PE, a new sulfated saponin from sea cucumber, Exhibits anti-angiogenic and anti-tumor activities in vitro and in vivo

Abstract

Here, we examined the in vitro and in vivo anti-angiogenesis and anti-tumor activities of PE, a new marine-derived compound. Inhibition of angiogenesis was assessed in vitro using proliferation, migration, adhesion, tube-formation and apoptosis assays in PE-treated HMECs and HUVECs. In vivo, CAM assays were used to assess inhibition effect of PE on physiological angiogenesis, and immunofluorescent microscopy was used to examine tumor microvessel density and apoptosis in PE-treated mouse tumor models. Finally, Western blotting analyses were performed to examine the effect of PE on VEGF signaling in HMECs. The results showed that PE inhibited proliferation of HMECs and HUVECs with IC50 values of 2.22 +/- 0.31 microM and 1.98 +/- 0.32 microM, induced endothelial cell apoptosis at concentrations <2 microM, induced dose-dependent suppression of cell migration, cell adhesion and tube formation in HMECs and HUVECs, and showed anti-proliferative activities against several tumor cell lines (IC50 values of approximately 4 microM). In vivo, PE (5 nM/egg) suppressed spontaneous angiogenesis in our CAM assay, and induced marked growth inhibition in mouse sarcoma 180 and hepatoma 22 models. Specifically, PE treatment reduced mouse sarcoma 180 tumor volume by triggering apoptosis of both tumor and tumor-associated endothelial cells, preferentially targeting on endothelial cells comparable with tumor cells. Finally, PE treatment suppressed the active (phosphorylated) forms of VEGFR2, Akt, ERK, FAK and paxillin, which are involved in endothelial cell survival, proliferation, adhesion and migration. Our results indicate that PE exerts an anti-angiogenic activity associated with inhibition of VEGFR2 signaling, and an anti-tumor activity associated with decreased proliferation of tumor cells and increased apoptosis of both endothelial cells and tumor cells.

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©2005 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
[Cancer B
iology & Therapy 4:8, 874-882, August 2005]; ©2005 Landes Bioscience
874
Cancer B
iology &
Therapy 2005; Vol. 4 Issue 8
Fang Tian
1,2
Xiongwen Zhang
1
Yunguang Tong
1,2
Yanghua Yi
3
Shilong Zhang
3
Ling Li
3
Peng Sun
3
Liping Lin
1
Jian Ding
1,
*
1
Division of Anti-Tumor Pharmacology; State Key Laboratory of Drug Research;
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Science;
Chinese Academy of Sciences; Shanghai P.R
. China.
2
Graduate School of Chinese Academy of Sciences; Beijing P
.R. China.
3
Research Center for Marine Drugs; School of Pharmacy; Second Military Medical
University; Shanghai 200433 P.R. China
*Correspondence to: Jian Ding; Division of Anti-tumor Pharmacology; State Key
Laboratory of Drug Research; Shanghai Institute of Materia Medica; Shanghai
Institutes for Biological Sciences; Chinese Academy of Sciences; 555 Zu Chong Zhi
Road; Zhangjiang Hi-Tech Park; Shanghai, 201203 P.R. China; Fax:
86.21.50806722; Email: jding@mail.shcnc.ac.cn
Received 05/12/05; Accepted 06/14/05
Previously published online as a
Cancer Biology & Therapy E-publication:
http://www
.landesbioscience.com/journals/cbt/abstract.php?id=1917
KEY WORDS
PE, marine, angiogenesis, endothelial cells,
VEGFR2, apoptosis, anti-tumor
ACKNOWLEDGEMENTS
High Tech Research and Development Program
(N
o. 2002AA2Z346A; 2001AA624100); the
Knowledge Innovation Program of Chinese Academy
of Sciences (No. KSCX2-SW-202, No. KSCX2-3-
07-8) and the N
ational Natural Science Foundation
(No. 30228032).
Research Paper
PE, a New Sulfated Saponin from Sea Cucumber, Exhibits Anti-Angiogenic
and Anti-Tumor Activities In Vitro and In Vivo
ABSTRACT
Here, we examined the in vitro and in vivo anti-angiogenesis and anti-tumor activities
of PE, a new marine
-derived compound. Inhibition of angiogenesis was assessed in vitro
using proliferation, migration, adhesion, tube
-formation and apoptosis assays in PE-treated
HMECs and HUVECs. In vivo, CAM assays were used to assess inhibition effect of PE on
physiological angiogenesis, and immunofluorescent microscopy was used to examine
tumor microvessel density and apoptosis in PE-treated mouse tumor models. Finally,
Western blotting analyses were performed to examine the effect of PE on VEGF signaling
in HMECs.The results showed that PE inhibited proliferation of HMECs and HUVECs with
IC
50
values of 2.22 ± 0.31 µM and 1.98 ± 0.32 µM, induced endothelial cell apoptosis
at concentrations <2
µM, induced dose-dependent suppression of cell migration, cell
adhesion and tube formation in HMECs and HUVECs, and showed anti-proliferative
activities against several tumor cell lines (IC
50
values of ~4 µM). In vivo, PE (5 nM/egg)
suppressed spontaneous angiogenesis in our CAM assay, and induced marked growth
inhibition in mouse sarcoma 180 and hepatoma 22 models. Specifically, PE treatment
reduced mouse sarcoma 180 tumor volume by triggering apoptosis of both tumor and
tumor-associated endothelial cells, preferentially targeting on endothelial cells comparable
with tumor cells. Finally, PE treatment suppressed the active (phosphorylated) forms of
VEGFR2, Akt, ERK, FAK and paxillin, which are involved in endothelial cell survival,
proliferation, adhesion and migration. Our results indicate that PE exerts an anti-angiogenic
activity associated with inhibition of VEGFR2 signaling, and an anti-tumor activity associated
with decreased proliferation of tumor cells and increased apoptosis of both endothelial
cells and tumor cells.
ABBREVIATIONS
ATCC, American type culture collection; CAM, chorioallantoic membrane; DMSO,
dimethylsulfoxide; EGF, epithelial growth factor; ELISA, enzyme-linked immunosorbent
assay; FAK, focal adhesion kinases; H–22, hepatuma 22; HMEC, human microvascular
endothelial cell; HUVEC, human umbilical vein endothelial cells; IC
50
, 50% inhibitor
y
concentration; KDR/Flk-1, kinase inser
t domain-dontaining receptor/fetal liver kinase;
MMP, matrix metalloproteinases; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium
bromide; OD, optical density; PBS, phosphate buffer saline; S–180, aSarcoma 180; SD,
standard deviation; SRB, sulforhodamine B; TUNEL, terminal deoxynucleotidyl transferase
mediated nick end labeling; VEGF, vascular endothelial growth factor; VEGFR, vascular
endothelial growth factor receptor
INTRODUCTION
Angiogenesis is formation of ne
w vessels from preexisting capillaries. Among the
known angiogenic growth factor and cytokines, VEGF and its corresponding receptors are
indispensable in regulating multiple facets of the angiogenic processes. As angiogenesis
plays a central role in tumor growth, progression, invasion and metastasis,
1
inhibition of
this process provides a potential strategy for cancer treatment.
2
Researchers are currently
seeking to dev
elop ne
w angiogenesis inhibitors
3
fr
om sour
ces such as cleav
ed proteins,
monoclonal antibodies, synthesized small molecules and natural products.
4
However,
recent studies have suggested that sole blockage of angiogenesis may not be sufficient to
fully suppress malignancies,
5,6
indicating that dual treatments may be more effective. For
example, the VEGF inhibitor Avastin was shown to extend the lives of colon cancer
patients when given intravenously in combination with standard chemotherapy drugs
(ironotecan, 5-FU and leucovorin), but was not effective when used as a single treatment

drug.
7
Thus, it would seem valuable to seek molecules that simulta-
neously confer both anti-angiogenic and anti-tumoral activities.
Marine-derived natural products contain a variety of chemother-
apeutic compounds that have been shown to prevent the development
of malignancies,
8
and several marine-derived molecules are currently
in or entering clinical trials in cancer therapy
,
9
pr
omising that
marine natural pr
oducts act as a rich sour
ce for cancer therapy
.
Notably, we discovered that serials marine-derived chemotherapeutic
agents exert anti-angiogenic properties. Of them, PE, a novel com-
pound isolated from the sea cucumber
pentacta quadrangularis, drew
our special inter
ests, due to its both anti-angiogenesis and
anti-tumor activity upon our screening in several in vitro models.
Here, we examined the ability of PE to suppress angiogenesis and
tumor development in vitro and in vivo, and examined possible
mechanisms of action. O
ur r
esults indicate that P
E has str
ong
anti-angiogenic activity, which may account in part for its
anti-tumor activities. The potent anti-angiogenic activity of PE at
least partly due to inhibition of VEGF receptor 2 signaling, and its
anti-tumoral effects was associated with decreased tumor cell prolif-
eration and increased tumor cell apoptosis. Thus, PE may be viewed
as a possible candidate molecule for cancer therapeutics.
MATERIALS AND METHODS
Materials. Soluble KDR/F
lk
-
1 was pur
chased fr
om Calbiochem.
Anti-pERK, Anti-pKDR and Anti-pAKT were obtained from Cell
Signaling Technology. The anti-CD-31, anti-VEGF, anti-pFAK and
anti-paxillin antibodies were obtained from Santa Cruz Biotechnology. The
Prolong anti-fade kit and the fluorescent Alexa Fluor 546-conjugated sec-
ondary antibody were purchased from Molecular Probes. The cell culture
mediums (M199 and MCDB131), endothelial cell growth supplement
(ECGS), VEGF165, MTT, Heparin, 5-FU, Suramin, and all other reagents
w
er
e pur
chased fr
om S
igma.
I
solation and pur
ification of PE.
P
E was isolated fr
om the sea cucumber
Pentacta quadrangularis. Specimens were collected near Guangdong
P
r
ovince, China, and identified by Prof. J. R. Fang of the Fujian Institute of
Oceanic Research (China). Air-dried body walls (5 kg, dried weight) of
P. quadrangulasis were cut into pieces and extracted twice with refluxing
ethanol.
The combined extracts w
ere evaporated in vacuo and further parti-
tioned between water and chloroform. The water layer was extracted with
n-butanol and the organic layer was evaporated in vacuo to yield n-butanol
extracts.
The n
-butanol extracts were concentrated, and the extracted residue
was dissolved in water. Samples were desalted with a DA101 resin column
(60 x 30 cm), with the inorganic salts and polar impurities eluted with
water
, and the crude glycoside fraction (8.1 g) subsequently eluted with
80% ethanol. The latter fraction was separated by flash chromatography on
silica gel (6/40 cm; CHCl
3
)/MeOH/H
2
O 7.0:3.0:0.2, 20 ml/min) to yield
a cr
ude PE-containing fraction. This faction was further separated by
HPLC (Zorbax 300 SB-C18, 9.4 mm x 25 cm, 55% MeOH/H
2
O, flow
rate 1.5 ml/min) to yield pure PE. The PE was dissolved in DMSO and
diluted to the desir
ed concentrations before use, with the final DMSO
concentration maintained <0.05% in the various treatment groups. The
chemical structure of PE was determined using
1
H and
13
C NMR spectra,
ESI-MS and the IR spectrum, as previously described (Fig. 1A).
Cell lines and cell culture. Human umbilical vein endothelial cells
(HUVECs) were isolated from human umbilical cord veins by collagenase
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Cancer B
iology & Therapy 875
P
E Inhibits Angiogenesis and Tumor Growth
Figure 1. Inhibitory effect of PE on endothelial and tumor cell proliferation. (A) The chemical structure of PE. (B) HMECs and HUVECs were treated with PE
for 72 h and the effect on cell growth was measured using the SRB method. (C) HMECs were treated with PE for 8 or 24 h and measured using the SRB
method. (D) IC
50
values for the effect of PE on endothelial and tumor cells.*P < 0.01 vs. PE treated HMECs. Each value is the mean ± SD of three independent
experiments.
A B
CD

876
Cancer B
iology &
Therapy 2005; Vol. 4 Issue 8
treatment as described previously.
10
Cells from passages 2–6 were grown in
Medium 199 containing 100 unit/ml penicillin, 10 ng/ml EGF, 3
µg/ml
endothelial cell growth supplement (ECGS), and 20% fetal bovine serum
(FBS). Human dermal microvascular endothelial cells (HMECs) were
obtained from the ATCC, and cultured in MCDB-131 containing 15% FBS,
1 ng/ml EGF and 1 mg/ml hydrocortisone. Gastric adenocarcinoma cell line
MKN-28 was obtained from the Japanese Foundation for Cancer Research
(JFCR) and cultured in 1640 culture medium containing 10% FBS. Colon
adenocarcinoma cell line HCT-116, and breast adenocarcinoma cell lines
MDA-MB-468 and MCF-7 were obtained from the ATCC and were cul-
tured in 5A, L-15 and DMEM media, respectively, containing 10% FBS.
Hepatocellular carcinoma cell line BEL-7402, lung adenocarcinoma cell
line SPC-A4 and ovarian epitheloid carcinoma cell line HO-8910 were
obtained from the Shanghai Institute of Biochemistry and Cell Biology
(SIBCB), and cultured in 1640 culture medium containing 10% FBS. All
cells were maintained at 37˚C under a humidified 95% and 5% (v/v) mixture
of air and CO
2
.
Cell proliferation assay. Endothelial cells were seed into 96-well plates
at a density of 8 x 10
3
cells/well. After the cells were incubated overnight,
the medium in each well was replaced by fresh MCDB131 medium containing
different concentrations of PE or 0.01% DMSO (v/v). After incubation for
72 h, the medium was removed from each well and the relative number of
cells was determined in triplicate wells using the SRB assay, as described
previously.
11
Endothelial cell differentiation assay. Matrigel was thawed at 4˚C, and
60
µl aliquots were quickly added to each well of a 96-well plate and allowed
to solidify for 30 min at 37˚C. Endothelial cells (2.5 x 10
4
cells/well) were
seeded onto the Matrigel and cultured in MCDB131 medium containing
different concentrations of PE or 0.01% DMSO (v/v) for 12 h. For analysis
of tube formation, the enclosed networks from five
randomly chosen fields were counted and photographed
under a microscope (IX70, Olympus, Japan). The total
length of the tube structure in each photograph were
measured using the Adobe Photoshop software,
12
and
inhibition of tube formation was calculated as [1 -
(tube length
tr
eated
/tube length
contr
ol
)] x 100%.
Endothelial cell migration assay. Briefly, the
chemotactic motilities of endothelial cells were
assayed using Transwell Boyden Chambers (Costar, MA,
USA) with 6.5-mm diameter polycarbonate filters (8
µm
pore size).
13
The lower surface of each filter was coated
with 10
µg of gelatin, and 600 µl M199 medium
containing 1% FBS and 10 ng/ml VEGF was placed
in each lower well. Endothelial cells were trypsinized
and suspended at a final concentration of 1 x 10
6
cells/ml in M199 containing 1% FBS, and 100 µl of
the cell suspension was loaded into each upper well,
along with various concentrations of PE or 0.01%
DMSO (v/v). The chambers were then incubated at
37˚C for 8 h, whereupon cells were fixed and stained
with crystal violet. Nonmigrating cells on the upper
surface of the filter were removed with a cotton swab,
and chemotaxis was quantified by counting the cells
that had migrated to the lower side of the filter, as
visualized under a microscope (IX70, Olympus, Japan).
Ten random fields were counted for each assay. The
inhibition of migration was calculated as [1 - (migrated
cells
treated
/migrated cells
control
)] x 100%.
Chicken embryo chorioallantoic membrane (CAM)
assay.
The CAM angiogenesis assay was performed as
described previously, with some modifications.
14
Briefly, fertilized chicken eggs were incubated in a
humidified egg incubator (Lyon, CA, USA) for eight
days, and then a small hole was punched into the
broad side of the egg and a window was carefully
created in the eggshell. Various amounts of PE or 0.01% DMSO (v/v) were
air-dried onto sterile glass coverslips of approximately 1 mm
2
. The coverslips
were placed onto well-vascularized sites of the CAM of the developing chick
embryos, and the eggs were returned to the humidified egg incubator.
Forty-eight hours later, the sites were evaluated and recorded with stereomi-
croscopic photography (MS5, Leica, Switzerland). Angiogenesis was quanti-
fied b
y counting the number of blood v
essel branch points in each photo
. A
positive anti-angiogenic effect was scored when the microvessels were obvi-
ously reduced under the coverslips. At least ten viable embryos were tested
for each treatment.
Cell adhesion assay. The efficiency of cell adhesion was determined by
measuring the number of cells that adhered to a given substrate.
15
F
ibronectin was diluted in sterile water (10 mg/ml), aliquotted to 96-well
plates 100
µl per well, and incubated overnight at 4˚C. The samples were
blocked with 1% BSA at 37˚C for 1 h, and then 0.5 x 10
5
cells in 100 µl of
pr
e
warmed serum-free medium were seeded into each well and allowed to
adhere at 37˚C for 1 h with various concentrations of PE or 0.01% DMSO
(v/v). Nonadherent cells were rinsed off with PBS and the remaining cells
w
er
e fix
ed with 4% paraformaldehyde for 10 min, then stained with 0.5%
crystal violet in 4% paraformaldehyde for 5 min and rinsed with water. Cells
were solubilized by the addition of 100
µl of 1% SDS and quantified in a
micr
otiter plate r
eader at 590 nm with a multi
-
well spectrophotometer
(
VERSAmax, Molecular Devices, CA, USA).
TUNEL assay. Cellular apoptosis was determined using the TUNEL
assay
.
16
E
ndothelial cells w
er
e gr
own to 75% confluence on coverslips and
incubated for 36 h with various amounts of PE or 0.01% DMSO (v/v). The
TUNEL (terminal deoxynucleotidyl transferase mediated dUTP-biotin nick
end
-
labeling) assay was per
formed with the I
n Situ Cell Death Detection Kit
(Roche Diagnostics, Barcelona, Spain), according to the manufacturer’s
instructions. Briefly, the cells were fixed in 4% paraformaldehyde, permeabilized
P
E Inhibits Angiogenesis and Tumor Growth
Figure 2. Effect of PE on endothelial cell apoptosis. (A) HMECs and HUVECs were treated with PE
for 36 h and TUNEL staining was used to examine apoptosis. (B) Fluorescence intensity of TUNEL
staining in HMECs and HUVECs undergoing PE treatment. Results are expressed as the mean ± SD
of three separate experiments. *p < 0.01 vs. control.
A
B

with 0.1% Triton X-100 and 0.1% sodium citrate (freshly prepared), and
labeled with fluorescein- 12-dUTP. Apoptosis was detected by fluorescence
microscopy (Olympus, BX51, Japan) and quantified by the Image-Pro Plus
5.0 image analysis software (Media Cybernetics Inc, Maryland).
Western blotting analysis. Confluent HMECs were incubated for 24 h
in MCDB131 containing 1% FBS, incubated for 1 h in MCDB131 without
FBS in the presence PE or DMSO control, and then stimulated by the addition
of VEGF (50 ng/ml) for 10 min. After stimulation, cells were lysed in lysis
buffer (20 mM Tris/HCl, pH 8.0, 2 mM EDTA, 137 mM NaCl, 1 mM
Na
3
VO
4
, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 1%
Triton X-100). Lysates were clarified by centrifugation at 15,000 x
g for
10 min, resolved by SDS-PAGE and transferred to polyvinylidene difluoride
membranes. The membranes were blocked with 5% nonfat milk for 1 h at
room temperature and then probed with primary antibody overnight at
4˚C. Immunoreactive bands were visualized by incubation with horseradish
peroxidase-conjugated second antibodies and application of an enhanced
chemiluminescent (ECL) system (Amersham Biosciences). Each experiment
was repeated at least 3 times, with representative blots presented.
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Cancer B
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P
E Inhibits Angiogenesis and Tumor Growth
Figure 3. Effect of PE on endothelial cell function. (A) Endothelial cells seeded in Transwell Boyden Chambers were incubated for 8 h with or without 1.25
µM PE. Migrated cells on the lower sur
face of the filter were stained with cr
ystal violet and counted manually from five random fields. (B) Overall rate of
PE-induced inhibition of endothelial migration. (C) Effect of PE on endothelial cell adhesion. HMECs were plated on fibronectin-coated wells with or without
the indicated concentration of PE for 1 h. The relative number of attached cells was assessed by staining with crystal violet, and the absorbance value was
determined at 590 nm. (D) Endothelial cells seeded in Matrigel-coated 96-well plates were incubated for 24 h with or without 1.25 µM PE. The enclosed
networks of tubes were photographed from five randomly chosen microscopic fields. (E) Overall rate of PE-induced inhibition of tube formation. Results are
expressed as mean ± SD of three separate experiments.
A B
C D
E

878
Cancer B
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In vivo tumor growth inhibition assay. Female KM mice (6–8 weeks of
age) were used to study inhibition of tumor growth in vivo. The use of lab
animals was in accordance with guidelines of the Experimental Animal
Association of China, certificate number: SCXK (Shanghai; 2003-0003).
Sarcoma 180 cells or hepatoma 22 cells (2.5 x 10
6
) were subcutaneously
implanted into the axilla of mice on day 0, and mice were randomly
grouped (8 mice per group) on day 1. Normal saline and dilutions of PE (1,
2 and 3 mg/kg, dissolved in normal saline) were delivered intravenously to
nonanaesthetized mice once daily for seven days. Animals were sacrificed
24 h after the last administration, and mice and tumor weights were measured.
The tumor growth inhibition rates were calculated as follows:
Tumor weight of control - Tumor weight of drug-treated
Tumor growth inhibition rate = x100%
Tumor weight of control
Immunofluorescence. Paraffin-embedded tumor tissue sections (5 µm-
thick) putted on slides were dewaxed by heating at 60˚C, washed in xylene
and rehydrated through a graded series of ethanol and double distilled water
washes. The slides were washed with PBS and permeabilized with 0.1%
Triton X-100 and 0.1% sodium citrate (freshly prepared). The samples were
blocked with PBS/5% BSA for 1 h, incubated with the anti-CD31 polyclonal
antibody for 1 h at room temperature, washed three times in PBS, and then
incubated for 1 h at room temperature with a secondary antibody conjugated
to fluorescent Alexa Fluor 546 (Molecular Probes). Samples were finally
washed three times in PBS and mounted with coverslips using Prolong
anti-fade medium (Molecular Probes). Immunofluorescent staining was
observed and photographed using a fluorescence microscope. For double
staining, TUNEL detection was performed as described above, followed by
anti-CD-31 detection. All fluorescence photographs were taken under a
fluorescence microscope (Olympus, BX51, Japan) and analyzed with the
Image-Pro Plus image analysis software (Media Cybernetics Inc, Maryland).
Fluorescence intensities were gathered from three random fields per slice
(from three random slices per tumor) and presented as the mean±SD.
Data analysis. All results were expressed as mean±SD, and statistical
significance was assessed by Student’s t-test.
RESULTS
PE inhibits proliferation of HMECs, HUVECs and tumor cell lines. As
angiogenesis involves local proliferation of endothelial cells, we initially
investigated the effects of PE on proliferation of HMECs and HUVECs.
Treatment with PE inhibited the growth of cultured HMECs and HUVECs
in a concentration-dependent manner (Fig. 1B), with IC
50
values of 2.22 ±
0.31
µM and 1.98 ± 0.32 µM, respectively. Treatment of HMECs with
5
µM of PE for 8 or 24 h did not have any detectable effect on HMEC
proliferation (Fig. 1C), so this concentration of PE was used for the HMEC
migration and tube formation assays, to ensure that the detected abilities of
PE to inhibit endothelial cell functions were specific effects rather than the
results of general cytotoxicity. In addition, PE treatment inhibited the
proliferation of all tested tumor cell lines, displaying slightly higher IC
50
values
ranging from 2.4 to 4.1
µM (Fig. 1D).
PE induces apoptosis of endothelial cells. Anti-proliferation and subse-
quent anti-angiogenesis have been correlated with several underlying mech-
anisms,
17
including induction of apoptosis.
18
Accordingly, we used TUNEL
staining to investigate whether PE treatment induced endothelial cell apop-
tosis. HMECs and HUVECs exposed to 0.625, 1.25, and 2.5
µM PE for
24 h showed a slight induction of apoptosis (data not shown), while treatment
for 36 h resulted in dose-dependent induction of apoptosis (Fig. 2).
PE hinders endothelial cell migration. As endothelial cell migration is a
prerequisite for angiogenesis, we explored the effect of PE on directional cell
motility using a Transwell Boyden Chamber assay. As shown in Figure 3A,
incubation of control HMECs or HUVECs in the chamber for 8 h resulted
in large
-
scale migration of endothelial cells to the lo
wer side of the filter. In
contrast, treatment with PE (0.313–5
µM) dose-dependently inhibited
HMEC and HUVEC migration, yielding IC
50
values of 1.36 ± 0.12 µM
and 1.09
±
0.01
µM, r
espectiv
ely
.
PE suppresses endothelial cell adhesion. Adhesion of endothelial cells to
the extracellular matrix enables the cells to respond to growth factors by
migrating, pr
oliferating and forming ne
w blood vessels. Therefore, we inves-
tigated whether PE affects adhesion of endothelial cells to fibronectin, a
common component of the extracellular matrix. Control HMECs attached
efficiently to fibr
onectin after plating for 1 h, wher
eas HMECs treated with
0.1–20 µM PE showed a dose-dependent decrease in cell adhesion (Fig. 3C),
with an IC
50
value of 2.84 ± 0.18 µM.
P
E disr
upts the capillary tube formation of endothelial cells.
As organ
-
ization of endothelial cells into a network of tubes is a late event during
angiogenesis, we used a Matrigel-induced tube formation assay to determine
whether P
E treatment inhibits endothelial cell differentiation. Matrigel-
cultured control HMECs migrated and organized into capillary-like
enclosed tubular networ
ks, wher
eas those tr
eated with v
arious concentra
tions
of P
E for 8 h showed dose-dependent inhibition of tube formation (Fig. 3D).
Furthermore, PE treatment disrupted tube structure, leading to the develop-
ment of incomplete tube morphologies. S
imilar r
esults w
er
e obtained in
P
E Inhibits Angiogenesis and Tumor Growth
Figure 4. Effect of PE on CAM. Fertilized eggs were incubated continuously
for 8 days, and then a window was opened to expose the CAM and PE was
added to a final concentration of A) 0 nM/egg (solvent control), B) 2.5
nM/egg, C) 5 nM/egg or D) 10 nM/egg. The eggs were incubated for
another 48 h, and then the treated CAMs were harvested and photographed.
E) Overall inhibition rate of PE on CAM angiogenesis, assessed by quan-
tification of the blood vessel branch points in each photograph. Each value
is the mean±SD from ten eggs.
A B
C D
E

HUVECs. PE at concentrations of 0.313, 0.625, 1.25, 2.5 and 5 µM
reduced tube formation by 3.28, 9.95, 41.03, 63.84, and 94.18% in
HMECs and by 11.10, 19.94, 53.66, 82.38 and 97.77% in HUVECs,
compared to control cells. The IC
50
values were 1.52±0.05 µM in HMECs
and 1.18±0.02
µM in HUVECs.
PE reduces neovascularization of the CAM. The CAM of the chicken
embryo provides a unique model for evaluating new blood vessel formation
and the response of new vessels to potentially anti-angiogenic agents. Using
this model, we examined the potential anti-angiogenic activities of PE in
vivo. In control eggs, blood vessels formed densely branching vascular net-
works (Fig. 4A). Treatment with PE caused a dramatic, dose-dependent
inhibition of blood vessel numbers and branching patterns, with PE con-
centrations of 2.5, 5 and 10 nM/egg yielding inhibition rates of 51, 70.6
and 92.6%, respectively (Fig. 4B). Notably, PE at 10 nM/egg suppressed
new blood vessel development not only in the treatment area, but also in the
surrounding area. Moreover, 5 nM/egg PE had a similar anti-angiogenic
effect to that of 100 nM/egg of suramin, a well-known angiogenesis
inhibitor
4
(69% inhibition).
PE counteracts tumor angiogenesis and induces apoptosis of endothelial
cells in vivo.
To further explore the anti-angiogenic and anti-tumor effects
of PE in vivo, we next examined the effect of PE in two mouse models,
those harboring tumors induced by implantation of sarcoma 180 cells, and
those harboring tumors induced by hepatoma 22 cells. Administration of 2,
3 and 4 mg/kg PE for 7 consecutive days following implantation of sarcoma
180 cells hindered tumor growth by 28.2, 55.6 and 60.7%, respectively
(Table 1), while administration of 1, 2 and 3 mg/kg PE for seven consecutive
days following implantation of hepatoma 22 cells suppressed tumor growth
by 20.6, 46.1 and 59.4%, respectively (Table 2). These results indicate that
PE has anti-tumoral activity in vivo.
Tumor specimens from PE-treated and control mice implanted with
sarcoma 180 model cells were subjected to immunofluorescent staining with
anti-CD31 for detection of endothelial cells and TUNEL staining for
visualization of apoptosis. Anti-CD31 immunofluorescent staining revealed
that PE dramatically decreased tumor microvessel density in a concentration-
dependent fashion (Fig. 5A), while TUNEL staining revealed that treatment
with 4 mg/kg PE induced a 9.8-fold increase in apoptotic tumor endothelial
cells (Fig. 5D) and a 5.6-fold increase in apoptotic tumor cells (Fig. 5B).
Collectively, these in vitro and in vivo results indicate that PE counteracts
tumor angiogenesis and induces apoptosis of tumor endothelial cells. In
contrast, while 5-FU (a representative chemotherapeutic agent) inhibited
tumor growth and induced tumor cell apoptosis, this drug exerted almost no
effect on tumor angiogenesis or endothelial cell apoptosis.
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Cancer B
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P
E Inhibits Angiogenesis and Tumor Growth
Figure 5. Immunofluorescent evaluation of tumor tissues. (A) Apoptosis detection in tumor tissues. Sequential staining for TUNEL and CD31 was performed
in mouse sarcoma 180 tumor tissue sections taken from the normal saline (NS)-, PE- and 5-FU-treated groups. (B) Effect of PE and 5-FU on apoptosis of tumor
cells. The data were gathered from three random fields per slice (from three random slices per tumor) and are presented as apoptotic fluorescence intensity
(mean ± SD). *p < 0.01 vs. control. (C) Effect of PE and 5-FU on tumor blood vessels. The data were gathered from three random fields per slice (from
three random slices per tumor) and are presented as endothelial cell fluorescence intensity (mean ± SD). *p < 0.01 vs. control. (D) Effect of PE and 5-FU on apop-
tosis of tumor-associated endothelial cells. The data were gathered from three random fields per slice (from threerandom slices per tumor) and are expressed
as the mean ± SD of the fluorescence intensity ratio (TUNEL/CD31). *p < 0.01 vs. control, p < 0.01 vs. 5-FU group.
AB
C
D

PE inactivates VEGF-induced tyrosine phosphorylation of VEGFR2
and arrests downstream signaling in HMECs.
Angiogenesis and subsequent
cancer growth and progression are often modulated by vascular endothelial
gr
owth factor (VEGF), a key mediator of angiogenesis that is produced by
tumor cells. VEGF stimulates endothelial cell growth via binding and
activation of the VEGF receptor 2 (KDR/Flk-1), which triggers downstream
sign
aling pathways. Accordingly, we examined the effect of PE on VEGF
165
-
induced cellular KDR phosphorylation using Western blot analysis. When
HMECs were stimulated with 50 ng/ml VEGF
165
for 5 min, KDR was
strongly tyrosine phosphorylated. In contrast, preincubation of HMECs
with 1.25–5
µΜ PE for 1 h prior to VEGF
165
stimulation reduced KDR
tyrosine phosphorylation in a dose-dependent manner (Fig. 6). To examine
signaling downstream of KDR, we further visualized the levels of activated
FAK (downstream of KDR), paxillin (which associates with FAK and plays
an important role in cell adhesion and migration,
19
), Akt (which regulates
cell survival) and ERK (which regulates mitogenicity
20
) in PE-treated cells.
We found that VEGF
165
treatment stimulated the phosphorylation of these
proteins in control cells, but that this phosphorylation was dose-dependently
inhibited by 1 hr pretreatment of cells with 1.25–5
µΜ PE. These data
suggest that PE can arrest multiple VEGF-associated signaling events in
endothelial cells.
DISCUSSION
Once focused solely on tumor-selective cytotoxicity, cancer therapy
research has branched out into considerations of anti-angiogenesis
drugs arising from both synthetic and natural backgrounds. Here,
we show that PE, a novel marine-derived compound, has both
anti-angiogenic and anti-tumoral activities in vitro and in vivo. We
found that noncytoto
xic concentrations of P
E are capable of inhibiting
the main steps involved in angiogenesis, including endothelial cell
proliferation, migration, adhesion and tube formation. In addition,
these same concentrations of P
E had a mar
ked anti
-
angiogenic effect
on our in vivo CAM model. Notably, our CAM assay revealed that
treatment with 5 nM/egg PE inhibited small vessel development
comparably to the effect of 100 nM/egg of suramine, a well-known
angiogenesis inhibitor.
4
Further in vivo investigations in sarcoma
180 mouse tumor models demonstrated that PE treatment reduced
blood vessel density in sarcoma tissues and dramatically increased
endothelial cell apoptosis in tumors. And finally, PE exhibited signif-
icant anti-tumor activities in vivo, characterized by inhibition of
sarcoma and hepatoma growth. Thus, our results show that PE is
capable of conferring both anti-angiogenic and anti-tumoral activities
in vitro and in vivo. In contrast, while 5-FU significantly inhibited
tumor growth and induced tumor cell apoptosis, this well-known
chemotherapeutic drug did punily affect endothelial cell apoptosis or
angiogenesis, indicating that these two drugs likely represent different
modes of action.
Induction of apoptosis in tumor cells and tumor-associated
endothelial cells is critical to anti-tumoral and anti-angiogenic activ-
ities,
18
making induction of apoptosis a favorable strategy for
anti-cancer therapeutics. Immunofluorescent analysis showed that
PE treatment reduced the volumes of mouse sarcoma 180-induced
tumors by triggering apoptosis of both tumor cells and tumor-asso-
ciated endothelial cells. Endothelial cells were targeted more highly
than tumor cells, indicating PE inhibited tumor growth mainly by
inducing endothelial apoptosis rather than by inducing tumor cells
apoptosis. This result consisted with other laboratory evidences that
cytot
o
xic chemotherapy and anti
-angiogenic therapy are each dependent
on endothelial cell apoptosis.
18
D
uring cytoto
xic chemotherapy
,
apoptosis of endothelial cells in the v
ascular bed of rumors precedes
apoptosis of tumor cells.
18,21
Administration of an angiogenesis
inhibitor can increase tumor cell apoptosis and inhibit tumor growth
by inhibiting endothelial proliferation and migration and/or by
inducing endothelial apoptosis.
18
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Cancer B
iology &
Therapy 2005; Vol. 4 Issue 8
P
E Inhibits Angiogenesis and Tumor Growth
T
able 1
Effect of PE on the in vivo growth of sarcoma 180 tumors
Tumor Treatment group Dosage Mice/number Body weight (g) Tumor weight (g) Inhibition rate (%) p value
(mg kg
-
1
d
-
1
) × d (Initial/End) (Initial/End)
Sarcoma 180 Normal Saline —— 16/16 21.5/31.5 1.17 ± 0.41 —— ——
PE 2 × 7 8/8 21.6/26.8 0.84 ± 0.42 28.2 >0.05
PE 3 × 7 8/8 21.3/20.9 0.52 ± 0.35 55.6 <0.01
PE 4 × 7 8/8 21.3/20.4 0.46 ± 0.27 60.7 <0.01
5-FU 75 × 2 8/8 21.5/24.3 0.19 ± 0.11 83.8 <0.01
Sarcoma 180 cells were implanted in mouse models, and the indicated drugs were administered once daily for seven days. On day eight after implantation of cells, mouse tumor tissues were harvested and weighed and
the tumor growth inhibition rates were calculated. The data were expressed as mean ± SD from three independent experiments.
T
able 2
Effect of PE on the in vivo growth of hepatoma 22 tumors
Tumor group Treatment Dosage number Mice/number Body weight (g) Tumor weight (%) Inhibition rate (%) p value
(mg kg
-
1
d
-
1
) × d (Initial/End) (Initial/End)
Hepatoma 22 Normal Saline —— 20/20 21.5/30.4 2.01 ± 0.43 —— ——
PE
1
× 7
10/10 21.5/29.3 1.31 ± 0.56 34.8 <0.01
PE
2
× 7
10/10
21.5/25.1 1.45 ± 0.45 27.9 >0.05
PE 3
× 7 10/10 21.6/23.5 0.96 ± 0.40 52.2 <0.01
5
-
FU
75
× 2
10/10
21.7/24.4
0.37
± 0.26 81.6 <0.01
Hepatoma 22 cells were implanted in mouse models, and the indicated drugs were administered once daily for seven days. On day eight after implantation of cells, mouse tumor tissues were harvested and weighed and
the tumor growth inhibition rates were calculated. The data were expressed as mean ± SD from three independent experiments.

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Cancer B
iology & Therapy 881
The angiogenesis of endothelial cells is mediated by a number of
impor
tant mitogenic factors, including
VEGF
, which binds to its
receptor (VEGF Receptor 2/KDR) to trigger proliferation and
migration of endothelial cells.
22,23,24
Inhibition of VEGF-triggered
cas
cades generally leads to pr
ofound anti
-
angiogenesis and subsequent
anti
-tumor activity.
25,26
H
ere, we show that PE inhibits VEGF
165
-
induced KDR phosphorylation in HMECs, and dramatically hinders
downstream KDR signaling by decreasing the activation (phospho-
rylation) of FAK, paxillin, Akt and ERK, which are required for the
mitogenic activities of VEGF in endothelial cells. Since Akt inhibition
is central to initiation of the apoptotic cascade, the apoptosis
-
induced action of PE on endothelial cells probably benefits its
inhibition on Akt activation. Similarly, since FAK and ERK
phos
phor
ylation is inv
olv
ed in
VEGF
-
induced endothelial cell
migration and cell adhesion, PE-induced decreases in FAK and ERK
activation may account for the observed effect of PE on endothelial
cell migration and adhesion. Although future work will be required
to fully elucidate the inv
olv
ed pathways, these r
esults seem to indi
cate
that PE exerts its anti-angiogenic effects (at least in part) by blocking
VEGF-induced KDR activation and downstream signaling.
R
ecent studies have indicated that in cancer therapy, anti-angiogenic
agents should be used in synergistic combinations with traditional
cytoto
xic or other molecular-targeting agents capable of providing
anti-tumoral activities. Thus, it would seem beneficial to utilize single
agents capable of acting against both angiogenesis and tumor growth.
It is demonstrated that some (not all) conventional anti-cancer drugs
possess dual cytotoxic and anti-angiogenic effects, including camp-
tothecin, docetaxel and cyclophosphosphamid.
27,28
On the other
hand, many cytotoxic anti-tumor drugs have not the anti-angiogenic
activities, even in the cytotoxic doses, such as carboplastin, mitomycin
and etoposide. Here, we show that the marine-derived compound,
PE, has both anti-tumoral and anti-angiogenic activities, suggesting
that it may fill the need for a dual-acting agent. Future work will be
required to identify the structureactivity relationship and structural
optimization of PE. However, the present study provides strong
evidence that this marine-derived compound may prove to be an
attractiv
e candidate for further preclinical testing as a new anti-neo-
plastic agent.
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E Inhibits Angiogenesis and Tumor Growth
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