20) antibody (1:400) (sc-816, Santa Cruz Biotechnology). Membranes were then washed with 1X TBST
containing 0.1% Tween 20 and incubated as required
with the HRP-conjugated anti-goat (Dako, Glostrup,
Denmark) or anti-rabbit IgG (Santa Cruz Biotechnology)
antibodies. Chemiluminescence detection was conducted
using the ECL prime Western blotting detection reagent
(GE Healthcare, Buckinghamshire, England). To quantify
the differences in protein phosphorylation, the ratio
Page 4 of 13
between non phosphorylated and phosphorylated protein expression was evaluated. Membranes were then
washed with 1X TBST containing 0.1% Tween 20 and
incubated as required with the HRP-conjugated antigoat (Dako, Glostrup, Denmark) or anti-rabbit IgG
(Santa Cruz Biotechnology) antibodies.
RNA isolation and real time PCR
Total RNA was isolated using Trizol Reagent (Ambion,
Life Technologies, Carlsbad, California, USA) according
to the manufacturer’s protocol, and quantified spectrophotometrically (Nanodrop ND-1000). For gene expression
analysis, quantitative real-time PCR was performed. Briefly,
first-strand cDNA was produced from 200 ng of total RNA
using the High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, Foster City, California, USA).
Quantitative Real-time PCR experiments were performed in 20-μl reaction mixture containing 5 ng of
cDNA template, the sequence-specific oligonucleotide
primers (purchased from MWG-Biotech, Gmbh, Eurofins
Genomics, Hamburg, Germany) and the Power SYBR
Green PCR Master Mix (Applied Biosystems). 18S was
used to normalize RNA inputs. Fold change expression
respect to HMEC was calculated for all samples. The
sequence-specific oligonucleotide primers used are: AR
(NM_000044.3) forward, 5′-GCAGGAAGCAGTATCC
GAAG-3′ (position 1709); reverse, 5′-CTCTCGCCTT
CTAGCCCTTT-3′ (position 2067); 18S ribosomal
RNA (18S, X03205) forward, 5′- CAGCTTCCGGGA
AACCAAAGTC-3′ (position 1132); reverse, 5′- AATT
AAGCCGCAGGCTCCACTC -3′ (position 1222). Primer amplification efficiencies were 100.2% for AR and
101.3% for 18S, and the slope values were −3.316 for
AR and −3.291 for 18 s respectively. The comparative
Ct method was adopted for relative quantification of
gene expression and 18 s was used to normalize RNA
inputs. Fold change expression respect to HMEC was
calculated for all samples.
Statistical analysis
Data are presented as means ± S.E.M. Statistical and significant differences were determined using one-way
ANOVA with Newmann-Keuls or Dunnett multicomparison tests (GraphPad Prism version 4.00, GraphPad
Software, San Diego, CA) or nonparametric unpaired
Wilcoxon-Mann–Whitney test, as appropriate. A p value
of < 0.05 was considered significant. KolmogorovSmirnov statistical analysis was used to test significant
differences in cytofluorimetric data.
Results
Characterization of PTEC lines
Endothelial cells were purified by prostate carcinomas
of three patients who underwent radical prostatectomy
Fiorio Pla et al. BMC Cancer 2014, 14:939
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(Table 2). Three cell preparations of PTEC (PTEC 1, 2,
3) were obtained from different tumors. Cells were
characterized by cytofluorimetric analysis on the basis
of positive expression of a panel of endothelial
markers, such as CD31, CD105 and the angiopoietin
receptor TIE-2 (Figure 1A and Table 2). PTEC
expressed VEGFR1 and VEGFR2 but low levels of
VEGFR3 (Figure 1), the lymphatic-associated VEGF receptor. Moreover, CD146 was also expressed at low
levels (Figure 1), as previous reported on murine PTEC
isolated from spontaneous prostate tumors [17]. The
expression of endothelial markers was tested every culture passages and remained constant during cell culture for the three cell lines (Table 2). No cytokeratin
positive cells were detected by immunofluorescence
analysis (not shown).
The endothelial nature of PTEC was also showed by
functional characteristics. PTEC display a migration rate
comparable to that of breast tumor-derived ECs (BTEC)
both in serum-free conditions and in endothelial
medium (EndoGRO 5% FCS), as detected in wound
healing assays by time-lapse microscopy (Figure 1B).
Moreover, PTEC were able to organize in pre capillarylike structures onto Matrigel and to form tubules within
18 hours from seeding (Figure 1C). To evaluate the behavior of PTEC in vivo, cells (passage 2–4) were injected
subcutaneously within diluted Matrigel in SCID mice.
After 7 days, plugs were recovered and processed for
histological analysis. All cell lines grew and spontaneously organized within 1 week in functional microvessels, connected to the mouse vasculature, as shown by
the presence of blood cells and leukocytes (Figure 1C).
Together, these data indicate that PTEC presents both
Table 2 Expression of different endothelial markers by
the three cell lines isolated in the study
Cell lines
Isotype
CD146
PTEC1
PTEC2
PTEC3
AVERAGE
5.32 ± 0.57
6.34 ± 0.77
5.69 ± 0.98
5.783 ± 0.83
6.598 ± 1.08
12.203 ± 2.87
14.727 ± 9.89
11.176 ± 4.93
CD105
91.614 ± 28.56 70.547 ± 17.45 65.707 ± 26.94 75.956 ± 19.62
CD31
27.942 ± 3.75
30.723 ± 4.46
24.54 ± 7.92
28.068 ± 5.89
TIE-2
23.555 ± 2.46
19.973 ± 4.77
29.13 ± 4.04
24.219 ± 4.84
VEGFR1
19.954 ± 5.35
8.68 ± 4.67
12.17 ± 2.78
13.575 ± 5.94
VEGFR2
20.782 ± 3.07
18.545 ± 3.71
25.83 ± 17.55
21.719 ± 8.32
VEGFR3
5.057 ± 2.69
7.811 ± 2.44
10.603 ± 3.92
7.823 ± 3.16
Levels of endothelial markers was detected by cytofluorimetric analysis after
cell staining with a specific conjugated Abs. An irrelevant isotypic Ab (isotype)
was used as control of aspecific binding. Data represent the median
fluorescent intensity (MFI ± SD) detected on the cell isolates at all culture
passages used (1–6). All markers were significantly different versus isotypic
control (p < 0.001), as evaluated using the Kolmogorov-Smirnov
statistical analysis.
phenotypical as well as functional properties of endothelial cells.
Effect of Sunitinib and Sorafenib on PTEC proliferation,
cytotoxicity, tubulogenesis and migration
We subsequently evaluated the effect of Sunitinib and
Sorafenib, two anti-angiogenic drugs currently in clinical
trial for prostate cancer [5,22-24] on PTEC functional
properties. Both drugs impaired proliferation of normal
endothelial cells with an IC50 at similar doses around
1.5 μM (1.4675 and 1.5329 μM respectively), as evaluated by MTT analysis (Figure 2A). PTEC were treated
with 1 and 2.5 μM of both drugs. Sunitinib impaired
survival and proliferation of PTEC at a concentration as
low as 1 μM. At variance with Sunitinib, Sorafenib
(1 μM) had no effect on both proliferation and survival
of PTEC while a cytotoxic effect was observed at 2.5 μM
(Figure 2B and C). On the other hand, both macrovascular (HUVEC) and microvascular (HMEC) endothelial
cells, used as control, showed high sensitivity to both
Sorafenib and Sunitinib (Figure 2B and C and Additional
file 1: Figure S1 A and B).
We also studied the effect of Sunitinib and Sorafenib
on PTEC organization in pre capillary-like structures
onto Matrigel. As shown in Figure 2D, the ability of
PTEC to organize in tube structures was strongly inhibited by Sunitinib, both a 1 μM and 2.5 μM at a similar
extent to HUVEC and HMEC (Figure 2D and Additional
file 1: Figure S1 C). In contrast, Sorafenib had no inhibitory effect on tubulogenesis both on PTEC and HUVEC,
even at the higher dose tested (Figure 2D) and a minor
effect on HMEC at higher doses (Additional file 1:
Figure S1 C).
The effect of Sorafenib and Sunitinib on PTEC was also
tested in wound healing migration assays at the noncytotoxic dose (1 μM). Figure 3A shows that both 1 μM
Sunitinib and 1 μM Sorafenib significantly decrease cell
migration of about 15-20% compared to control conditions starting from 4 to 6 hours after treatment in all of
the three lines. However, 1 μM Sorafenib has significantly
less effect than 1 μM Sunitinib in two cell lines out of
three (Figure 3B and C), in line with the resistant behavior
of PTEC showed in the previous biological assays.
Sunitinib and Sorafenib reduce VEGFR2 phosphorylation
PTEC expressed the VEGFRs, known to be target of
anti-angiogenic tyrosine-kinases inhibitors Sunitinib and
Sorafenib (Figure 1A) whereas they did not express
other known targets such as the PDGFRβ and cKIT
(Figure 1A), suggesting VEGFR2 as a main extracellular target for both Sorafenib and Sunitinib. In order to
investigate whether the different sensitivity to Sorafenib observed for PTEC was due to a reduced inhibition
of the main target, we evaluated VEGFR2 activity by
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Figure 1 Characterization of PTEC lines. (A): Representative cytofluorimetric analysis of a PTEC line at the first passage. Specific antibodies are
shown as black line, isotypic controls as red line. (B): Percentage of migration of PTEC (blue line) compared to endothelial tumor cells isolated
from breast cancer (BTEC, black line) in EndoGRO plus 5% FCS (CNTRL+) or in DMEM without FCS (CNTRL-). (C-E): Representative images showing
PTEC organization in vitro and in vivo. In vitro, PTEC plated on Matrigel-coated wells organized in capillary-like structures (18 h) (C). When injected
in SCID mice within Matrigel, PTEC, organized in functional vessels containing red blood cells (arrows) (D and E, hematoxylin and eosin staining).
Original magnification D: × 200, E: × 400. All the isolated lines showed a similar marker expression and functional properties in vitro and in vivo.
Western blot analyses in the presence of the two antiangiogenic drugs. Sunitinib or Sorafenib treatment (1 μM,
10 or 30 minutes) significantly reduced one of the major
sites for VEGFR2 phosphorylation sites (Tyr951) [25] in
both PTEC and HUVEC, used as control (Figure 4A). In
particular, p-VEGFR2(Tyr951) levels decreased as soon as
10 minutes after treatment in PTEC, while in HUVEC the
decrease was evident only at 30 minutes after treatment.
Similarly, cell treatment with 1 μM Sorafenib decreased
the relative expression of p-VEGFR2(Tyr951) both in
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Figure 2 Cytotoxicity resistance, proliferation and tubulogenesis of Sunitinib and Sorafenib treated PTEC and HUVEC. (A): Relative
vitality of HUVEC treated with different concentrations (x-axis, logarithmic scale) of Sunitinib and Sorafenib, showing the IC50 (1.4675 μM for
Sunitinib and 1.5329 μM for Sorafenib, dotted line). (B and C): Cell survival and proliferation of PTEC (black columns) and HUVEC (white columns)
after 48 h incubation with 1 μM and 2.5 μM Sunitinib or Sorafenib. Cytotoxicity was detected as MTT assay, proliferation as BrdU assay. (D):
Capillary-like organization of PTEC (black columns) and HUVEC (white columns). Cells were seeded on Matrigel and observed at different times
points. Images at 18 hours of treatment were analyzed and total tubule length was measured for each field. Data are the mean ± S.E.M. of a
minimum of three independent experiments performed with three (PTEC1, 2 and 3) or five (HUVEC) different cell lines in triplicate. Statistical
significance *p < 0.05.
PTEC and HUVEC after 10 or 30 minutes of treatment
respectively (Figure 4B). The data therefore indicate that
VEGFR2-activated signaling pathway is impaired by both
Sunitinib and Sorafenib in PTEC at a similar extent as in
HUVEC.
Combination of anti-androgen and anti-angiogenic drugs
In the attempt to decrease the observed resistance of
PTEC to Sorafenib, we evaluated the effect of a combined treatment with the anti-androgen Casodex and
the anti-angiogenic drugs. Indeed, functional ARs were
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Figure 3 Wound healing migration in PTEC lines. (A): percentage of migration of PTEC after treatment with 1 μM Sunitinib or 1 μM Sorafenib
compared to positive control. (B): representative images of PTEC migration at time 0 and 8 hours. (C): quantification of relative cell migration at
8 hours for the three PTEC cell lines. 1 μM Sunitinib showed a higher inhibition of cell migration compared to 1 μM Sorafenib. Data are
expressed as mean ± S.E.M of three independent experiments performed with PTEC1, 2 and 3 and migration was normalized to control: Statistical
significance *p < 0.05.
Figure 4 Effect of Sorafenib and Sunitinib on VEGFR phosphorylation. Western blot analysis showing basal VEGFR2 and phosphorylated
p-VEGFR2(Tyr951) in PTEC and HUVEC after 10’ or 30’ treatment with 1 μM Sunitinib (A) and 1 μM Sorafenib (B). The relative expression of
p-VEGFR2(Tyr951) was normalized to basal VEGFR2. Values are expressed as mean ± S.E.M. relative to the control of three independent experiments
performed with three (PTEC1, 2 and 3) and two (HUVEC) different cell lines. Statistical significance *p < 0.05.
Fiorio Pla et al. BMC Cancer 2014, 14:939
http://www.biomedcentral.com/1471-2407/14/939
described in endothelial cells from benign prostate and
prostate cancer [4,26]. All PTEC lines expressed the AR
at mRNA level, being the expression in the PTEC2 line
the highest (Figure 5A). The AR expression was confirmed on PTEC2 by means of Western blot, as compared
with positive controls such as the AR overexpressing HEK
or the prostate LNCaP cells, (Figure 5B).
Cell treatment with Casodex alone (10 μM) significantly reduced cell proliferation of PTEC (Figure 5C)
while no effect was observed on HMEC, in accordance
with the lower receptor level expression (Figure 5A).
The reduction of cell proliferation induced by Sunitinib
was not modified by combination with Casodex on both
PTEC and HMEC. Interestingly, addition of Casodex
was able to counteract the resistance of PTEC to Sorafenib (Figure 5C). No additional effect on reduction of
proliferation was observed on HMEC (Figure 5C).
We subsequently analyzed the signal transduction
mechanism involved in PTEC resistance and in its rescue by Casodex. For this purpose, we first tested the effect of both anti-angiogenic drugs on Akt and p44/
42MAPK (ERK1/2) activation, important signaling pathways in the effect of anti-angiogenic drugs [27-29]. Sunitinib treatment reduced Akt phosphorylation (Ser473)
Page 9 of 13
whereas Sorafenib did not promote any effect (Figure 6A).
The inhibitory effect was observed at early times
(2 minutes), and quickly reverted by longer treatments
(5, 10 and 30 minutes, data not shown). On the other
hand, p44/42MAPK (ERK1/2) phosphorylation (Thr202/
Tyr204) was not affected by Sorafenib and only slightly
by Sunitinib (Figure 6B). This pattern did not change for
longer drug treatments (data not shown).
Finally, we evaluated the effect of the combination of
Casodex and anti-angiogenic drugs on the intracellular
signal transduction pattern observed in PTEC. Cell treatment with Casodex alone did not decrease Akt phosphorylation while a marked effect was detected on p44/
42MAPK (ERK1/2) phosphorylation (Figure 6C). When
cells were treated with Casodex and Sunitinib, Akt phosphorylation was farther reduced as compared to Sunitinib alone. Interestingly, the combined treatment of
Casodex and Sorafenib was able to strongly inhibit Akt
phosphorylation in respect to Sorafenib alone. On the
other hand, the effect observed with combined treatments
of both anti-angiogenic drugs and Casodex maintained the
inhibitory phosphorylation effect of the Casodex alone. We
can therefore speculate that the Akt intracellular pathway
plays a role in the observed resistance of PTEC to
Figure 5 Androgen inhibition impairs Sorafenib resistance in PTEC. (A): Analysis of Androgen receptor (AR) mRNA expression levels by qPCR
in PTEC cell lines. Data were normalized to 18S rRNA and to 1 for HME. Data are mean ± S.E.M. of three different experiments. (B): AR and actin
protein expression by Western blot in wild type HEK, HEK overexpressing AR (HEK-AR), LNCaP, HMEC and PTEC2 line. (C): Cell proliferation of PTEC
(black columns) and HUVEC (white columns) after 48 h incubation with 1 μM Sunitinib or Sorafenib in the presence or absence of 10 μM
Casodex. Data are the mean ± S.E.M. of a minimum of three independent experiments performed with two PTEC cell lines (PTEC2 and 3) in
triplicate. Statistical significance *p < 0.05.
Fiorio Pla et al. BMC Cancer 2014, 14:939
http://www.biomedcentral.com/1471-2407/14/939
Page 10 of 13
Figure 6 Effect of Sorafenib and Sunitinib on Akt and ERK1-2 phosphorylation. Western blot analysis showing basal Akt and phosphorylated Akt
(Ser473) (p-AKT) (A) or basal ERK1/2 and phosphorylated ERK1/2 (Thr202/Tyr204) (pERK1-2) (B) in PTEC2 after 2’ treatment with 1 μM Sunitinib and
1 μM Sorafenib in the presence or absence of 10 μM Casodex. The relative expression of phospho-Akt or phospho-pERK1/2 were normalized to basal
Akt or ERK1/2. Values are expressed as mean ± S.E.M. relative to the control of three independent experiments performed with three PTEC2 cell lysate.
Statistical significance *p < 0.05.
Sorafenib. The inhibition of Akt phosphorylation by the
combined treatment of Casodex and Sorafenib can therefore explain the rescue observed on cell proliferation.
Discussion
Taken together, the results of this study show a different
sensitivity of endothelial cells isolated from prostate tumors to the anti-angiogenic drugs Sunitinib and Sorafenib. Whereas normal endothelial cells showed similar
responses to both drugs in term of proliferation, survival
and motility, PTEC were affected by Sunitinib whereas
they were more resistant to Sorafenib. However, combined treatment with the anti-androgen Casodex was
able to enhance the susceptibility of PTEC to Sorafenib
likely trough inhibition of the Akt intracellular pathway.
Several evidences of the literature showed that TEC
present in different tumors, including prostate carcinoma, are different from normal endothelial cells at genetic, epigenetic and functional levels [9,17,30,31]. In
particular, recent studies of transcriptome and methylome analysis of endothelial cells from healthy or patients
affected by prostate cancer showed a wide spectrum of
differences in gene expression and methylation patterns in
endothelial cells between malignant and normal prostate
tissues [30,31]. In addition, murine endothelial cells from
spontaneous prostate tumors were reported to display a
mesenchymal differentiative ability [17]. In the present
study we isolated and cultured endothelial cells from
prostate tumors from patients without androgenic ablation therapy. PTEC were able to migrate, organize in
capillary-like structures in vitro and in vessel structures
in vivo, connected with the mouse vasculature, indicating
their endothelial phenotype. As previously reported [26],
PTEC expressed higher AR levels than normal endothelial
cells indicating the persistence of the phenotype of origin.
The ability of TEC to organize into functional vessels
in vivo has been previously described to be characteristic
of TEC isolated from human tumors, at variance with
HUVEC that undergo apoptosis [10,17,18].
PTEC may therefore represent a suitable model to assess the response to anti-angiogenic drugs and the related cell signal mechanisms. Indeed, although the
results of anti-angiogenic therapy in preclinical models
of prostate cancer provided promising results, some discrepancy between these data and those obtained in clinical trials were observed [32]. In particular, monotherapy
treatment of patients with advanced prostate cancer partially failed the endpoints [27,33-36]. A phase III study
comparing Sunitinib versus placebo showed a progression free survival but not an overall survival improvement in Sunitinib treated patients, although phase II
studies showed a PSA decline in plasma [33,37]. Phase II
studies with Sorafenib showed a regression of metastases
but not PSA decline [34,36]. Is therefore evident that
additional knowledge on endothelial characteristics in
prostate cancer is required.
Fiorio Pla et al. BMC Cancer 2014, 14:939
http://www.biomedcentral.com/1471-2407/14/939
In the present study, we showed that both drugs had a
cytotoxic effect on normal endothelial cells with a similar IC50 at 48 hour around 1.5 μM. This is in line with
previous observations showing that both Sorafenib and
Sunitinib are well known inhibitors of pro-angiogenic
functions in normal endothelial cells [16,38]. Accordingly, Sunitinib induced a dose dependent reduction of
proliferation and survival in PTEC as well as in HUVEC
and HMEC. In contrast, Sorafenib only partially affected
PTEC proliferation and survival. Both drugs slightly reduced cell motility, with a consistent lower effect of Sorafenib. In addition, Sunitinib, but not Sorafenib, affected
tubulogenesis in both HUVEC and PTEC. As Sunitinib
and Sorafenib share common targets [5,6], the differential
effect of these drugs on PTEC appears unexpected and of
interest.
Studies comparing the in vitro activity of Sorafenib
and Sunitib on endothelial cells are limited in the literature. A similar effect of the drugs on cell viability was
previously reported in neuroblastoma and corneal epithelial cells [39,40]. At variance, our results, showing
that PTEC were less sensible to Sorafenib than to Sunitinib, are in line with the reported resistance to Sorafenib
of endothelial cell from hepatocellular carcinoma [16].
As PTEC only expressed the VEGFRs, and not other
surface targets of Sunitinib and Sorafenib, such as
PDGFRβ and cKIT, we reasoned that the differential response to these drugs could depend on a differential effect on VEGFR2 phosphorylation [6]. However, VEGFR2
phosphorylation was inhibited by both drugs, excluding
this possibility. On the other hand, these drugs were reported to affect different intracellular pathways, including the Ras/Raf/MEK/ERK, JAK/STAT and the PI3K/
AKT pathways [27-29]. It is conceivable that the increased resistance to Sorafenib observed in PTEC may
be due to its activity on intracellular pathways differentially activated in normal and tumor endothelial cells, as
reported [10,41]. In PTEC, we observed that Sorafenib
and Sunitinib treatments differentially modulated Akt
phosphorylation, as no inhibitory effect of Sorafenib was
observed on Akt activation. These data correlate with
the functional resistance to the effect of Sorafenib observed on PTEC behavior in vitro. On the contrary, no
major effect of these drugs was observed on the p44/
42MAPK (ERK1/2) pathway. In this regard, Kharazhia
and co-workers recently described an increased Sorafenib resistance of highly-metastatic compared with non-metastatic
prostate cancer cells which was due to constitutively active
PI3K/AKT pathway targeted by Sorafenib [29].
Due to the important role of ARs in sustaining prostate
cancer progression, second line hormone therapy is frequently employed in prostate cancer to target persistent
AR activation. In addition, AR has been described to play
a role in the regulation of endothelial cell proliferation
Page 11 of 13
[26]. Moreover, the association of Sorafenib with antiandrogen therapy (Casodex) in a recent phase II clinical
trial induced PSA decline and stable disease [42], improving the effect of the anti-angiogenic monotherapy. Accordingly, our results combining Sorafenib and Casodex
successfully overcame the PTEC resistance to Sorafenib
both at the functional level and on the Akt pathway activation. Therefore, it can be inferred that the resistance to
Sorafenib treatment involves the Akt pathway; which is in
turn affected by the combined treatment with Casodex.
Conclusions
In conclusion, the results of the present study clearly
demonstrate a resistant behavior of endothelial cells isolated from prostate cancer to specific anti-angiogenic
drugs compared to normal endothelial cells. Indeed, it
appears that TEC are more appropriate for studying
angiogenesis mechanisms of tumors and exploring antiangiogenic drugs, compared with normal endothelial
cells. Finally, strategies to combine multi-targeted kinase
inhibitors with hormonal therapies may be of interest in
the context of prostate cancer.
Additional file
Additional file 1: Figure S1. Cytotoxicity resistance, proliferation and
tubulogenesis of Sunitinib and Sorafenib treated PTEC and HMEC. (A and
B): Cell survival and proliferation of PTEC (black columns) and HMEC
(white columns) after 48 h incubation with 1 μM Sunitinib or Sorafenib.
Sorafenib significantly affected HMEC, but not PTEC. Cytotoxicity was
detected as MTT assay, proliferation as BrdU assay. (C): Capillary-like
organization of PTEC and HMEC. Cells were seeded on Matrigel and
observed at different times points. A decrease in tube formation was
observed for both PTEC (black columns) and normal HMEC (white
columns) after treatment with 2.5 μM Sunitinib, while Sorafenib (2.5 μM)
only affected tubule formation in HMEC. Data are the mean ± S.E.M. of a
minimum of three independent experiments in triplicate. Statistical
significance *p<0.05.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AFP, NP and BB designed the research; AV and XL carried out the
prostatectomy and collected patients’ data; AB isolated the cells, carried out
flow cytometry, proliferation and cytotoxicity assays, protein and RNA
isolation, Real Time PCR; BB and AB carried out immunoflorescence analysis
and in vivo tubule formation. MB and AFP carried out in vitro tubule
formation and cell migration experiments; TG and GG performed Western
blotting; MB carried out the statistical analysis; BB, AFP, AB, MB and DG wrote
the paper. All of the authors have been involved in revising the manuscript
and have given final approval of the version to be published.
Acknowledgements
This study was supported by Italian Ministry of University and Research
(MIUR) Prin08 to BB and by grants from Ministère de l’Education Nationale,
Inserm, France to DG. MB is supported by the Vinci program 2012-Université
Franco Italienne.
Author details
1
Department of Molecular Biotechnology and Health Sciences, Molecular
Biotechnology Centre, University of Torino, via Nizza 52, 10126 Torino, Italy.
Fiorio Pla et al. BMC Cancer 2014, 14:939
http://www.biomedcentral.com/1471-2407/14/939
2
Department of Life Science and Systems Biology, University of Torino,
Torino, Italy. 3Nanostructured Interfaces and Surfaces Centre of Excellence
(NIS), University of Turin, Torino, Italy. 4Inserm U1003, Equipe labellisée par la
Ligue Nationale contre le cancer, Université des Sciences et Technologies de
Lille (USTL), Villeneuve d’Ascq, France. 5Laboratory of Excellence, Ion
Channels Science and Therapeutics, Université de Lille 1, Villeneuve d’Ascq,
France. 6Department of Urology, CHU Lille, University Lille Nord de France,
F-59000 Lille, France. 7Institute of Pathology, Centre de Biologie-Pathologie,
CHRU de Lille, Faculté de Médecine Henri-Warembourg, Université de Lille 2,
Lille, France.
Received: 17 June 2014 Accepted: 4 December 2014
Published: 12 December 2014
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doi:10.1186/1471-2407-14-939
Cite this article as: Fiorio Pla et al.: Differential sensitivity of prostate
tumor derived endothelial cells to sorafenib and sunitinib. BMC Cancer
2014 14:939.
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