80169 (343)

Biosci. Biotechnol. Biochem., 72, 80169-1–5, 2008

Note
Propolis Suppresses Tumor Angiogenesis by Inducing Apoptosis
in Tube-Forming Endothelial Cells
Toshiro O HTA,y Kazuhiro K UNIMASA, Tomomi K OBAYASHI,
Miwa S AKAMOTO, and Kazuhiko K AJI
Graduate School of Nutritional and Environmental Sciences, University of Shizuoka,
52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan

Received March 17, 2008; Accepted May 22, 2008; Online Publication, September 7, 2008
[doi:10.1271/bbb.80169]

We have reported that propolis suppresses tumor- cells (3:0
106 cells) and implanted into a subcutaneous
induced angiogenesis in vivo and in vitro, but antiangio- dorsal air sac, which was created by injecting air into the
genic mechanism of propolis at cellular level remains back of each mouse. The mice were fed a control diet
unclear. In this study, we observed that propolis not (20% casein)6) with and without 5% propolis (calculated

Adv
only inhibited tube formation but also induced apoptosis
of endothelial cells. These results suggest that propolis
by dry weight) for 12 d. They were implanted with the
chamber on day 8. On day 13, the implanted chambers

anc
exerts its antiangiogenic effects at least in part through were removed from the subcutaneous area, and the
induction of apoptosis. angiogenic response was assessed by the angiogenesis
index by determining the number of newly formed blood
Key words:
lial cell; tube formation
eV
propolis; angiogenesis; apoptosis; endothe- vessels greater than 3 mm in length and 0.075 mm in
diameter. In cellular analysis, capillary tube-like struc-

iew
Propolis is a resinous substance collected by honey-
tures formed by HUVECs were prepared and quantified
as previously described, with slight modifications.5,7)
Briefly, HUVECs (6:0
104 cells/cm2 ) were seeded

Pro
bees from buds and exudates of certain trees and plants.
It is used in folk medicine to treat various ailments.1) It
is also used in foods and beverages to improve health
and prevent disease.2) Since propolis is generally used as
an alcohol or water extract when applied to humans, it is
very important to evaluate its biological activities in
extracted form.
ofs
In this study, the antiangiogenic effects of propolis
were evaluated using in vivo and in vitro angiogenesis
models. Angiogenesis, or new blood vessel growth, is
defined as a process in which a network of new blood
vessels emerges from pre-existing vessels. It has been
found that angiogenesis is essential for tumor growth
and metastasis, and that tumor angiogenesis can be a
very effective target in cancer prevention and treat-
ment.3) We investigated to determine how propolis
exerts its antiangiogenic effects at the cellular level.
Propolis and all the other chemicals were purchased
from Sigma (St. Louis, MO) unless otherwise noted.
Mouse dorsal air sac assay was performed as previously
described, with slight modifications.4,5) Female ICR
mice were purchased at 5–8 weeks old from Japan SLC
(Shizuoka, Japan). A chamber covered with Millipore
filters (Millipore, Billerica, MA) of 0.45 mm pore size on
both sides was filled with either saline or S180 tumor
between two layers of collagen gels and incubated
in MCDB-104 with 0.5% FBS supplemented with 10
ng/ml of bFGF, 8 nM PMA, and 25 mg/ml of ascorbic
acid with various concentrations of propolis or vehicle
(dimethylsulfoxide) for up to 48 h. Observation and
quantification of apoptosis were carried out as previ-
ously described.8,9) Briefly, cells were fixed with 1%
glutaraldehyde overnight at 4  C and stained with
500 ng/ml of DAPI overnight at room temperature.
Cells exhibiting chromatin condensation and/or nuclear
fragmentation were counted as apoptotic cells. Results
were expressed as means  SE. Differences were ascer-
tained by analysis of variance (ANOVA). Multiple
comparisons for angiogenesis and tube formation ex-
periments were checked by the Fisher-PLSD test. A
comparison of the two treatments for the apoptosis
experiment was performed using Student’s unpaired
t-test ( P < 0:05,  P < 0:01).
We observed that oral administration of the propolis
purchased from Sigma suppressed tumor-induced angio-
genesis (Fig. 1A). In the negative control group, little or
no indication of neovessel formation was observed. In
the positive control group, a drastic induction of new
blood vessel formation, characterized by zigzagging

y
To whom correspondence should be addressed. Tel/Fax: +81-54-264-5571; E-mail: ohtat@u-shizuoka-ken.ac.jp
Abbreviations: bFGF, basic fibroblast growth factor; DAPI, 40 ,6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; PMA,
phorbol 12-myristate 13-acetate; FBS, fetal bovine serum
80169-2 T. OHTA et al.

A Negative control Positive control

5% Propolis

Adv
anc
B
8 eV
7
iew
Angiogenesis index

6
5
Pro
4
3
2
**
ofs **

1
0
Negative control Positive control 5% Propolis
Fig. 1. Suppressive Effect of Propolis on Tumor Cell-Induced Angiogenesis in a Mouse Dorsal Air Sac Assay.
A, Chambers filled with S180 cells or saline were inoculated subcutaneously into ICR mice. Arrowheads point to newly formed vessels of a
zigzagging character. S180-treated positive control mice induced strong angiogenic responses as compared to the saline-treated negative control.
Oral administration of 5.0% propolis reduced the number of newly formed blood vessels. Representative photographs are shown. The bar
indicates 3 mm. B, The angiogenesis index was defined as the number of newly formed blood vessels above 3 mm in length and 0.075 mm in
diameter. Propolis significantly reduced the angiogenesis index. Values are expressed as means  SE (n ¼ 4 or 5).  P < 0:01 (Fisher-PLSD
test) vs. the positive control group.

lines of vessels, was observed. In the 5% propolis group,
substantial reduction in such new blood vessel formation
was observed. None of the treatments exhibited any
notable effect on pre-existing vasculature. The angio-
genesis indexes were 0.75, 6.5, and 2.0 for the negative
control, the positive control, and 5% propolis respec-
tively (Fig. 1B). The number of newly formed blood
vessels in the mice treated with the diet containing 5.0%
propolis was suppressed to 22% as compared to the
positive control group. In addition, no signs of toxicity
were observed in any ICR mice (body weight change
during the feeding period, data not shown). Thus it was
found that the propolis from Sigma suppressed tumor-
induced angiogenesis in vivo. Normal (pre-existing)
vessels are known to be stable, since they are protected
by pericytes and smooth muscle cells, as compared to
newly formed vessels, which lack such protecting cells.
Such differences might be the reason propolis failed to
affect normal vessels while it significantly suppressed
neovessel formation.
 Propolis Induces Apoptosis in Endothelial Cells 80169-3

A
0 µg/ml 12.5 µg/ml

50 µg/ml

Adv
anc
B 40
eV
30 iew
Tube area (%)

20 Pro *

10 ofs **

0
0 12.5 50

Propolis (µg/ml)

Fig. 2. Inhibitory Effect of Propolis on Tube Formation of Endothelial Cells.

A, HUVECs were sandwiched between two layers of collagen gel and induced to form blood vessel-like tubes. The cells were treated with the
indicated concentrations of propolis for 48 h. Representative photographs are shown. The bar indicates 100 mm. B, The areas of formed tubes
(area ratios of the tubes per pictured field) were quantified. Values are expressed as means  SE (n ¼ 4).  P < 0:05 and  P < 0:01 (Fisher-
PLSD test) vs. control group.

We then observed that propolis inhibited tube for-
mation of HUVECs cultured in a 2-D system in a
concentration-dependent manner (Fig. 2). It slightly
reduced the width of the tubes at 12.5 mg/ml, and
completely inhibited elongation of the tubes at 50
mg/ml. Inhibition of tube formation by propolis was also
accompanied by partial fragmentation of endothelial
cells, an indication of cell-death induction. Thus, it was
found that propolis inhibited tube formation of endo-
thelial cells and induced cell death at the same time.
We then observed the morphology of the cell nuclei to
determine whether the cell death induced by propolis
was apoptosis. Propolis induced chromatin condensa-
tion, a morphological marker of apoptosis (Fig. 3A).
The rates for apoptotic cells were 10.8 and 57.9% for
control and propolis (50 mg/ml) respectively, calculated
to be a 5.4-fold increase for propolis as compared to the
control group (Fig. 3B). Thus it was confirmed that
propolis caused cell death by inducing apoptosis in tube-
forming HUVECs.
80169-4 T. OHTA et al.

A
Control 50 µg/ml Propolis

B 70
**

Adv 60

50

anc
Apoptosis (%)

40

30
eV
20
iew
Pro
10

0
Control
ofs
Fig. 3. Apoptosis Inducing Effect of Propolis on Tube-Forming Endothelial Cells.
Propolis

A, HUVECs were sandwiched between two layers of collagen gels and induced to form blood vessel-like tubes. The cells were treated with the
indicated concentrations of propolis for 24 h, fixed, and stained with DAPI to observe cell nuclei. Representative photographs are shown. The bar
indicates 100 mm. B, The rates of apoptosis (percentages of condensed and fragmented cell nuclei against total cell nuclei) were quantified.
A total of more than 500 cells from six fields were counted for each datum. Values are expressed as means  SE (n ¼ 6).  P < 0:01 (Student’s
t-test) as compared to the control group.

In this study, we found for the first time that propolis
can induce apoptosis in tube-forming endothelial cells.
Such induction of apoptosis appears to be a likely
mechanism of angiogenesis suppression by propolis.
Apoptosis induction in endothelial cells is also known to
be an important mechanism of antiangiogenic drugs.10)
Several propolis components have been detected in the
plasma after oral administration to rats.11) Among these,
kaempferol and galangin have been reported to possess
antiangiogenic activities.12) We intend to further inves-
tigate how propolis induces apoptosis in endothelial
cells at the molecular level, and to determine which
propolis constituents are responsible for induction. Since
propolis inhibited elongation of HUVECs during tube
formation, it is likely that it is capable of affecting
endothelial cell migration. We also observed that
propolis inhibited proliferation of HUVECs (data not
shown). Such antiangiogenic aspects of propolis should
also be further investigated. We hope our findings on the
antiangiogenic effects of propolis will help the research
community improve medical prevention and treatment
of human cancer in the near future.
Acknowledgments
We are grateful to Hiroyuki Sakakibara of the Univer-
sity of Shizuoka for his advice on statistical analysis. This
work was supported by a grant-in-aid from the Japan
Society for the Promotion of Sciences (JSPS, to T.O.).
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