Carcinogenesis vol.32 no.6 pp.904–912, 2011
doi:10.1093/carcin/bgr052
Advance Access publication March 22, 2011
1#-Acetoxychavicol acetate suppresses angiogenesis-mediated human prostate tumor
growth by targeting VEGF-mediated Src-FAK-Rho GTPase-signaling pathway
Xiufeng Pang1,, Li Zhang1, Li Lai1, Jing Chen1, Yuanyuan
Wu1, Zhengfang Yi1, Jian Zhang2, Weijing Qu1, Bharat
B.Aggarwal3 and Mingyao Liu1,4
1
Institute of Biomedical Sciences and School of Life Sciences, East China
Normal University, 500 Dongchuan Road, Shanghai 200241, China, 2Institute
of Medical Science, School of Medicine, Shanghai Jiaotong University,
Shanghai 200025, China, 3Cytokine Research Laboratory, Department of
Experimental Therapeutics, The University of Texas MD Anderson Cancer
Center, Houston TX 77030, USA and 4Department of Molecular and Cellular
Medicine, Institute of Biosciences and Technology, Texas A&M University
Health Science Center, Houston, TX 77030, USA
To whom correspondence should be addressed. Tel: þ86 21 54345016;
Fax: þ86 21 54344922;
Email: [email protected]
Correspondence may also be addressed to Mingyao Liu. Tel: 713 677 7505;
Fax: 713 677 7512; Email: [email protected]
Introduction
Angiogenesis plays a major role in cancer metastasis (1), which has
been shown to contribute to .90% of cancer deaths (2). Tumor vascularity as measured by microvessel density, which is a feature of
angiogenesis, has also been determined to be a prognostic factor in
a variety of solid tumors (3). Src family kinases represent the largest
family of non-receptor tyrosine kinases. Overexpression and hyperactivation of Src are correlated with advanced malignancy and poor
Abbreviations: ACA, 1#-acetoxychavicol acetate; FAK, focal adhesion
kinase; FITC, fluorescein isothiocyanate; GTPase, guanosine triphosphatase;
HUVEC, human umbilical vascular endothelial cell; MCP-1, monocyte chemotactic protein-1; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; TUNEL, terminal
deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling;
VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial
growth factor receptor.
Materials and methods
Reagents and antibodies
A 50 mmol/l stock solution of ACA (Figure 1A; LKT Laboratories, St Paul,
MN) was prepared in dimethyl sulfoxide and frozen at 20°C in small aliquots
until needed. Bacteria-derived recombinant human VEGF (VEGF165) was a
gift from the Experimental Branch of the National Institutes of Health (NIH,
Bethesda, MD). Growth factor-reduced Matrigel was purchased from BDBiosciences (San Diego, CA). Antibodies against RhoA, Rac1 and Cdc42 were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against
CD31, Ki-67, and Src were bought from Epitomics (Burlingame, CA). The
antibodies anti-VEGFR2, anti-FAK, phospho-specific anti-VEGFR2 (Tyr1175),
anti-Src (Tyr416) and anti-FAK (Tyr397) were purchased from Cell Signaling
Technology (Beverly, MA). The ECM cell adhesion array kit (colorimetric), in
situ terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end
labeling (TUNEL) apoptosis detection kit and fluorescein isothiocyanate
(FITC)-Annexin V apoptosis kit were obtained from Millipore (Temecula,
CA). The FITC-BrdU flow kit was from BD Biosciences (San Jose, CA).
Our non-radioactive cell proliferation kit was from Promega (Madison, WI).
Ó The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Cancer therapeutic agents that are safe, effective and affordable
are urgently needed. We describe that 1#-acetoxychavicol acetate
(ACA), a component of Siamese ginger (Languas galanga), can
suppress prostate tumor growth by largely abrogating angiogenesis. ACA suppressed vascular endothelial growth factor (VEGF)induced proliferation, migration, adhesion and tubulogenesis of
primary cultured human umbilical vascular endothelial cells
(HUVECs) in a dose-dependent manner. ACA also inhibited
VEGF-induced microvessel sprouting from aortic rings ex vivo
and suppressed new vasculature formation in Matrigel plugs in
vivo. We further demonstrated that the mechanisms of this chavicol were to block the activation of VEGF-mediated Src kinase,
focal adhesion kinase (FAK) and Rho family of small guanosine
triphosphatases (GTPases) (Rac1 and Cdc42 but not RhoA) in
HUVECs. Furthermore, treatment of human prostate cancer cells
(PC-3) with ACA resulted in decreased cell viability and suppression of angiogenic factor production by interference with dual
Src/FAK kinases. After subcutaneous administration to mice
bearing human prostate cancer PC-3 xenografts, ACA (6 mg/kg/
day) remarkably inhibited tumor volume and tumor weight and
decreased levels of Src, CD31, VEGF and Ki-67. As indicated by
immunohistochemistry and TUNEL analysis, microvessel density
and cell proliferation were also dramatically suppressed in tumors from ACA-treated mice. Taken together, our findings suggest that ACA targets the Src-FAK-Rho GTPase pathway, leading
to the suppression of prostate tumor angiogenesis and growth.
prognosis in a variety of cancers, both solid and hematological (4,5).
Src directly interacts or co-operates with tyrosine kinase receptors
such as epithelial growth factor receptor and vascular endothelial
growth factor receptor (VEGFR), G protein-coupled receptors, integrins, actins, guanosine triphosphatase (GTPase)-activating proteins
and non-receptor focal adhesion kinase (FAK) (6). These interactions
affect such processes as proliferation, differentiation, cell shape,
motility, angiogenesis and survival in normal and tumor cells (7).
Interestingly, Src has been reported to participate in tumor angiogenesis by regulating the gene expression of pro-angiogenic growth
factors, such as fibroblast growth factor, vascular endothelial growth
factor (VEGF), monocyte chemotactic protein-1 (MCP-1) and interleukin 8 (8–10). In fact, Src has been strongly associated with solid
tumor metastasis through its ability to promote the epithelialto-mesenchymal transition (6,11). On the basis of these important
functions of Src kinase, small molecule Src inhibitors have been
developed and are undergoing early phase clinical trials for tumors,
including prostate cancer (7).
Although these inhibitors targeting tumor angiogenesis, such as
bevacizumab, have been approved for cancer treatment (12,13), their
cost, toxicity and lack of effectiveness are major roadblocks. In contrast, it is estimated that from 1981 to 2002, 48 (74%) of 65 drugs
approved for cancer treatment were either natural products or mimics
of natural products (14). One such compound, 1#-acetoxychavicol
acetate (ACA) (Figure 1A), derived from the rhizomes of the commonly used ethnomedicinal plant Languas galanga (Zingiberaceae),
has been shown to exhibit inhibitory actions on osteoclastic differentiation (15) and HIV replication (16,17). Existing evidence has already proved that ACA has antioxidant and anti-inflammatory
activities by suppressing xanthine oxidase (18), superoxide anion
generation (19) and inducible nitric oxide synthase gene expression
(20). Other reports have shown that ACA possesses antitumor properties against a wide variety of chemically induced tumors, such as skin
(19), oral (18), colon (21) and esophageal (22) carcinogenesis in mice
and rats. Potentiation of a dual mitochondrial- and Fas-mediated caspase (23), reduction of cellular sulfhydryl groups (24) and inhibition
of nuclear factor-kappaBactivation (25,26) have been implicated in
ACA-induced tumor apoptosis in breast cancer, colon cancer and
leukemia cells. However, the mechanism of ACA action on tumor
inhibition remains unknown.
In this study, we tested whether ACA has antiangiogenic activity
that contributes to its antitumor function. We demonstrate that ACA
suppressed the angiogenesis in vitro and in vivo and inhibited human
prostate cancer growth in mouse xenograph tumor models by targeting the Src-FAK-Rho GTPase-signaling pathways.
ACA inhibits prostate tumor angiogenesis
The human angiogenesis antibody array was purchased from RayBiotech
(Norcross, GA).
Cell lines and cell culture
Primary human umbilical vascular endothelial cells (HUVECs) were cultured
in endothelial cell growth medium as described previously (27), and M199
medium served as the basal medium. Human prostate PC-3 cancer cells were
purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum
(HyClone Laboratories, Logan, UT). HUVECs and PC-3 cells were cultured
at 37°C under a humidified 95%: 5% (vol/vol) mixture of air and CO2.
Cell viability analysis
HUVECs and PC-3 cancer cells (5 103 cells per well) were directly treated
with ACA for 48 h. To determine cell viability, we used a3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt (MTS) kit from Promega and a VERSAmax microplate reader
(Molecular Devices, Sunnyvale, CA).
Cell cycle analysis and apoptosis detection
Cell cycle analysis (28) was performed in HUVECs synchronized overnight in
starvation medium consisting of M199 medium with 2% fetal bovine serum, 25
lg/ml porcine heparin and 20 mmol/l N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid buffer. Cells were then stimulated with 50 ng/ml VEGF and
ACA for additional 24 h in the starvation medium. Where noted, cells were
pulsed with 10 lmol/l 5#-bromo-2#-deoxyuridine (BrdU; BD Biosciences, San
Jose, CA) by incubation for 1 h prior to harvest. Endothelial cells were then
fixed and stained following the protocol of the FITC-BrdU flow kit. Seven
amino-actinomycin D staining was chosen to determine total DNA.
Apoptosis was examined in PC-3 prostate cancer cells. Normal cultured PC3 cells were directly treated with indicated concentrations of ACA for 24 h.
Cells were fixed and stained according to the instructions of the FITC-Annexin
V apoptosis kit (Millipore). Propidium iodide staining was chosen to determine
basal DNA. Data were collected using a fluorescence-activated cell sorting
Calibur flow cytometer (Becton Dickinson) and analyzed using CELLQuest
software (Becton Dickinson, San Jose, CA).
Endothelial cell migration assay
Cell motility was examined using the wound-healing migration and Boyden
chamber migration assays. In the wound-healing migration assay, wounds were
made by sterile pipette tips on a monolayer of HUVECs in six-multiwell plates
(Becton Dickinson). After being refreshed with medium with or without the
addition of 50 ng/ml VEGF and indicated concentrations of ACA for 8–10 h,
the cells were fixed and photographed. The Boyden chamber assay was performed using a Transwell filter (8.0 lm pore size polyethylene terephthalate
membrane with Falcon cell culture insert; Becton Dickinson). The Transwell
filter was first precoated with gelatin. Then, 2 to 5 104 cells per treatment in
100 ll of serum-free medium were pretreated with different concentrations of
ACA for 1 h and added directly to the upper chamber, whereas 500 ll of
medium with 30 ng/ml VEGF was added to the lower chamber. Transwells
were incubated for 4–6 h at 37°C. Cells on the inner inside of the Transwell
inserts were removed with a cotton swab, and cells on the underside of the
insert filter were fixed and stained. Photographs of three random fields were
taken, and the cells were counted to calculate the average number of cells that
had transmigrated.
Cell adhesion assay
For de novo cell attachment experiments (29), HUVEC were collected in 2–5
mM ethylenediaminetetraacetic acid/phosphate-buffered saline buffer and resuspended in serum-free M199 medium. After being incubated for 2 h at 37°C,
the cells were subsequently plated at a density of 2 105 cells per well on a 96well plates (Millipore) that had been precoated with collagen, fibronectin,
tenascin or vitronectin in phosphate-buffered saline for 1 h at 37°C. Wells
were washed, fixed and stained. The absorbance of each well was read at
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Fig. 1. ACA inhibits VEGF-induced cell proliferation, motility and adhesion of HUVECs in vitro. (A) The chemical structure of D, L-1#-acetoxychavicol acetate.
(B) ACA inhibited VEGF-triggered endothelial cell cycle progression by decreasing the proportion of BrdU-positive cells in the S phase. (C) ACA inhibited
VEGF-induced cell motility in endothelial cells. The migrated cells were counted manually. (D) ACA demolished cell adhesion in a de novo cell attachment assay.
Columns, mean; bars, standard deviation; P , 0.05; P , 0.01 versus VEGF alone.
X.Pang et al.
560 nm in a plate reader (Packard Spectra Count, Meriden, CT). Results are
expressed as mean value of triplicate determination ± standard deviation.
cells, immunohistochemical analysis was also performed with anti-CD31
antibody.
Capillary-like tube formation assay
Tube formation was assessed as described previously (30). Briefly, HUVECs
were pretreated with ACA for 1 h and then seeded onto the Matrigel layer in
48-well plates at a density of 4 to 5 104 cells. After 6 h, the tubular structure
of endothelial cells was photographed using an inverted microscope (original
magnification, 100; Olympus, Center Valley, PA). Three independent experiments were performed.
Immunoblot and GTPase activation assay
HUVECs were starved, pretreated with ACA for 20 min and then stimulated
with 50 ng/ml VEGF to activate kinases and GTPases. After that, cells were
lysed with lysis buffer (20 mmol/l Tris–HCl, 2.5 mmol/l ethylenediaminetetraacetic acid, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate,
40 mmol/l NaF, 10 mmol/l Na4P2O7 and 1 mmol/l phenylmethylsulfonyl fluoride) supplemented with proteinase inhibitor cocktail (Calbiochem, San Diego,
CA). For western blotting, 40 lg of cellular protein from each treatment was
applied to 8–12% sodium dodecyl sulfate–polyacrylamide gels and probed
with specific antibodies. For the GTPase pull-down experiment, 30 lg of
GST-PBD (Pak Rac/Cdc42-binding domain) or GST-RBP (Rho-binding domain) attached to beads (Santa Cruz Biotechnology) was added to the cell
lysates. After incubation at 4°C for 60 min, the beads were washed five times
with lysis buffer and boiled at 90°C for 10 min with 2 sodium dodecyl sulfate
sample buffer. Immunoblotting for Rac1, Cdc42 and RhoA was done with
specific antibodies (Santa Cruz Biotechnology). For detecting pro-angiogenic
cytokines produced by tumor cells, PC-3 cells were directly treated with
20 lmol/l ACA for 24 h. Cell culture medium was harvested and analyzed
according to the manual of the human angiogenesis array kit (RayBiotech).
Animal studies
All experimental animals used in the present study were purchased from
the National Rodent Laboratory Animal Resources, Shanghai Branch
(Shanghai, China) and maintained in a laminar airflow cabinet under
specific-pathogen-free conditions and a 12 h light–dark cycle. Mice were
maintained according to the NIH standards established in the Guidelines for
the Care and Use of Experimental Animals, and all the protocols were
approved by East China Normal University.
Rat aortic ring assay
The rat aortic ring assay was performed as described previously (31). Aortas
isolated from Sprague–Dawley rats were cleaned and cut into rings 1–1.5 mm
in circumference. These aortic rings were randomized into a Matrigel-coated
48-well plates and sealed with a 100 ll overlay of Matrigel. VEGF (30 ng/ml)
with or without ACA was added to the wells. Fresh medium was changed every
other day. After a week, microvessel sprouting was fixed and photographed
using an OLYMPUS inverted microscope (magnification, 100; Olympus).
The assay was scored from 0 (least positive) to 5 (most positive) in a doubleblinded manner.
In vivo Matrigel plug assay
The Matrigel plug assay was performed as described elsewhere (32). Matrigel
containing 100 ng of VEGF and 20 U of heparin with or without indicated
amounts of ACA was subcutaneously injected into the ventral area of C57/BL/
6 mice (n 5 6 each group). After a week, intact Matrigel plugs were carefully
exposed, fixed and embedded in paraffin. To identify infiltrating endothelial
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Molecular modeling
The co-ordinates of human proto-oncogene tyrosine-protein kinase Src were
obtained from the refined X-ray crystal structure of 2H8H.pdb (33), which is
available from the Protein Data Bank. The protein structure was prepared in
Protein Preparation Wizard, and ACA was prepared in LigPrep encoded in
Glide (34). A set of docking grids was generated for the binding site using
the default parameters, and ACA was then docked into Src using the standard
precision mode.
Human prostate tumor xenograft mouse model
The human prostate mouse xenograft model was constructed as described
previously (30). Human prostate cancer PC-3 cells (5 106 cells per mouse)
were subcutaneously injected into 6-week-old male BALB/cA nude mice.
After tumors grew to 100 mm3, the mice were selected and randomly divided
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Fig. 2. ACA inhibits capillary structure formation of HUVECs in vitro and microvessel sprouting ex vivo. (A) ACA inhibited the VEGF-induced tube formation of
endothelial cells in Matrigel. After incubation, endothelial cells were fixed, and tubular structures were photographed (original magnification, 100). (B) Aortic
segments isolated from Sprague–Dawley rats were placed in the Matrigel-covered wells and treated with VEGF in the presence or absence of different
concentrations of ACA. Representative photographs of sprouts from the margins of aortic rings are shown. (C) Sprouts were scored from 0 (least positive) to 5
(most positive) in a double-blinded manner. Columns, mean; bars, standard deviation; P , 0.01 versus VEGF alone.
ACA inhibits prostate tumor angiogenesis
into two groups of 6–7 each. Tumor-bearing mice were then treated with or
without ACA (dissolved in dimethyl sulfoxide) via intralesional injection
(6 mg/kg/day) for an additional 20 days. The mice of control group were
administrated with the same amount of dimethyl sulfoxide. The body weight
of each mouse was recorded, and tumor volume was determined by Vernier
caliper every day. On day 20, the mice were euthanized, and solid tumors were
removed and fixed for in situ TUNEL staining and immunohistochemical
analysis with anti-CD31, anti-VEGF, anti-Src or anti-Ki-67 antibodies. Images
were taken using a Leica DM 4000B photomicroscope (magnification, 400;
Solms, Germany).
Statistics
All data are presented as mean ± standard deviation, and statistical comparisons between groups were performed using one-way analysis of variance
followed by Student’s t-test. P values 0.05 were considered statistically significant.
Results
ACA inhibits VEGF-induced cell proliferation, chemotactic motility
and adhesion of HUVECs in vitro
To assess the antiangiogenic function of ACA in vitro, we first evaluated its inhibitory effects on endothelial cell proliferation, migration
and adhesion, all of which are key steps in angiogenesis (35). For
measuring cell cytotoxicity and antiproliferative effect of ACA, we
used an MTS assay and BrdU incorporation analysis. Our results
demonstrated that ACA effectively inhibited VEGF-induced cell viability at 5 lmol/l (data not shown) and potently decreased the proportion of BrdU-positive cells in the S phase (indicated as R4) from
18.88 to 7.53% at 10 lmol/l (Figure 1B), indicating that ACA
blocked endothelial cell cycle progression and arrested cells in the
G1 phase.
Next, we examined the inhibitory effects of ACA on the chemotactic motility of endothelial cells using wound-healing migration and
Boyden chamber invasion assays. The results showed that 5 lmol/l
ACA significantly decreased the number of migrated cells into the
scratched gap (P , 0.01; Figure 1C1) and dramatically reduced the
number of cells passing through the chamber membrane (Figure 1C2).
Both inhibitory effects of ACA were concentration dependent
(Figure 1C).
Integrins are a major family of cell adhesion receptors. Integrins
play fundamental roles in cell trafficking, differentiation and extracellular matrix assembly (36) and are thus critical for angiogenesis. To
test whether ACA inhibited endothelial cell adhesion through the
integrin pathway, we used a cell adhesion plate coated with different
integrin ligands. Our results demonstrated that ACA significantly suppressed avb3-dependent HUVEC adhesion to vitronectin (P , 0.01),
fibronectin (P , 0.01) and tenascin (P , 0.05) but not b1-dependent
adhesion to collagen (Figure 1D).
ACA inhibits capillary-like structure formation in vitro and
microvessel sprouting ex vivo
Angiogenesis is a very complex process; however, tubulogenesis of
endothelial cells is a fundamental step for angiogenesis (37). Here, we
used a two-dimensional Matrigel assay and a rat aortic ring assay to
examine the potential effects of ACA on capillary-like structure formation in vitro and ex vivo. When seeded on Matrigel, HUVECs
become elongated and form tube structures, mimicking the in vivo
process of angiogenesis (38). As shown in Figure 2A, 6 h after seeding, HUVECs exhibited a clear capillary-like network formation
(Figure 2A, control). However, treatment of ACA dramatically decreased the formation of capillary-like network, with a complete
inhibitory effect at the concentration of 10 lmol/l ACA (Figure 2A).
To further explore whether ACA inhibited VEGF-induced angiogenesis ex vivo, we examined the sprouting of microvessels from
aortic rings in the presence of VEGF. As shown in Figure 2B, VEGF
(30 ng/ml) alone significantly triggered microvessel sprouting,
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Fig. 3. ACA inhibits VEGF-induced angiogenesis in vivo. Six-week-old C57/BL/6 mice were injected with Matrigel containing ACA, 100 ng of VEGF and 20 U
of heparin into the ventral area (n 5 6 per group). After 1 week, the skin of treated mice was pulled back to expose intact Matrigel plugs. (A) Representative
Matrigel plugs in mice were photographed. (B) ACA inhibited new blood vasculature formation. The Matrigel plugs were fixed, embedded in paraffin and
sectioned; 5 lM plug sections were immunostained with specific anti-CD31 antibody for blood vessels. Images were taken using a Leica DM 4000B
photomicroscope (magnification, 400). (C) Infiltrating CD31-positive cells in different treatments were manually counted. Columns, mean; bars, standard
deviation; P , 0.01 versus VEGF alone.
X.Pang et al.
leading to the formation of a complex network of microvessels around
the aortic rings, whereas treatment with ACA dramatically antagonized the sprouting at 10 lmol/l, suggesting that ACA suppresses
angiogenesis ex vivo.
Fig. 4. ACA blocks Src/FAK/Rho GTPases signaling in HUVECs. (A) ACA had little effect on VEGFR2 activity at tested concentrations. (B) The
phosphorylation of the Src and FAK kinases were markedly inhibited by ACA in a concentration- (B1) and time-dependent (B2) manner. (C) Src prediction
docking model revealed that ACA inserted itself into the hydrophobic pocket of Src kinase. For protein, carbon atoms of the human Src were shown in green,
oxygen atoms in red and nitrogen atoms in blue. Side chains of crucial residues in the binding site are shown as sticks and labeled. Hydrogen bonds between ACA
and Src are depicted as yellow dotted lines. (D) ACA inhibited the activation of Rac1- and Cdc42-GTPases but had little effect on RhoA activation.
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ACA inhibits VEGF-induced angiogenesis in vivo
In order to validate ACA-mediated inhibitory actions on angiogenesis
in a whole animal model, we used the mouse Matrigel plug assay, a
powerful angiogenesis in vivo assay. When embedded subcutaneously
into mice, Matrigel plugs containing VEGF alone developed a dark
red appearance (Figure 3A, VEGF), indicating that new vasculatures
had formed inside the Matrigel through VEGF-triggered angiogenesis. In contrast, the addition of ACA (15 or 30 lg/plug) to the Matrigel
plugs dramatically inhibited the generation of infiltrating vasculature,
leading to the formation of much paler Matrigel plugs in color (Figure
3A, VEGF þ ACA), suggesting that ACA blocked the formation of
new vasculatures in the Matrigel plugs.
We next performed immunohistochemical analysis with anti-CD31
antibody, a specific-endothelial marker, to identify the new vasculature content in the plugs. We found that CD31-positive endothelial
cells in plugs with VEGF alone polarized and formed linage around
the vasculature (Figure 3B, VEGF), whereas those in ACA-treated
plugs were significantly decreased in vasculature (P , 0.01; Figure
3B, VEGF þ ACA and Figure 3C), suggesting that ACA potently
suppressed angiogenesis in vivo.
ACA blocks Src/FAK kinases, followed by the inactivation of Rac1and Cdc42-GTPases in HUVECs
To further delineate the mechanism that underlies the antiangiogenic
effect of ACA, we examined the signaling pathways using western
blot analysis. As shown in Figure 4A, VEGFR2 activity appeared to
be little affected, whereas the phosphorylation of Src and FAK kinases
was markedly inhibited by ACA in a concentration- (Figure 4B1) and
time-dependent manner (Figure 4B2).
To determinehowACAaffectedSrcactivity,weperformedadocking
simulation of how ACA binds to Src protein. As shown in the model of
Figure 4C, ACA formed two hydrogen bonds with Asp404 and
Met341 of Src protein, which has been reported to be key residues
interacting with the adenine ring nitrogen of adenosine triphosphate
(33). One terminal methyl group of ACA inserted itself into a hydrophobic pocket composed of the residues Met314, Ile336, Phe405,
and Leu407 near the adenosine triphosphate-binding site, and the
other terminal substituent formed a hydrophobic interaction with
residues Leu273, Val281 and Leu393.
A cell’s ability to polarize and move is controlled by remodeling
of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases, which specify the
peripheral localization of Src (39). As shown in Figure 4D, we
demonstrate that ACA dramatically suppressed the activation
of Rac1- and Cdc42-GTPases (GTP-bound form) but not of
RhoA-GTPase.
ACA inhibits prostate tumor angiogenesis
ACA suppresses cell viability and pro-angiogenic factor production of
prostate cancer cells
Although ACA possesses antitumor properties against a wide variety
of cancer, such as leukemia, oral, colon and esophageal carcinogenesis (18,21,25), its inhibitory action on human prostate cancer remained unknown. We found that when PC-3 cancer cells were
directly exposed to ACA for 48 h, cell survival was concentration
dependently inhibited by ACA, with IC50 at 50 lmol/l
(Figure 5A). Furthermore, we analyzed apoptosis of PC-3 cells after
24 h of incubation with ACA and found that both early- (11.39%) and
late-stage (29.54%) apoptosis were induced by ACA at 50 lmol/l
(Figure 5B), suggesting that ACA inhibited prostate cancer cell
survival partially through an apoptosis-dependent pathway at higher
concentration (50 lmol/l).
Based on the information provided by the docking model
(Figure 4C), we postulated that ACA could also suppress constitutive
Src and FAK kinases in prostate cancer cells. As shown in Figure 5C,
direct treatment of 20 lmol/l ACA led to concentration-dependent
inhibition of Src/FAK in PC-3 cells, suggesting that dual Src/FAK
kinases might be the potential target of ACA in both normal
endothelial cells and tumor cells.
Angiogenesis is a complex process that is tightly controlled by
numerous pro-angiogenic cytokines and by an array of inhibitory
factors (1). To examine the ability of ACA to inhibit angiogenesis,
we used a human antibody array to screen pro-angiogenic cytokines in
the culture medium of PC-3 cancer cells. The results demonstrated
that VEGF and MCP-1 were significantly suppressed after ACA treatment (Figure 5D), indicating that ACA affect the profile of angiogenic
cytokine production by cancer cells.
ACA inhibits tumor angiogenesis and tumor growth in the xenograft
mouse model
To investigate whether ACA inhibits angiogenesis-mediated tumor
growth, we constructed a xenograft mouse model with human prostate
PC-3 cancer cells. We found that administration of 6 mg/kg/day of
ACA for 20 days substantially suppressed tumor volume (Figure 6A)
and reduced tumor weight (Figure 6B). The average tumor volume in
the control mice increased from 107.59 ± 35.80 to 255.64 ± 105.51
mm3 after 20 days, whereas that in the ACA-treated mice decreased
from 104.24 ± 45.78 to 26.48 ± 22.41 mm3, a significant inhibition of
tumor growth (P , 0.01). At the end of the experiment, the average
body weight of untreated mice was 28.28 ± 0.83 g, and that of ACAtreated mice was 27.98 ± 1.58 g (Figure 6C), suggesting that ACA
exerted little toxicity at the test dosage and conditions. In our experimental system, low dosage of ACA at 3 mg/kg/day was also tested;
however, little effect was observed in mice.
To further examine blood vessel formation and cell viability in the
solid tumors, we performed TUNEL and immunohistochemical analyses using anti-CD31, anti-VEGF, anti-Src and anti-Ki-67 antibodies
on tumor sections from xenografted mice. The results showed that
expression of Src and VEGF, microvessel density and cell proliferation in solid tumors were remarkably inhibited by the treatment of
ACA (Figure 6D). Where noted, apoptotic cells were increased in
ACA-treated group as indicated by TUNEL analysis. Together, our
results demonstrate that this chavicol acetate dramatically inhibited
tumor growth of human prostate cancer xenografts by blocking tumor
angiogenesis and inducing tumor cell apoptosis.
Discussion
Tumor angiogenesis, as a hallmark of cancer (35), is induced by a
variety of angiogenic factors, of which the best characterized is VEGF
(40). Thus, we used VEGF to construct a series of models to test the
antiangiogenic activities of ACA in vitro, ex vivo and in vivo. We
found, to our knowledge for the first time, that ACA inhibits
VEGF-induced proliferation, migration, invasion, adhesion and tubulogenesis of endothelial cells (Figures 1 and 2) and induced the apoptosis of human prostate cancer cells (Figure 5). Furthermore, our
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Fig. 5. ACA induces apoptosis and inhibits angiogenic factor production in prostate cancer cells. (A) ACA inhibited the cell survival of human PC-3 prostate cells.
MTS was used to quantify cancer cell viability. Columns, mean; bars, standard deviation; P , 0.01 versus control. (B) ACA exerted apoptotic action on human
PC-3 prostate cells. PC-3 cells were directly exposed to different concentrations of ACA for 24 h. Cells were then harvested, and apoptosis was detected using a
FITC-Annexin V apoptosis kit. (C) ACA inhibited constitutive activity of Src/FAK kinases in human cancer cells in a concentration-dependent manner. (D) ACA
suppresses the expression of VEGF and MCP-1 in PC-3 cancer cells. PC-3 cells were directly treated with 20 lmol/l of ACA for 24 h. Cell culture medium were
collected, centrifuged and applied to a human angiogenesis antibody array.
X.Pang et al.
studies have demonstrated that ACA suppresses the angiogenesis and
growth of human prostate cancer in xenograft mice. Based on these in
vitro and in vivo observations, we determined that the Src/FAK/Rho
GTPase-signaling pathway is the potential target of ACA.
A large number of previous studies have shown that Src kinase is
strongly involved in VEGF-induced angiogenesis (41,42). Various
angiogenic-signaling molecules and transcriptional factors, such as
p125FAK, endogenous nitric oxide synthase and signal transducers
and activators of transcription have been shown to be frequently activated upon the interaction of Src kinase and surface receptors (6,42).
Blockage of this pathway by either pharmacologic inhibitors (e.g.
dasatinib and bosutinib) or a dominant-interfering mutant of Src
(41) suppressed angiogenesis in vitro and in vivo, which confirms
Src kinase as a therapeutic target (10). Considering that ACA was
unable to inhibit VEGFR2 autophosphorylation at concentrations
,20 lmol/l (Figure 4A) but efficiently blocked the functional events
of endothelial cells at only 5–10 lmol/l (Figure 1), we thus postulate
that ACA enters cells as a small molecule and blocks intracellular
non-receptor tyrosine kinases. In our study, we found that ACA exerted concentration- and time-dependent inhibition of dual Src/FAK
kinases (Figure 4B). Interestingly, our direct predicted binding model
showed that ACA docked into the hydrophobic pocket of Src kinase at
the catalytic site (Figure 4C), which suggests some degree of specificity of ACA toward this enzyme. The ability of a cell to polarize and
move depends on the remodeling of the cellular adhesion/cytoskeletal
network, which is controlled by the Rho family of small GTPases
910
(43). In our study, we observed that because the Src/FAK-signaling
pathway was inactivated, the active forms of Rac1- and Cdc42GTPases were concentration dependently inhibited by ACA in endothelial cells (Figure 4D). In accord with these molecular results, in
vitro VEGF-stimulated motility, adhesion and capillary-like structure
formation of endothelial cells, all of which require Src activity, were
greatly suppressed by ACA.
During tumor angiogenesis, when angiogenic growth factors or
cytokine ligands bind to receptors on the endothelial cell surface,
receptors are consecutively activated, triggering a cascade of downstream signaling events (44). It has been proved that repression of Src/
FAK signaling leads to a downregulation of VEGF expression (10,45)
and that sequential activation of Src results in increased MCP-1 expression (46), indicating that both VEGF and MCP-1 are regulated by
Src tyrosine kinase. Notably, in our study, we found that ACA inhibited VEGF and MCP-1 levels (Figure 5D). Consistent with these in
vitro results, Src and VEGF expression in ACA-treated solid tumors
was also inhibited by ACA (Figure 6D). When these angiogenic
growth factor and kinase are inhibited in cancer cells, the cascade
of signaling events in the tumor microenvironment (e.g. angiogenesis)
would be substantially decreased. This may explain why ACA exerted
such potent inhibitory actions on tumor angiogenesis and tumor
growth in vivo.
Endothelial cells, as the major components making up blood vessels, proliferate much more rapidly during angiogenesis than under
normal conditions (47). Our results demonstrated that ACA blocked
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Fig. 6. ACA inhibits tumor angiogenesis-mediated tumor growth in a prostate cancer xenograft mouse model. Human prostate PC-3 cells were injected into
BALB/cA nude mice. After solid tumors established, the mice were subcutaneously administrated with dimethyl sulfoxide (control; n 5 6) or ACA (6 mg/kg/day;
n 5 7). (A and B) ACA inhibited tumor growth as measured by tumor volume and tumor weight. (C) ACA had little toxicity at the tested dose. (D) Ki-67, Src,
VEGF and CD31 immunohistochemical and TUNEL analysis revealed that ACA inhibited microvessel density and cell proliferation in human prostate xenografts.
Blots and columns, mean; bars, standard deviation; P , 0.01 versus control.
ACA inhibits prostate tumor angiogenesis
Funding
Chenguang Program from Shanghai Municipal Education Commission (10CG25); Innovation Program from East China Normal University (78210021); National Institute of Health (1R01CA106479 and
1R01CA134731); Research Platform for Cell Signaling Networks
(06DZ22923); Pujiang Program from the Science and Technology
Commission of Shanghai Municipality (09PJ1403900).
Acknowledgements
We thank Virginia M.Mohlere from MD Anderson’s Department of Scientific
Publications for editing.
Conflict of Interest Statement: None declared.
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VEGF-triggered endothelial cell cycle progression at the G1 phase by
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18.88 to 7.53% at 10 lmol/l (Figure 1B); however, such low concentration of ACA had little effect on cell viability and apoptosis of PC-3
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Received November 5, 2010; revised March 7, 2011;
accepted March 14, 2011
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