Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 16
doi:10.1111/j.1745-7270.2008.00370.x
Dihydrotanshinone I inhibits
angiogenesis both in vitro and in vivo
Weipeng Bian1,2, Fei Chen2*, Ling Bai3, Ping Zhang2, and Wenxin Qin2
1 School of Life Science, East China Normal
University, Shanghai 200062, China
2 National Laboratory for Oncogenes and Related
Genes, Shanghai Cancer Institute, Shanghai Jiaotong University, Shanghai
200240, China
3 Institute of Mechanobiology and Medical Engineering,
Shanghai Jiaotong University, Shanghai 200240, China
Received: September
3, 2007
Accepted: October
8, 2007
This work was
supported by the grants from the Foundation of Shanghai Municipal Health Bureau
(No. 287), and the National High Technology Research and Development Program
of China (No. 2006AA020501)
*Corresponding
author: Tel/Fax, 86-21-34206022; E-mail, [email protected]
Dihydrotanshinone
I (DI), a naturally occurring compound extracted from Salvia miltiorrhiza
Bunge, has been reported to have cytotoxicity to a variety of tumor cells.
In this study, we investigated its anti-angiogenic capacity in human umbilical
vein endothelial cells. DI induced a potent cytotoxicity to human umbilical
vein endothelial cells, with an IC50 value of
approximately 1.28 mg/ml. At 0.25–1 mg/ml, DI
dose-dependently suppressed human umbilical vein endothelial cell migration,
invasion, and tube formation detected by wound healing, Transwell invasion and
Matrigel tube formation assays, respectively. Moreover, DI showed significant in
vivo anti-angiogenic activity in chick embryo chorioallantoic membrane
assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 mg/egg. Taken
together, our results showed that DI could inhibit angiogenesis through
suppressing endothelial cell proliferation, migration, invasion and tube
formation, indicating that DI has a potential to be developed as a novel
anti-angiogenic agent.
Keywords dihydrotanshinone I; angiogenesis; human umbilical vein
endothelial cell
Angiogenesis is a process of new blood vessel formation by
endothelial cells that plays a critical role in normal physiology, such as
development and wound healing [1]. At the pathological level, angiogenesis is
regulated by numerous pro-angiogenic factors leading to the induction of
several diseases such as spreading of tumor, diabetic retinopathy, and
rheumatoid arthritis [2]. Most primary solid tumors are dependent on
angiogenesis for survival, growth, invasion, and metastasis. Therefore,
targeting the angiogenesis process has become one of the important strategies
in treating tumors [3]. In the angiogenesis process, vascular endothelial cells
are activated to migrate out from the parental vessels, invade through the
matrix, proliferate, and get together to format capillary tubes. Each step is
tightly controlled by pro- and anti-angiogenic factors [4]. In tumor tissues,
vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and
other pro-angiogenic factors are usually overexpressed [5], whereas
anti-angiogenic factors such as interleukin-12 (IL-12) and g-interferon have
few functions [6]. Thus, the anti-angiogenic therapy strategy is to suppress
the functions of pro-angiogenesis factors and/or promote the functions of
anti-angiogenesis factors. Currently, there are a variety of angiogenesis
inhibitors being used in clinical trials [6,7], many of which are natural
products. Drug development from natural products has become a rapidly emerging
and highly promising strategy to identify novel anti-angiogenic and anticancer
agents.Dihydrotanshinone I (DI) is a tanshinone extracted from a well-known
traditional Chinese medicinal plant, Salvia miltiorrhiza Bunge. The dry
root of this plant, called Danshen, has been widely used in China to treat
haematological abnormalities, heart disease, hepatitis, hemorrhage, menstrual
abnormalities, and collagen-induced platelet aggregation [8,9]. DI was
reported to possess the function of cytotoxicity in vitro [10] and
showed inhibitory effects on mast cell degranulation [11],
lipopolysaccharide-induced nitric oxide generation [12], osteoclast
differentiation [13], and production of IL-12 and g-interferon in immune cells
[14]. However, its anti-angiogenic capacity has not been well studied.
Considering IL-12 and nitric oxide both play important roles in mediating
angiogenesis [15,16], we hypothesized that DI might have an anti-angiogenic
action. In this study, we used different assays to identify and characterize
whether DI could induce inhibitory effects on angiogenesis in vitro and in
vivo.
Materials and Methods
Drugs and reagents
DI was bought from the National Institute for the Control of Pharmaceutical
and Biological Products (Beijing, China). It was dissolved with ethanol for the
stock concentration of 5 mg/ml, and stored at –20 ?C. The stock solution
was further diluted immediately before use. Dimethyl
thiazolyl-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St.
Louis, USA). Dulbcco? modified Eagle? medium (DMEM), Medium 199, fetal bovine serum, and bovine calf
serum were obtained from Gibco (Rockville, USA).
Isolation of human umbilical vein
endothelial cells (HUVECs) and cell cultures
HUVECs were isolated from fresh human umbilical veins by established
methods as described previously [17]. Briefly, endothelial cells were harvested
from human umbilical veins by adding 0.1% collagenase (Gibco) for 30 min. The
cells were grown to confluence in Medium 199 supplemented with 20%
heat-inactivated fetal bovine serum, 100 mM HEPES, 100 U/ml penicillin, 100 mg/ml
streptomycin, 2 mM glutamine, 5 ng/ml acidic fibroblast growth factor (aFGF)
(Sigma), and 5 U/ml heparin. Cells were assessed for endothelial cell phenotype
by morphology, the typical monolayer cobblestone growth pattern, and the
expression of von Willebrand factor antigen. Cells between passages 1 and 2 and
cell populations with more than 95% purity were used in all experiments.The hepatocellular carcinoma SMMC-7721 cell line was obtained from
the Committee of Type Culture Collection of the Chinese Academy of Sciences
(Shanghai, China). Cells were maintained in DMEM supplemented with 10% bovine
calf serum, incubated in a humidified atmosphere of 5% CO2 at 37 ?C, and subcultured every 2 d.
MTT assay
Cells were seeded in 96-well tissue culture plates (4000
cells/well), incubated for 24 h, then treated with various concentrations of DI
for another 24 h. Then 20 ml MTT was added to each well (final concentration of 0.5 mg/ml) for
another 4 h incubation. The medium was pipetted out from each well, 200 ml dimethyl
sulfoxide was added and the optical density was read at 570 nm. The IC50 values were calculated and defined as the concentration of drug
causing 50% inhibition in absorbance compared with control cells [18].
Wound healing assay
Wound healing assay
The wound healing assay was carried out according to previous studies
[19]. Briefly, HUVECs were seeded on 24-well tissue culture plates. When the
cells reached 90% confluence, they were wounded with a tip from the center of
each well and marked at the injury line. After wounding, the cultures were
washed with serum-free medium and further incubated with different
concentrations of DI for 24 h. Then cells in each well were stained with
Wright-Giemsa, and photographed under an inverted microscope. The total
migrated cells in each group were quantified by counting the number of cells
that moved beyond the reference lines in five views.
Cell invasion assay
Cell invasion assay was carried out using Transwell chambers
(Corning, New York, USA) with an 8 mm pore polycarbonate filter insert (Corning)
according to a previous report [20]. Briefly, the upper side of every insert
was coated with 10 ml Matrigel (3 mg/ml; Becton Dickinson, Mountain View, USA). HUVECs
(3104 cells/well) were seeded in the upper inserts, and SMMC-7721 cells
were seeded in the lower chamber 24 h beforehand. After 6 h of incubation, the
medium in the inserts was pipetted out and 150 ml medium with different
concentrations of DI was added. Chambers were incubated for 24 h. Cells on the
lower surface of the filters were fixed and stained with Wright-Giemsa and
sealed on slides. Stained filters were photographed under a microscope.
Invasiveness was determined by counting the cell number. Five visual fields
were chosen randomly for each filter. The average number of the invaded cells
in the five fields was taken as the mean of cell invasion numbers of the
group.
In vitro tube formation assay
Unpolymerized Matrigel (17 mg/ml) was placed in a 96-well plate
(0.32 cm2/well) at 50 ml/well and polymerized for 1 h at 37 ?C. HUVECs (2104 cells/well) in 200 ml medium, as well as in the presence or absence of DI (0.25, 0.5,
and 1 mg/ml) were layered onto the Matrigel surface. After 24 h of
incubation, cell growth and 3-D organization were observed under a microscope
[21].
Chick embryo chorioallantoic
membrane (CAM) assay
The modified CAM assay was used to evaluate the inhibitory effects
on angiogenesis in vivo on a chicken embryo model according to a
previous report [22]. Fertilized chick eggs were incubated at 37 ?C and with
60% humidity for 6 d. Then a square window was opened on the egg’s shell,
exposing the CAM and ensuring that the yolk sac membrane remained intact and
the embryo was viable. A methylcellulose membrane loaded with various
concentrations of DI was placed in areas between vessels of the 6-d-old egg but
never onto any large vessels. Then the window was covered with tape. After
another 48 h of incubation, the CAMs were fixed in situ, excised from
the eggs, placed on slides, and left to air dry. Pictures were taken through a
stereoscope equipped with a digital camera. Local vessel density was measured
and the inhibitory effects on CAM angiogenesis were evaluated. Assays were
repeated three times and each experiment group included 10 eggs.
Statistical analysis
All of the experiments were carried out at least in triplicate. The
results are expressed as mean±SD. The statistical differences between means
were evaluated using Student’s t-test. Differences between two groups
were determined using a P-value. A value of P<0.05 was regarded as being statistically significant.
Results
DI induced cytotoxicity in
HUVECs and SMMC-7721 cells
Cells were treated with increasing concentrations of DI for 24 h, then
cell viability was determined by MTT assay. The cell viability was shown as the
DI dose-dependent reduction in both HUVECs and SMMC-7721 cells (Fig. 1).
The IC50 values were 1.28 and 5.02 mg/ml for HUVECs and SMMC-7721 cells,
respectively. We set the working concentrations of DI as 0.25, 0.5, and 1 mg/ml for the
following experiments.
DI inhibited HUVEC migration
The wound healing migration assay is an established and widely-used
procedure that allows an examination of cell migration in response to an
artificial wound produced on a cell monolayer. DI showed a dose-dependent
inhibitory effect on the wound healing ability of HUVECs (Fig. 2). The
control group produced a distinguished cell migration in the wound area 24 h
after wounding, whereas DI-treated groups (0.25, 0.5 and 1 mg/ml) showed
dose-dependent inhibitory effects on wound healing under the same conditions [Fig.
2(A)]. The mean migratory cell number in five fields in the control group
was 504±23, but in experimental groups treated with DI at 0.25, 0.5, or 1 mg/ml, the cell
numbers were 349±42, 295±22, and 172±28, respectively, with inhibitory rates of
30.75% (P<0.05), 41.81% (P<0.05), and 65.87% (P<0.01), respectively. These results indicated that sub-cytotoxicity of DI inhibited the motility of HUVECs.
DI suppresses HUVEC invasion
in a dose-dependent way
The invasion of endothelial cells is also one of the critical
features in the formation of new blood vessels and in the repair of injured vessels.
We investigated the invasion ability of HUVECs through Transwell inserts.
Results showed that the average number of invaded cells in the control was
159.33±5.68, and the numbers were 135.33±4.04, 82.00±10.54, and 41.00±3.61 in
0.25, 0.5, and 1 mg/ml groups, respectively, with inhibitory rates of 15.06% (P<0.05), 48.53% (P<0.05), and 74.27% (P<0.01), respectively. In agreement with the results of the wound healing assay, DI at all three tested doses significantly inhibited HUVEC invasion (P<0.05 or P<0.01). The inhibitory effect was dose-dependent (Fig. 3).
DI inhibited in vitro
tube formation on Matrigel
The production of tubular structures is another important step in angiogenesis.
We therefore investigated the effects of DI on HUVEC tube formation. As shown
in Fig. 4, HUVECs plated on Matrigel and incubated with control medium
aligned to form lumen-like structures and anastomotic tubes with multicentric
junctions [Fig. 4(A)]. When HUVECs were treated with various
concentrations of DI for 24 h, the cells formed fewer tubes, as well as fewer
and weaker anastomoses, in a dose-dependent manner [Fig. 4(B–D)].
DI reduced in vivo
angiogenesis in CAM model
CAM assay is a widely-used model to determine angiogenesis in
vivo. The average vessel numbers under the methylcellulose membrane in the
four groups (vehicle, ethanol, 0.1 mg DI/egg, and 0.2 mg DI/egg) were 69.2529.64,
70.0036.23, 31.1711.94, and 27.209.09, respectively (Fig. 5). The vessel
density between the vehicle and ethanol groups had no significant difference (P>0.05).
Experimental groups both represented distinguished differences from the
vehicle and ethanol groups (P<0.05). This result suggested that DI had an inhibitory effect on in vivo angiogenesis in CAM model.
Discussion
Angiogenesis plays a critical role in tumor invasion and metastasis
[23]. In the initial process of tumor invasion, angiogenesis is activated by
the pro-angiogenesis factors produced by environmental cells. Notably, VEGF
plays a key role in the whole angiogenesis processes. VEGF can be expressed and
secreted by various solid tumor cells mediated by hypoxia inducible factor 1
(HIF-1) [4]. With a high invasion and metastasis capacity, human hepatocellular
cells secrete a certain level of VEGF and express VEGF receptors [5]. Based on
this and other studies, VEGF monoclonal antibodies have been used in the clinic
to treat metastasis cancers [24]. Other angiogenesis inhibitors suppress
angiogenesis by blocking signal transduction induced by VEGF [25]. In the HUVEC invasion assay in this study, we put SMMC-7721 cells on
the lower chamber of the Transwell, and found DI treated groups significantly suppressed
the invasion of HUVECs to the lower chamber (Fig. 3), suggesting DI
might block the signal transduction of VEGF produced by SMMC-7721 cells. In
addition, DI directly induced cytotoxicity in HUVECs, with the IC50 3.92-fold less than that in SMMC-7721 cells (Fig. 1). This
result indicates that, in treating solid tumors like hepatocellular carcinoma,
DI might preferentially target vascular endothelial cells to tumor cells. The anti-angiogenesis capacity of DI might be underlined by its
chemical structure. The anti-angiogenesis capacity of cryptotanshinone was
determined by the double bond at the C-15 position of the dihydrofuran ring
[26]. DI is another tanshinone with such a key structure [27]. In our previous
study, we found both cryptotanshinone and DI could inhibit HIF-1 activity by
HIF-1 receptor using the screening method (data not shown), and this finding
was verified by Dat et al [28]. Previous studies have shown that
cryptotanshinone could inhibit vascular endothelia cell invasion, tube
formation in vitro, and CAM angiogenesis in vivo [22,29]. In this
study, we illustrated that DI (0.25–1 mg/ml) possessed the capacity to suppress
HUVEC migration (Fig. 2), invasion (Fig. 3), and tube formation (Fig.
4). The mechanism might be that DI blocked the signal transduction of the
effects of some growth factors, like VEGF, produced by tumor cells, but this
remains to be further investigated. Also, DI could suppress microvessel density
in vivo in the CAM model in a dose-dependent way (Fig. 5). These
results lay the critical foundation for further research into the antitumor
ability of DI, and suggest that DI has a potential to be developed as a novel
anti-angiogenic agent.
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