SYP-5, a novel HIF-1 inhibitor, suppresses tumor cells invasion and angiogenesis

Article abstract of DOI:10.1016/j.ejphar.2016.09.027

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in carcinogenesis. The overexpression of HIF-1 induced by hypoxia is closely associated with metastasis, poor prognosis and high mortality. In this study, a novel HIF-1 inhibitor SYP-5 was first observed by the luciferase reporter assay. Western blots results showed SYP-5 inhibited hypoxia-induced upregulation of HIF-1. Moreover, the proteins of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 that are targets of HIF-1, were down-regulated by SYP-5. Furthermore, in the tube formation assay, SYP-5 suppressed angiogenesis induced by hypoxia and VEGF in vitro. Additionally, using Transwell and RTCA assays, we found that SYP-5 also retarded the Hep3B and Bcap37 cells migration and invasion induced by hypoxia and FBS. Last, we also detected the upstream pathways related to HIF-1 and found both PI3K/AKT and MAPK/ERK were involved in the SYP-5 mediated invasive inhibition of Bcap37 cells. These results indicates that SYP-5 inhibits tumor cell migration and invasion, as well as tumor angiogenesis, which are mediated by suppressing PI3K/AKT- and MAPK/ERK-dependent HIF-1 pathway. It suggests that SYP-5 might be a potential HIF-1 inhibitor as an anticancer agent.

Full text of DOI:10.1016/j.ejphar.2016.09.027

Author’s Accepted Manuscript
SYP-5, a novel HIF-1 inhibitor, suppresses tumor
cells invasion and angiogenesis
Li-Hui Wang, Xiao-Rui Jiang, Jing-Yu Yang, Xue-
Fei Bao, Jun-Li Chen, Xing Liu, Guo-Liang Chen,
Chun-Fu Wu
www.elsevier.com/locate/ejphar
PII:
S0014-2999(16)30615-X
http://dx.doi.org/10.1016/j.ejphar.2016.09.027
EJP70852
DOI:
Reference:
To appear in: European Journal of Pharmacology
Received date: 20 April 2016
Revised date: 12 September 2016
Accepted date: 19 September 2016
Cite this article as: Li-Hui Wang, Xiao-Rui Jiang, Jing-Yu Yang, Xue-Fei Bao,
Jun-Li Chen, Xing Liu, Guo-Liang Chen and Chun-Fu Wu, SYP-5, a novel HIF
1
inhibitor, suppresses tumor cells invasion and angiogenesis, European Journa
of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.09.027
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SYP-5, a novel HIF-1 inhibitor, suppresses tumor cells invasion and angiogenesis
a
Li-Hui Wang , Xiao-Rui Jiang , Jing-Yu Yang , Xue-Fei Bao , Jun-Li Chen , Xing
a
a
b
a
a
Liu , Guo-Liang Chen , Chun-Fu Wu
b**
a*
a
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua
Road, 110016 Shenyang, PR China
b
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of
Education, Shenyang Pharmaceutical University, 103 Wenhua Road, 110016
Shenyang, PR China
wucf@syphu.edu.cn(CF Wu)
guoliang222@gmail.com (GL Chen)
*
Corresponding author˖ Department of Pharmacology, Shenyang Pharmaceutical
University, Shenyang, China, Tel: 86 24 23986011; fax: 86 24 23986339.
*
*
Corresponding author: Key Laboratory of Structure-Based Drugs Design and Discovery of
Ministry of Education, Shenyang Pharmaceutical University, Shenyang, PR China. Tel: 86 24
23986412; fax: 86 24 31306459.
1

Abstract
Hypoxia-inducible factor-1 (HIF-1) plays an essential role in carcinogenesis. The
overexpression of HIF-1 induced by hypoxia is closely associated with metastasis,
poor prognosis and high mortality. In this study, a novel HIF-1 inhibitor SYP-5 was
first observed by the luciferase reporter assay. Western blots results showed SYP-5
inhibited hypoxia-induced upregulation of HIF-1. Moreover, the proteins of vascular
endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 that are
targets of HIF-1, were down-regulated by SYP-5. Furthermore, in the tube formation
assay, SYP-5 suppressed angiogenesis induced by hypoxia and VEGF in vitro.
Additionally, using Transwell and RTCA assays, we found that SYP-5 also retarded
the Hep3B and Bcap37 cells migration and invasion induced by hypoxia and FBS.
Last, we also detected the upstream pathways related to HIF-1 and found both
PI3K/AKT and MAPK/ERK were involved in the SYP-5 mediated invasive inhibition
of Bcap37 cells. These results indicates that SYP-5 inhibits tumor cell migration and
invasion, as well as tumor angiogenesis, which are mediated by suppressing
PI3K/AKT- and MAPK/ERK-dependent HIF-1 pathway. It suggests that SYP-5 might
be a potential HIF-1 inhibitor as an anticancer agent.
Keywords: HIF-1, SYP-5, angiogenesis, invasion, PI3K/AKT pathway, MAPK
pathway.
2

1. Introduction
Oxygen is essential for survival, while lack of oxygen will result in a strong stress. In
order to adapt to hypoxia, cells regulate the expression of certain proteins and genes
via oxygen sensor and signal transduction pathway (Hochachka et al., 2001). The
existence of micro-environmental hypoxia is an important and continuous
pathological feature of solid tumors (Gillies et al., 2007). Hypoxia in solid tumors is
associated with resistance to radiation therapy and chemotherapy, selection of more
invasive and metastatic clones and poor survival (Karakashev et al., 2015;Semenza et
al., 2012; Masoud et al., 2015).
Semenza et al (Semenza et al., 1991) discovered for the first time that
hypoxia-inducible factor 1 (HIF-1) could be induced by hypoxia and possess DNA
binding activation. This transcription factor, which consists of HIF-1α and HIF-1β, is
a master regulator for hypoxic adaptation (Semenza et al., 2003). As a result of
insufficient blood supply caused by the rapid proliferation of tumor cells in hypoxia
environment, HIF-1 is usually high expressed in tumor tissues. The overexpression of
HIF-1α has been reported in numerous tumor types, including breast, liver, gastric,
lung, prostate and colon (Kaya et al., 2012; Gaballah et al., 2014; Bhattacharyya et al.,
2010; Wang et al., 2014; Ranasinghe et al., 2015; Nagaraju et al., 2015). HIF-1 plays a
pivotal role in the regulation of key survival pathways in cancer cells. It activates the
transcription of genes that are involved in angiogenesis, cell survival, glucose
3

metabolism, migration, and invasion (Nagaraju et al., 2015; Yang et al., 2015). HIF-1,
therefore, has been identified as a potential drug target for cancer treatment.
Currently, there are a fraction of small molecules originally designed or discovered as
HIF-1 inhibitors (Hu et al., 2013), including a few chalcone-based compounds.
Recently, the results of ours and others showed that chalcone is a privileged template
to develop HIF-1 inhibitor (Srinivasan et al., 2011; Wang et al., 2015a). In the present
study, we showed that the novel chalcone-based compound SYP-5 is a potential
HIF-1 inhibitor. We selected the HUVEC, Hep3B and Bcap37 cell lines to investigate
the antitumor activity of SYP-5 targeting HIF-1. It obviously suppressed angiogenic
and invasive potential. The above results indicate that SYP-5, a novel chalcone-based
compound, might be a potential antitumor drug targeting HIF-1.
2
. Materials and methods
2
.1 Preparation of SYP-5
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(thiophen-2-yl)prop-2-en-1-one(
SYP-5) was synthesized as shown in Scheme 1. A key intermediate (4) was prepared
according to our previous work (Wang et al., 2015a). Chlorination of the
commercially available 2-methyl-3-butyn-2-ol(1) with HCl in presence of copper
powder afforded to 3-chloro-3-methylbut-1-yne(2), which was then reacted with
1
-(2,4-dihydroxyphenyl)ethanone in the presence of potassium carbonate and
potassium iodide to yield the 4-position alkylation product(3). The cyclisation of
intermediate(3) refluxed in N,N-dimethylaniline produced only 3-position product
4

1
rather than 5-position one which can be confirmed by H-nuclear magnetic resonance
(NMR). The target compound(SYP-5) was obtained by reacting intermediate(4) with
thiophene-2-formaldehyde catalyzed by piperidine in toluene with moderate
o
yield(43%, m.p. 105-107 C). The structure of SYP-5 was confirmed by the nuclear
1
resonance magnetic ( H-NMR) and shown in Fig. 1A.
Scheme 1 The synthetic route of target compound.
2
.2 Biology Regents
The primary antibodies against HIF-1β, MMP-2, p-EGFR, EGFR, p-AKT, AKT,
p-ERK, ERK, p-4E-BP1, 4E-BP1 and β-actin were got from Cell Signaling
Technology (Danvers, MA). The primary antibodies against HIF-1α and VEGF were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The PD98059, MEK
inhibitor, and LY294002, AKT inhibitor, were purchased from Beyotime
Biotechnology (CHN).
5

2
.3 Cell Culture and Exposure to Hypoxia
Hep3B (human hepatocellular carcinoma cell line, American Type Culture Collection,
ATCC) and Bcap37 (human breast cancer cell line, Institute of Biochemistry and Cell
Biology, Shanghai, CHN) were routinely cultured in DMEM (high glucose) with 10 %
heat-inactivated FBS (Gibco BRL, Grand Island, NY) and maintained at 37°C in a
2
humidified incubator with 5% CO . Primary human umbilical vascular endothelial
cells (HUVECs) were purchased from ATCC and were cultured in Roswell Park
Memorial Institute 1640 supplemented with 10% fetal bovine serum (FBS). For cells
culture under hypoxia, cells were grown in a microprocessor-based oxygen controller
(
Coy Laboratory Products) containing 1% O
.4 Luciferase reporter assay
2 2 2
, 5% CO and 94% N at 37 ć.
2
U251-HRE cells (human glioma cell line U251 stably expressing a hypoxia regulatory
element (HRE)-luciferase pGL2 construct) and U251-pGL3 cells (U251 cells stably
expressing a pGL3 construct) were kindly provided by Dr. Giovanni Melillo (NCI,
USA). The cells were grown in RPMI 1640 with 5% heat-inactivated FBS, 200 μg/ml
G418. The experiments were performed according to previous published paper
5
Rapisarda et al., 2002). Briefly, the cells (1×10 cells/ml) were seeded into 96-well
(
Opti-plates (Packard Instrument, Inc., Meriden, CT). After overnight incubation, the
cells were pretreated with various concentrations of agents for 1 h. Then cells were
grown in the microprocessor-based oxygen controller for 16 h. The Luciferase
reporter assays were tested by Bright Glo luciferase assay reagents (Promega Inc,
Madison, WI).
6

2
.5 Cell viability assay
5
The cells (1×10 cells/ml) were seeded into 96-well culture plates. After overnight
incubation, the cells were treated with various concentrations of SYP-5 for 24 h. Then
1
0 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution
(
2.5 mg/ml in PBS) was added to each well, and the plates were incubated for an
o
additional 4 h at 37 C. After centrifugation (2500 rpm, 10 min), the medium
containing MTT was aspirated, and 100 μl DMSO was added. The optical density of
each well was measured at 570 nm with a SpectraMax Paradigm Reader(Molecular
Devices).
2
.6 Western blot assay
7
About 1×10 Hep3B and Bcap37 cells were gathered after treatment by SYP-5
concurrently under hypoxia. In order to avoid interaction with SYP-5, the inhibitors
were pretreated for 1 h. Western blot was performed as previously described (Wang et
al., 2014). In brief, equal amount from cultured cells were fractionated by 8-15%
SDS-PAGE and electrically transferred onto polyvinylidene difluoride (PVDF)
membranes. Mouse or rabbit primary antibodies and horseradish peroxidase
(
HRP)-conjugated appropriate secondary antibodies were used to detect the
designated proteins. Membrane-bound secondary antibodies were reacted with ECL
detection reagents (Thermo Scientific, MA, USA) and exposed in dark room. Results
were normalized to the internal control β-actin.
2
.7 Tube formation assay
Tube formation assay was performed as previously described (Wang et al., 2015). In
7

brief, 50 μl matrigel was placed into each well of the 96 well-plate and allowed to
polymerize by incubation at 37 ć for 30 min. The HUVECs were seeded onto the
4
matrigel layer at a density of 5 × 10 cells with or without SYP-5, followed by
hypoxia treatment or addition 20 ng/ml VEGF. After 12 h, tubular structure of
endothelial cells was stained by Calcein-AM (Molecular Probes, Eugene, OR) and
photographed using high content screening system ImageXpress Micro (Molecular
Devices, CA, USA), and tube formation (branches/field) was examined and quantified.
Three independent experiments were performed.
2
.8 RTCA (Real-time cell analysis) migration assay
RTCA was performed as previously described (Wang et al., 2015b). In brief, The
CIM-plate contains 16 modified Boyden chambers, which can be used independently
to measure cell migration in real time through 8 μm pores of a polyethylene
terephthalate membrane onto gold electrodes on the underside of the membrane using
the xCELLigence analyser system (ACEA Biosciences, CA, USA). Experiments were
set up according to the manufacturer's instructions. A chemotactic signal for
movement was provided by inoculating various concentrations of SYP-5 treated
5
2
0,000 cells in serum-free medium in the upper chamber and supplying 15% FBS or
0 ng/ml VEGF in the lower chamber. Cell index (electrical impedance) was
monitored every 5 min for the duration of the experiment. The cell index reflects the
tumor cell’s migratory capacity. And the slope of the curve can be related to the
migration velocity of tumor cells.
2
.9 Transwell Assay
8

Transwell Assay was performed as previously described (Wang et al., 2015b). Cells
5
1-2×10 ) were plated in RPMI 1640 medium in the upper chamber of the Trans-well
(
(
8 μm pore, Corning Costar, Corning, NY), which were pre-coated with or without
matrigel (Becton-Dickinson, Bedford, MA). Different concentrations of SYP-5 were
added to the lower chamber followed by incubating under normoxia or hypoxia for 16
hours at 37 ć. The non-migrating cells and the collagen gel from the interior side of
the inserts were gently removed using a cotton-tipped swab. The cells that migrated
through the gel insert to the lower surface of the membrane were stained by
Calcein-AM and photo- graphed using a high-content screening system (Molecular
Devices). The number of migrated cells was quantified by counting cells number at X
4
objectives.
2
.10 Statistical analysis
Statistical analysis was performed using SPSS11.5 software package for Windows
SPSS, Chicago, IL). Data were presented as the mean±s.e.m. Statistical significance
(
was calculated using a student’s t-test, with a probability level of P<0.05 considered
to be statistically significant.
3
. Results
3
.1 SYP-5 inhibits HIF-1 and downstream gene expression in Hep3B and Bcap37
cells
U251-HRE and U251-pGL3 cells (a HIF-1α inhibitor screening model) were used to
detect the ability of SYP-5 to inhibit HIF-1 activity. In this model, U251-HRE cell
9

line, which stably transfected with pGL2 plasmid with 3 copies of the
hypoxia-responsive element (HRE), was used to measure the HIF-1 activity within the
cells under various conditions. Parallel U251-pGL3 cell line, which stably transfected
with pGL3 plasmid, was used to exclude compounds that inhibited luciferase
expression in a nonspecific fashion. As shown in Fig. 1B, SYP-5 specifically inhibited
hypoxic induction of luciferase expression in U251-HRE but not in U251-pGL3. To
evaluate how SYP-5 inhibits HIF-1α activity, we investigated the expressions of
HIF-1α and HIF-1β in protein levels using western blot analysis. SYP-5 was found to
inhibit HIF-1α and HIF-1β protein levels under hypoxic condition in a
concentration-dependent manner in both Hep3B and Bcap37 cells (Fig. 1C).
As a transcription factor, the activation of HIF-1 would lead to upregulation of target
genes that are involved in many aspects of cancer progression, such as vascular
endothelial growth factor (VEGF) which is the key factor in angiogenesis (Ahluwalia
et al., 2012). HIF-1 also regulates the expression of genes encoding matrix
metalloproteinase 2 (MMP2), which plays an established role in the pathophysiology
of invasion (Zhao et al., 2014).To investigate whether the inhibitory effect of SYP-5
on HIF-1α affects VEGF and MMP2 expression, we assessed the level of VEGF and
MMP2 in both cells. We found that treatment with SYP-5 displayed significant
inhibition on hypoxia-induced overexpression of VEGF and MMP2 in both cell lines
(
Fig. 1C). Taken together, our results showed that SYP-5 could inhibit the activation
of HIF-1 through downregulating HIF-1α and HIF-1β protein levels, and suppress its
target genes.
1
0

3
.2 SYP-5 inhibits microvessel formation induced by hypoxia (95%N2+5%CO2) and
VEGF in vitro
Angiogenesis, the formation of new blood vessel, is essential for cancer progression.
Under hypoxia, HIF-1 has been proved to be a direct transcriptional activator of
VEGF (Ahluwalia et al., 2012). HUVECs can spontaneously form three dimensional
capillary-like tubular structures when cultured on matrigel. Tube formation assay
represents a classical model for studying inhibitors of angiogenesis. Next we studied
the effects of SYP-5 on angiogenesis in HUVECs. Firstly, we have detected the cell
viability using MTT assay before conducting tube formation assay. The data was
showed in the Supplementary material Fig. 1. After treated with HUVEC cells with
various concentrations of SYP-5 for 24 h, the concentrations lower than 50 μM of
SYP-5 have no significant effect on cell viability (Cell viability more than 80%). Thus,
to exclude the effect of cell cytotoxicity of SYP-5 on tube formation assays, we
choose 50 μM as the maximum concentration of SYP-5. As shown in Fig. 2, SYP-5
was confirmed to significantly inhibit HUVECs tube formation induced by hypoxia.
To further address whether SYP-5 inhibits angiogenesis by targeting VEGF, tube
formation induced by VEGF was performed. Treatment with SYP-5 resulted in
significant reduction in the number and the continuity of microvessel formation of
HUVECs induced by VEGF (10 ng/ml) in a concentration-dependent manner. The
above data suggesting that SYP-5 could retard angiogenesis induced by either
hypoxia or VEGF.
3
.3 SYP-5 suppresses migratory and invasive potential of HUVEC induced by VEGF
11

in vitro
Endothelial cell migration is one of the most important and early event during the
process of angiogenesis (Zecchin et al., 2015). To explore the effect of SYP-5 on
HUVECs in migration and invasion, RTCA and transwell assay were performed. As
shown in Fig. 3, SYP-5 was confirmed to significantly inhibit HUVECs migration
and invasion induced by VEGF (10 ng/ml) at a concentration dependent manner,
suggesting SYP-5 displayed a potential to inhibit endothelial cell migration.
3
.4 SYP-5 inhibits Hep3B and Bcap37 cells migration and invasion induced by
hypoxia and FBS
The occurrence of tumor cell metastasis is the primary factor causing poor prognosis
of patients with cancer. When tumors encounter low oxygen tension, overexpression
of HIF-1α enhances tumor invasion and metastasis through up-regulating MMPs
(
Ding et al., 2009). In the present study, we found that SYP-5 at un-cytotoxic
concentrations (Supplementary materials Fig. 1) not only suppressed the expression of
MMP-2 under hypoxic conditions (Fig. 1B), but also significantly inhibited
hypoxia-induced migration and invasion of Hep3B and Bcap37 cells (Fig. 4A).
Furthermore, our data showed that FBS-induced cell migration and invasion of both
cell lines could be also dramatically suppressed by SYP-5(Fig. 4B and C). Taken
together, our data demonstrates that SYP-5 owns the potential against multiple
factors-induced migration and invasion of cancer cells.
3
.5 SYP-5 inactivates hypoxia-induced PI3K-AKT and MAPK-ERK1/2 pathways
Previous studies have shown that growth factors related pathways activation, mainly
1
2

including PI3K-AKT and MAPK-ERK1/2, is necessary to regulate VEGF and HIF-1
expression in human cancer cells(Masoud et al., 2015; Jiang et al., 2008), so we next
detect the effect of SYP-5 on both pathways. Our data indicated that hypoxia could
induced the activation of EGFR, which is consistent with previous report (Swinson et
al., 2008), and its downstream protein phosphorylation, including AKT, 4E-BP1, and
ERK, whereas, administration of SYP-5 inhibited the activation of EGFR and its
downstream pathways in Bcap37 cells (Fig. 5). These results demonstrated that
hypoxia related PI3K-AKT and MAPK-ERK1/2 pathways might also be regulated by
SYP-5.
3
.6 PI3K-AKT and MAPK-ERK pathways involve into the inhibitory effect of SYP-5
on cell invasion
In order to explore the role of PI3K-AKT and MAPK-ERK1/2 pathways in SYP-5
caused inhibitory effect on cell invasion, we applied the specific inhibitors for
MAPK/ERK and PI3K/AKT pathways and analyzed cell invasion. As shown in Fig. 6
A and B, single treatment with PD98059 (a specific inhibitor of MAPK/ERK) and
LY294002 (a specific inhibitor of PI3K/AKT) induced a slight inhibition of
hypoxia-induced invasion in Bcap37 cells. Interestingly, in contrast to PD98059,
pretreatment with LY294002 combined with SYP-5 resulted in a dramatically
suppression of invasive cells induced by hypoxia, indicating that the PI3K/AKT
pathway play a synergistic role in regulating SYP-5-mediated invasive inhibition of
Bcap37 cells.
Furthermore, the effects of SYP-5, LY294002, and PD98059 alone or combination on
1
3

the signaling pathways and invasion related protein MMP-2 were also assessed in
Bcap37 cells. As expected, single treatment with LY294002 or PD98059 could result
in the inhibition of AKT pathway and ERK pathway, respectively. In consistent with
the cell invasion data, LY294002 and PD98059 treatment also led to the
downregulation of MMP2. Interestingly, LY294002 treatment slightly inhibited ERK
phosphorylation, meanwhile the addition of PD98059 also resulted in the inhibition of
AKT pathway, suggesting the crosstalk between ERK and AKT pathways. Importantly,
we found the combination of SYP5 and LY294002 or PD98059 displayed an
enhanced inhibition of MMP2 expression, suggesting both pathways are involved in
the inhibitory effect of SYP-5 on cell invasion.
4
. Discussion
Hepatocellular carcinoma (HCC) and breast cancer share the character of tissue
hypoxia, especially when the tumor grows quickly but angiogenesis fails to catch up
with the speed of tumor growth. The key hypoxic regulator HIF-1α, has been shown
to be highly expressed in HCC and breast cancer. Meanwhile, evidence suggests that
HIF-1 is involved with invasion, metastasis and angiogenesis (Tu et al., 2014; Bos et
al., 2005).
In recent decades, the discovery and development of novel small molecules targeting
HIF-1 has been an exciting area of developmental therapeutics (Xia et al., 2012). The
role of HIF-1 inhibitors in cancer therapy remains speculative. It has been reported
that quite a number of chalcone-based compounds reveal moderate HIF-1 inhibitory
1
4

activity and many chalcone-based compounds prove promising anticancer activities in
various animal models (Srinivasan et al., 2011; Wang et al., 2015). It indicates that
chalcone might be a template to develop HIF-1 inhibitor. Here, we identified a novel
chalcone-based compound, SYP-5, which displayed specifically inhibit HIF-1
activity.
In this study, our results show that SYP-5 decreased the HIF-1α and HIF-1β protein
levels under hypoxic condition. As HIF-1 regulates multiple genes expression
involved in tumorigenesis, we further investigated its downstream gene expression.
The complex process of angiogenesis begins when cells respond to hypoxia by
increasing generation of VEGF, which has been widely known as one of the most
important factors stimulating angiogenesis in solid tumors. In our study, protein of
VEGF was up-regulated in both Hep3B and Bcap37 cells under hypoxic condition.
We speculated that SYP-5 suppressed the hypoxia-mediated VEGF expression by
down-regulating HIF-1 expression. Furthermore, we found that SYP-5 could reduce
the HUVECs tube formation induced by hypoxia and VEGF in vitro, which further
confirmed the inhibitory action of SYP-5 on HIF/VEGF signaling axle.
MMP-2, a critical protease, degrades many kinds of extracellular matrix and plays a
critical role in invasion. HIF-1α enhances tumor invasion and metastasis through
up-regulating MMP-2(Jing et al., 2012). Presently, we observed a significant
reduction of MMP-2 after treatment with SYP-5. Moreover, we discovered that
SYP-5 inhibited hypoxia-induced migration and invasion in Hep3B and Bcap37 cells,
indicating that SYP-5 could influence the both cell lines migration and invasion
1
5

through HIF-1 pathway.
It has been reported that various signaling pathways are involved in the expression of
HIF-1α. Among them, EGFR related PI3K/AKT and MAPK/ERK pathways appears
to play a major role in HIF-1α expression (Masoud et al., 2015; Bos et al., 2005).
Here we found that administration of SYP-5 could reverse hypoxia induced activation
of EGFR and its downstream pathways, PI3K/AKT and MAPK/ERK, in Bcap37 cells.
Further confirming results showed that treatment with PI3K/AKT inhibitor combined
with SYP-5 resulted in an enhanced suppression of migratory cells induced by
hypoxia. Additionally, western blot data showed that the combination of SYP5 and
LY294002 or PD98059 displayed an enhanced inhibition of migration related protein
MMP2. These results demonstrate that both PI3K/AKT pathway and MAPK/ERK
pathway play a role in regulating SYP-5-induced migratory inhibition.
In summary, this study shows for the first time that a novel chalcone-based compound
SYP-5 inhibited the tumor angiogenesis and cell invasion in vitro. Mechanistically,
SYP-5 decreased HIF-1α and HIF-1β expression in Hep3B and Bcap37 cells through
inhibiting EGFR/PI3K/AKT and MAPK/ERK pathways.
Acknowledgements:
The authors gratefully acknowledge financial support from the Excellent Talents Plan
in Liaoning Province (No: LJQ2014111) and Shenyang Pharmaceutical University
Science Foundation (No. ZCJJ2013401). We thank Dr. Giovanni Melillo
(
Developmental Therapeutic Program, NCI, Frederick, MD) for generously providing
1
6

the U251-HRE and U251-PGL3 cells.
Conflict of interest
The authors have declared no conflict of interest.
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Fig. 1 Effects of SYP-5 on HIF-1 activity and downstream gene expression. (A)
The structure of SYP-5. (B) Hypoxic induced luciferase expression in U251-HRE
cells was detected by luciferase assay. U251-pGL3 cells was considered as control,
and the luciferase unit in normoxia U251-pGL3 cells was considered as 100%. (C)
The HIF-1 and VEGF, MMP2 proteins were assessed by western blot analyses in
Hep3B and Bcap37 cell line after SYP-5 (2, 10, 50 μM) treated for 48 h under
hypoxic condition. β-actin expression was used as a loading control. All error bars are
###
S.E.M. Denotes a significant difference compared to normoxia treated U251-HRE
cells, P<0.001; ***Denotes a significant difference compared to hypoxia treated
U251-HRE cells, P<0.001.
Fig. 2 The effects of SYP-5 on hypoxia or VEGF-induced tube formation of
HUVEC cells. (A) HUVECs tube formation induced by hypoxia or VEGF (20 ng/ml)
was measured after SYP-5 (2, 10, 50 μM) treated for 12 h. The experiments were
performed as described in Materials and Methods. (B) Quantification of the effect of
SYP-5 on tube formation is shown in column diagram. Con represents the group
without treatment hypoxia or VEGF. All error bars are S.E.M. *Denotes a significant
difference compared to hypoxia or VEGF treated control, P<0.05; ***Denotes P
<0.001.
Fig. 3 The effects of SYP-5 on VEGF-induced migration and invasion of HUVEC
cell. (A) HUVEC cells migration was measured by RTCA. The cells were treated
SYP-5 at concentrations of 2, 10, 50 μM, as well as VEGF(20 ng/ml). (B) HUVEC
2
1

cells invasion was measured by transwell assay after SYP-5 (2, 10, 50 μM) treated for
6 h. VEGF(20 ng/ml) was added in the lower chamber. (C) Quantification of the
effect of SYP-5 on cell invasion is shown in column diagram. All error bars are S.E.M.
1
*
*
*Denotes a significant difference compared to VEGF single treated control, P<0.01;
**Denotes P<0.001.
Fig. 4 The effects of SYP-5 on hypoxia or FBS-induced migration and invasion of
Hep3B and Bcap-37 cells. (A) Hep3B cells and Bcap37 cells migration and invasion
induced by hypoxia were measured by transwell assay after SYP-5 (2, 10, 50 μM)
treated for 16 h. (B) Hep3B cells and Bcap37 cells migration induced by FBS were
measured by RTCA after SYP-5 (2, 10, 50 μM) treated for 24 h. (C) Hep3B cells and
Bcap37 cells invasion induced by FBS were measured by transwell assay after SYP-5
(
2, 10, 50 μM) treated for 16 h.
Fig. 5 The effects of SYP-5 on EGFR, PI3K/AKT and MAPK/ERK pathways.
The p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, p-4E-BP1, and 4E-BP1 proteins
were assessed by western blot analyses in Bcap37 cell line after SYP-5 (2, 10, 50 μM)
treated for 48 h under hypoxic condition. β-actin expression was used as a loading
control.
Fig. 6. The effects of specific inhibitor of PI3K/AKT or MAPK/ERK on SYP-5
mediated invasive inhibition in Bcap37 cells. (A) Bcap37 cells invasion was
measured by transwell assay after treatment with SYP-5(10 μM) and/or specific
inhibitors (PD98059 10 μM; LY294002 10 μM). (B) Quantification of cell invasion is
shown in column diagram. (C) The p-ERK, ERK, p-AKT, AKT, and MMP-2 proteins
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were assessed by western blot analyses in Bcap37 cell line as the above described
drug treatment condition. β-actin expression was used as a loading control. Con
represents the group without hypoxia treatment. All error bars are S.E.M. *Denotes a
significant difference compared to hypoxia control, P<0.05.
Supplementary materials Fig. 1. The effects of SYP-5 on cell viability in HUVEC,
Hep-3B and Bcap37 cell lines. The cells were treated with SYP-5 for 24 h, and
detected by MTT assay.
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