Selenadiazole Derivatives Inhibit Angiogenesis-Mediated Human Breast Tumor Growth by Suppressing the VEGFR2-Mediated ERK and AKT Signaling Pathways

Article abstract of DOI:10.1002/asia.201800110

Selenadiazole derivatives (SeDs) have been found to show promise in chemo-/radiotherapy applications by activating various downstream signaling pathways. However, the functional role of SeDs on angiogenesis, which is pivotal for tumor progression and metastasis, has not yet been elucidated. In the present study, we have examined the antiangiogenic activities of SeDs and elucidated their underlying mechanisms. The results showed that the as-synthesized SeDs not only enhanced their anticancer activities against several human cancer cells but also showed more potent inhibition on human umbilical vein endothelial cells (HUVECs). The in vitro results suggested that SeDs, especially 1 a, dose-dependently inhibited the vascular endothelial growth factor (VEGF)-induced cell migration, invasion, and capillary-like structure formation of HUVECs. Compound 1 a also significantly suppressed VEGF-induced angiogenesis in a Matrigel plug assay as part of a C57/BL6 mice assay by means of down regulation of VEGF. Furthermore, we found that 1 a significantly inhibited MCF-7 human breast tumor growth in nude mice without severe systematic cytotoxicity. Compound 1 a was more effective in inhibiting cell proliferation and induced a much more pronounced apoptosis effect in endothelial cells than MCF-7 cells, which implies that endothelial cells might be the primary target of 1 a. Further mechanistic studies on tumor growth inhibition effects and neovessel formation suppression demonstrated that 1 a inhibited cell viability of MCF-7 and HUVECs by induction of cell apoptosis, accompanied by poly(adenosine diphosphate ribose)polymerase (PARP) cleavage and caspase activation. Additionally, the 1 a-induced antiangiogenesis effect was achieved by abolishing the VEGF-VEGFR2-ERK/AKT (ERK=extracellular signal–regulated kinases; AKT=protein kinease B) signal axis and enhanced the apoptosis effect by triggering reactive oxygen species (ROS)-mediated DNA damage. Taken together, these results clearly demonstrate the antiangiogenic potency of SeDs and the underlying molecular mechanisms.

Full text of DOI:10.1002/asia.201800110

CHEMISTRY
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Accepted Article
Title: Selenadiazole derivatives inhibit angiogenesis-mediated human
breast tumor growth through suppressing VEGFR2-mediated
ERK and AKT signaling pathway
Authors: Haoqiang Lai, Xiaoyan Fu, Chengcheng Sang, Liyuan Hou,
Pengju Feng, Xiaoling Li, and Tianfeng Chen
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10.1002/asia.201800110
Chemistry - An Asian Journal
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Selenadiazole derivatives inhibit angiogenesis-mediated human
breast tumor growth through suppressing VEGFR2-mediated
ERK and AKT signaling pathway
Haoqiang Lai #,a, Xiaoyan Fu#,a, Chengcheng Sang a, Liyuan Hou,a Pengju Feng,a Xiaoling Li,*b and
Tianfeng Chen*a
Abstract: Selenadiazole derivatives (SeDs) have been found to
show promising application in chemo-/radiotherapy through
activating various downstream signaling pathways. However, the
functional role of SeDs on angiogenesis, which is pivotal for tumor
progression and metastasis, has not been elucidated. In the present
study, we have examined the antiangiogenic activities of SeDs and
elucidated their underlying mechanisms. The results showed that the
as-synthesized SeDs not only enhanced their anti-cancer activities
against several human cancer cells, but also showed more potent
inhibition on human umbilical vein endothelial cells (HUVECs). The
in vitro results suggested that, SeDs, especially 1a, dose-
dependently inhibited vascular endothelial growth factor (VEGF)-
induced cells migration, invasion and capillary-like structure
formation of HUVECs in vitro. 1a also significantly suppressed
VEGF-induced angiogenesis in Matrigel plug assay in C57/BL6 mice
assay via down-regulation of VEGF. Furthermore, we found that 1a
significantly inhibited MCF-7 human breast tumor growth in nude
mice without severe systematic cytotoxicity. 1a was more effective in
inhibiting cells proliferation and induced much more pronounce
apoptosis effect in endothelial cells than MCF-7 cells, which implying
that endothelial cells might be the primary target of 1a. Further
mechanic studies on tumor growth inhibition effects and neovessel
formation suppression demonstrated that 1a inhibited cell viability of
MCF-7 and HUVECs by induction of cell apoptosis, accompanying
by PARP cleavage and caspases activation. Additionally, 1a-induced
antiangiogenesis effect was achieved by abolishing VEGF-VEGFR2-
ERK/AKT signal axis and enhanced the apoptosis effect through
triggering reactive oxygen species (ROS)-mediated DNA damage.
Taken together, these results clearly demonstrate the antiangiogenic
potency of SeDs and the underlying molecular mechanisms.
AKT, ERK, STAT3 and FAK can be activated and than result in
angiogenesis stimulating signaling amplification, which
coordinately leading to neovascular formation[4]. Consequently,
molecules involved in these cascades could be exploited as
ideal targets in angiogenesis restriction therapy. Moreover,
extensive evidences have supported that anti-angiogenesis
therapy has become one of the most promising strategy in
cancer treatments. In recent decades, many antiangiogenic
agents have been identified as potent anticancer drugs, and
some of them have been applied into clinic, including
monoclonal antibodies, small molecule tyrosine kinase inhibitors
and signal pathway molecules inhibitor. However, toxicities,
substantial costs and some severe side effects successively
occurred[5]. Therefore, searching novel antiangiogenic agents
with high performance and low toxicity and more affordable
agents are urgently needed.
Selenium (Se), as essential trace element for humans, has
been disclosed to display critical regulation role in physiological
and pathological conditions. Moreover, recent studies revealed
that selenium also could be sued in cancer prevention and
cancer treatments when combined with other anticancer agents
or radiation[6]. In the past decades, different chemical forms and
dose of selenium have been investigated as promising
anticancer agent in cancer treatment applications[7]. Among
them, organoselenium compounds exert fascinating application
in chemoprevention and chemotherapy for their pharmacological
potential and interesting chemical properties when compared to
inorganic selenium compounds[8]. Moreover, MeSe, and
Methylselenol exhibited as antiangiogenic agents in cancer
chemoprevention through inducing HUVEC cells apoptosis and
cell cycle perturbation, which indicated the potential application
of organoselenium compounds as antiangiogenesis agents[9].
Recently selenadiazole derivatives (SeDs) have triggered
intense research interest due to their excellent anticancer
biological activities, such as benzo[c]-[1,2,5]selenadiazole[10]5-
methylbenzo[c][1,2,5]selenadiazole[11]5-nitrobenzo[c]-
Introduction
Angiogenesis, as one of the critical hallmarks of cancers,
has been recognized to be the essential process in sustaining
tumour initiation, growth, progression and metastasis[1].
Angiogenesis is a tightly regulated process involving multiple
[1,2,5]selenadiazole[12]
,
1,2,3-thiadiazole
and
1,2,3-
factors, and comprehensively triggered by different stimulation
selenadiazole derivatives[13]. In our previous works, we have
rational synthesized a series of SeDs by chemical modification.
We found that SeDs exhibited as strongly apoptosis inducer in
A375 cells and MCF-7 cells through inducing mitochondria
dysfunction[14], SeDs potently sensitized X-ray induced cancer
2]
signaling pathway[1c,
. Nowadays, several pro-angiogenic
molecules and regulatory have been characterized. VEGF as
one of the most important pro-angiogenic molecules stimulates
endothelial cells proliferation, migration and formation of
neovessels through interaction with VEGFR-2[3]. Upon activation,
various downstream molecules in cascades such as Src kinase,
cells apoptosis through targeting TrxR[15]
,
antagonize
hyperglycemia-induceddrug resistance in HepG2 cells and
inhibited bladder cancer cell proliferation through activated ROS-
mediated pathway[16]
selenadiazole should be
.
These results all indicate that
good activity group in
[a]
[b]
*
Department of Chemistry, Jinan University, Guangzhou 510632,
China.
Institute of Food Safety and Nutrition, Jinan University, Guangzhou
510632, China, tlxlli@jnu.edu.cn.
Department of Chemistry, Jinan University Guangzhou 510632
(China) E-mail: tchentf@jnu.edu.cn.
a
pharmaceuticals designing. However, little information of SeDs
in antiangiogenesis is available. Therefore, searching for
#
Authors contributed equally to the work
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10.1002/asia.201800110
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effective and safety SeDs as angiogenesis antagonizer have
kindled great interest.
Table 1. Growth inhibitory effect of SeDs.
In our previous studies, we found that introduction of electron
donating group(–CH3) was able to increase the complex
lipophilicity and cellular uptake[15a, 17], however, the introduction
of electron-withdrawing group (–NO2) reduced these
properties[15a]. Moreover, the introduction of electron donating
group (-CH3, -OCH3, -OH) and electron-withdrawing group (-NO2,
-Cl) display important role in anticancer activity of selenadiazole
derivatives. For instance, the introduction of a nitro group (–NO2)
into SeD significantly enhanced the anticancer activity of SeDs,
however, SeDs exerted more potent anticancer activities after
the introduction of -OCH3 into the modifies selenadiazole
16,
(SeDs)[12,
18]. These results all indicate the critical role of
a. Cancer cells were exposed to different concentration of SeDs for
72 h and then cell viability was measured by MTT assay.
b. Normal cells.
electron donating group and electron-withdrawing group, and
selenadiazole should be good activity group in
a
pharmaceuticals designing. Consequently, in order to searching
better solubility and stability of SeDs as angiogenesis inhibitors,
other electron donating groups(-N(CH3)2, -NH2, -NHCOCH3 and -
OH) were used for SeDs designing. The results showed that
SeDs significantly inhibited VEGF-induced angiogenesis in vitro
and in vivo. Furthermore, we found that the introduction of –CH3
group into the complex (1a) significantly enhanced the
anticancer efficacy of SeDs. 1a dramatically decreased the
tumor growth of MCF-7 xenografts without severe systematically
cytotoxicity. The studies on the underlying molecular
mechanisms in HUVECs and MCF-7 cells revealed that SeDs
strongly inhibited the activation of VEGF and VEGFR-2
phosphorylation, which blocked the transmission of the
mitogenic signal through Akt and ERK1/2 pathways. 1a also
found to dose and time-dependently trigger reactive oxygen
species (ROS) generation and induced DNA damage.
Additionally, we found that 1a dose-dependent suppressed the
proliferation of MCF-7 cells through triggering apoptosis by
inducing DNA damage and upregulating p-ATM, p-ATR and
pp53 expression. Taken together, this study clearly
demonstrated that SeDs targeted VEGF-VEGFR2-ERK/AKT
signaling pathway, which resulted in suppression of tumor
growth and angiogenesis. These results clearly demonstrate the
antiangiogenic potency of SeDs and the underlying molecular
mechanisms.
Herein in this study,
a
series of phenylbenzo
[1,2,5]selenadiazole derivatives were tested for their anticancer
activities. First, in order to investigate the anticancer activities of
these SeDs, we carried out MTT assay to evaluate the
cytotoxicity of the SeDs on various cancer cells. As shown in
Table 1, the complexes exhibited splendid broad-spectrum
anticancer activity against series of human cancer cells, with 1a
exerted formidable inhibition effects in MCF-7 cells viability, as
evidenced by the half maximal inhibitory concentration of 1a is
7.45 μM, which is much lower than other cells. Intriguingly, we
found that these SeDs also effectively suppressed endothelial
cells proliferation. Furthermore, HUVECs seems to be more
susceptible to these complexes when compared to the tested
cancer cell lines, which indicated the potential antiangiogenesis
capacity of these SeDs, especially for 1a, 3.75 μM treatment of
1a for 72 h was enough to cause more than half of HUVECs
death. Meanwhile, we found that the synthesized analoguous O
compound 3a (Figure S1-S4) exhibited less potent inhibition
effects towards HUVECs at the same concentration and the IC50
is 14.5 μM, which is much higher than 1a (Figure S15). These
results indicated that the Se burried in the compound makes
special contributions to its antiangiogenesis activities. Moreover,
we found that the as-synthesized complexes displayed less
cytotoxicity to HK-2 human normal kidney cells, suggesting the
specific cytotoxicity towards human cancer cells. Together,
these results demonstrated the selective anticancer activities of
organoselenium complexes, and the possible antiangiogenic
activities of SeDs.
Results and Discussion
Organoselenium 1a inhibits tumor growth in vivo.
Synthesis and anticancer efficacy of SeDs
To evaluate the anticancer activities of organoselenium
complexes, we selected 1a for further evaluation through
establishing a human breast tumor xenograft model in nude
mice. After administrated with 1a, we found that the tumor
volume and tumor weight were dramatically inhibited when
compared with the vehicle treatments (Fig. 2A, B and 2C). For
instance, the tumor weight and tumor volume in the control
group was about 1.45 ± 0.23 g and 2090 ± 0.12 mm3, however,
after treated with 2.5 and 5 mg/kg for 14 days, the tumor volume
decreased to 1520 ± 0.22 mm3 and 1370 ± 0.1 mm3, and the
tumor weight reduced to 0.98 ± 0.13 g and 0.62 ± 0.24 g
respectively. Moreover, there is neglectable effect on the mouse
1a R=CH3; 1b R=Cl; 1c R=H; 1d R=OH;1e R=NO2;
1f R=N(CH3)2; 1g R=NH2; 1h R=NHCOCH3
2a R=H; 2b R=Cl; 2c R=CH3; 2d R=OH
Figure 1. Chemical structure of 1a-1h and 2a-2d.
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A
B
B
A
Control
2.5 mg/kg
5 mg/kg
2.0
1.5
1.0
0.5
0.0
**
**
C
E
D
F
(mg/kg)
0
2.5
5
C
D
25
20
15
10
5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Control
2.5 mg/kg
5.0 mg/kg
Control
2.5 mg/kg
5.0 mg/kg
0
(days) 0
2
4
6
8
10 12 14
(days)
0
2
4
6
8
10 12 14
E
F
Figure 3. 1a inhibited VEGF-induced in vitro cell migration (A), invasion (C)
and tube formation (E) in HUVECs. Quantitative analysis of migrated cells (B),
invasived cells (D) and the length of tubular structure (F). Three independent
experiments were carried out for these assays. The basal control groups were
treated with 0.5% DMSO and 50 ng/mL of VEGF treatment was used as
positive control group. Data in these experiments were expressed as means ±
SD of triplicates. Bars with different characters (a-e) are statistically different at
P<0.05 level. Scale bar: 100 nm.
G
H
models in vitro were employed to investigate the
antiangiogeneiss activities of 1a. Primarily, in vitro migration
assay activities of 1a. Primarily, in vitro migration assay was
employed to detect the suppression effects of 1a on HUVECs
migration. As shown in Figure 3A and 3B, enhanced migration
of HUVECs and complete wound closure by 48 h was observed
in VEGF-treated group when comparing with the basal medium
treatments. However, these effects were significantly inhibited
by 1a in a time- and dose- dependent manner, as convinced by
the uncovered area. Moreover, 1a (10 μM) dramatically
suppressed the migrated effects of HUVECs stimulated by
VEGF (50 ng/mL). Similarly suppression effects were also
observed in the analoguous O compound 3a treatments (Figure
S16), but much less efficiency than 1a.
Figure 2. 1a induced MCF-7 tumor growth suppression in vivo. 1a inhibited
MCF-7 tumor growth as measured by tumor weight (A and B) and tumor
volume (C) in MCF-7 xenografts models (D). (E-H) Hematological analysis of
healthy and SeDs treatment mice (72 h). **P < 0.01 vs control.
body weight (Fig. 2D). In order to further evaluate the potential
safety of 1a in vivo, we conduct hematological analysis of 1a as
previously described[19]. Aspartate aminotransferase (AST) and
alanine aminotransferase (ALT) are important indicators of liver
function. blood ureannitrogen (BUN) and lactate dehydrogenase
(LDH) are important indicators of kidney and heart function,
respectively. The results of hematological analysis revealed that
the mice treated with 2.5-5 mg/kg dose of 1a showed no
significant increased levels of AST, ALT, BUN, and LDH than
the control group (Figure 2E-2H) as well as other evalution
paramaters (Figure S18). Therefore, these results demonstrate
that 2.5-5 mg/kg dose of 1a might exerted slightly in vivo
toxicity.Together, these data indicated that organoselenium 1a
had a potent therapeutic effect to suppress tumor growth in vivo,
which is not attributed by its severe toxicity
Secondly, the effects of 1a on HUVECs invasion were
examined by transwell assay. As shown in Fig.3C and
3D,notable invasived cells in the control groups were observed
in the lower side of transwell chamber after 24 treatments.
VEGF treatment increased the invasived cells to the bottom of
the chamber after 24
h treatment. However, 1a dose-
dependently restrained the cell invasion in a marginally manner,
as evidenced by the decreased cell number. Similarly
suppression effects were also observed in the co-treatment of
1a (10 μM) and VEGF (50 ng/mL). The two models both
demonstrated the potent inhibitory effect of 1a on VEGF-induced
HUVECs motility.
Two-dimensioned (2D) Matrigel assay was further used to
detect the antiangiogenic effect of 1a on HUVECs. As shown in
Figure 3E and 3F, tubular structure was formed in the basal
treatments and VEGF (50 ng/mL) stimulation. However, the
structure was significantly destroyed after 1a incubation for 8 h.
Organoselenium 1a inhibits VEGF-induced cell migration,
invasion and capillary structure formation of HUVECs in
vitro.
Endothelial cell migration, invasion and tubular structure
formation are all necessary steps for angiogenesis, tumor
growth and tumor metastasis. Therefore, three well-established
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neovascularization in CAM assay (Figure S19). These results
clearly indicated that 1a can inhibit VEGF-induced angiogenesis
in vivo.
Organoselenium 1a induces apoptosis more effectively in
HUVECs than that in MCF-7 cells.
Tumor growth depends on angiogenesis and the formation
of new vessels requires angiogenic stimulation derived from
tumor cells paracrine. However, whether the tumor suppression
ccurs through affecting the endothelial cells function or cancer
cells, or both was not ascertainable. To further characterize the
molecular mechanism of growth inhibition in MCF-7 xenograft
tumor induced by 1a. We first examined the suppression effects
of 1a in HUVEC and MCF-7 cells. As illustrated in Figure 5A
and 5B, 1a inhibited HUVEC and MCF-7 cells proliferation in a
dose-dependent manner. However, HUVECs were more
susceptible to 1a than MCF-7 cells, as indicated by the different
cell viability suppression effects under the same concentration.
We next characterized the antiproliferative molecular
mechanism by using PI flow cytometry assay. As shown in
Figure 5C, exposure of cells to 1a resulted in a remarkable
concentration-dependent elevation in the proportion of apoptotic
cells in HUVECs, as can be seen in the increasing sub-G1
Figure 4. 1a restrained VEGF-induced new blood vessel formation in vivo. (A).
Representive matrigel macroscopic appearance, HE staining and IHC of
Matrigel plugs. Hemoglobin content (B) and VEGF expression (C) were
employed to confirm the antiangiogenesis effect of 1a in vivo. All data here are
expressed as means±SD of triplicates. Bars with different characters (a-d) are
statistically different at P<0.05 level.
A
B
MCF-7
HUVEC
100
75
50
25
0
100
75
50
25
0
*
Obvious antiangiogenic effects of 1a on HUVECs in vitro was
also observed in the co-incubation of VEGF (50 ng/mL) and 1a
(10 μM). These results indicate that 1a displayed strongly
antiangiogenic activities in vitro. Additionally, we found that 1a
and 3a slightly inhibited the survival of endothelial cells during
the treatments, which suggested that 1a and 3a inhibited VEGF-
induced angiogenesis under non-toxic or subtoxic doses in vitro.
(Figure S17).
**
**
**
**
0
2.5
5
10
0
2.5
5
10
Concentration (μM)
Concentration (μM)
1a (μM)
C
Control
2.5
5
10
Sub-G1: 21.7 %
G0/G1: 43.6 %
S: 52.9 %
Sub-G1: 3.2 %
G0/G1: 45.3 %
S: 48.1 %
Sub-G1: 59.4 %
G0/G1: 46.2 %
S: 50.4 %
Sub-G1: 89.0 %
Organoselenium 1a inhibits VEGF-induced angiogenesis
G2/M: 3.5 %
G2/M: 6.6 %
G2/M: 3.4 %
in vivo.
Sub-G1: 84.5 %
Sub-G1: 2.4 %
G0/G1: 40.9 %
S: 45.0 %
Sub-G1: 10.5 %
G0/G1: 48.7 %
S: 50.1 %
Sub-G1: 48.2 %
To further evaluate the potential suppression effects of 1a
on angiogenesis in vivo, Matrigel plug assay was employed by
using a C57BL/6 mice model. After 21-day administration,
notable in vivo antiangiogenic effect of 1a was observed. As
shown in Figure 4A (top), when compared with the control
groups, dark red matrigel plugs were obtained in VEGF (200
ng/mL) treatments, indicating the formation of a functional
vasculature filled with intact red blood cells (RBCs). Histologic
analysis of the Matrigel pellets using HE staining identified more
and thicker erythrocyte-containing vesselsin group treated with
VEGF alone than that of untreated group. In contrast, treatment
of 1a in combination with VEGF (200 ng/mL) potently inhibited
angiogenesis in vivo, especially that 10 μM of 1a treatment
strongly inhibited the neovascularization in Matrigel plugs as
evidenced by the pale color and a few infiltrating single cells
(Fig.4A, top and middle). Quantification of angiogenesis by
measuring hemoglobin content further confirmed the
antiangiogenesis activities of 1a (Figure 4B). Furthermore,
VEGF as one of the most important pro-angiogenic molecules
was also found to be significantly inhibited by 1a as indicated by
IHC assay (Figure 4C). Additionally, we found that 1a exhibited
G2/M: 14.1 %
G2/M: 1.2 %
DNA Content
HUVEC
2.5 10 (μM)
D
E
MCF-7
0
5
0
2.5 5 10 (μM)
PARP
1.0 0.9 0.7 0.1
0.7 0.8 1.0
PARP
C-PARP
0.7 0.8 0.9 1.0
1.0 0.9 0.9 0.8
0
Caspase-3
Caspase-3
Caspase-7
C-caspase-3
0.5 0.8 0.9 1.0
0
0
0.9 1.0
Caspase-8
Caspase-9
β-actin
1.0 0.6 0.8 0.3
1.0 0.9 0.8 0.2
1.0 0.9 0.3
0.1 0.6 1.0
1.0 0.8 0.1
0.8 1.0
0
C-caspase-7
0
Caspase-9
0
C-caspase-9
0
0
β-actin
Figure 5. 1a inhibited cell viability of HUVEC and MCF-7 cells by induction of
apoptosis. Cytotoxicity of 1a towards HUVECs (A) and MCF-7 cells (B). C. 1a-
induced apoptosis in HUVEC and MCF-7 cells. 1a-induced PARP cleavage
and caspases activation in HUVEC (D) and MCF-7 cells (E). The proteins
expression was examined by western blotting methods. All data were obtained
from three independent experiments. *P < 0.05 vs untreated control. **P <
0.01 vs untreated control.
strongly
suppression
effects
in
VEGF-induced
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peaks from 3.2 % in control group to 21.7 %, 59.4 % and 89 %
in 1a treatments. Similarly, 1a also induced concentration
dependent apoptosis effect in MCF-7 cells, however, the
apoptosis effect was less effective than HUVECs at the same
concentration, suggesting the more potent apoptosis-inducing
effects of 1a in HUVECs, and the anti-angiogenesis mediated by
1a on endothelial cells might be earlier than a direct cytotoxic
effect on tumour cells. To further explore the underlying
mechanism of apoptosis, fluorometric assay was conduct to
investigate the caspases activation, which has been postulated
to display essential role in apoptosis. Notably, 1a treatment
dramatically activated the caspase-3, caspase-9 and caspase-8
in HUVECs and MCF-7 cells (Fig. S20), indicating the activation
of cell death mediated and mitochondria mediated apoptosis
pathways. Moreover, activation of caspase-9, as thepredominant
initiator in the mitochondria mediated apoptotic pathway, is more
prominent than that of caspase-8 in HUVECs (Fig. S20A), which
implys the mainly role of mitochondria-mediated apoptosis
pathway induced by 1a. Additionally, we found that 1a triggered
caspases activation was more formidable in HUVEC cells than
in MCF-7cell, as can be seen in the different fluorescence
intensity at the same dosage treatments. The apoptosis effects
in HUVECs and MCF-7 cells induced by 1a were further
confirmed by western blotting assay (Figure 5D and 5E). For
instance, 1a treatment remarkably induced cleavage of PARP
and caspases-3/-7/-8/-9. Taken together, these results all
indicated that 1a inhibited cell viability in HUVECs and MCF
cells by induction of apoptosis and HUVECs were more
sensitive to 1a when compared to MCF-7cells.
the angiogenesis procession. Such as, RAF/MEK/ERK and
PI3K/AKT pathways are closely involved in endothelial cell
proliferation, cell survival and angiogenesis[1c]
activation of Raf/MEK/ERK and PI3K/Akt pathways often occurs
in human cancer due to mutation or aberrant expression[21]
.
Abnormal
.
Therefore, cancer therapies of targeting components of this
signal axis represent a promising tactic in recent years. To test
the possibility of whether 1a-induced inhibition of angiogenesis
was associated with the suppression of VEGF-VEGFR2-
ERK/AKT signalling axis, western blotting was employed to
examine the changes of proteins in this axis. As shown in
Figure 6A, treatment of HUVECs with 1a dose-dependently
decreased the protein expression of VEGF, VEGFR2, p-ERK
and p-AKT. To further evaluate the role of ERK and AKT, the
upstream inhibitor (U0126 and LY2294002) were used to
examine 1a-induced cell growth inhibition in HUVECs (Figure
6B). As illustrated in Figure 6B, enhancement of cell growth
inhibition was observed in the pretreatment of LY2294002 and
U0126, which indicated that 1a induced anti-proliferation activity
of HUVECs in ERK- and AKT-dependent manner. Taken
together, these results indicated that VEGF/VEGFR2/AKT/ERK
signaling pathway blockade was involved in 1a antiangiogenesis
activities.
Organoselenium 1a triggers DNA damage through ROS
overproduction.
Triggering DNA damage has been postulated to be one of
the most critical anticancer strategies in clinic. In response to
DNA damage, cells may directly repaired DNA breaks or
adducts as well as by halting cell cycle progression or triggering
apoptosis. DNA damage results in autoactivation of ATM and
ATR, which activates downstream substrates such as
checkpoint kinase Chk1/Chk2, and H2AX and multi-functional
transcription factor p53. P53 has been identified as a critical
regulator in cell cycle progression and apoptosis through
different mechanisms[22]. In our previous studies, SeDs are
capable of inducing DNA damage through triggering ROS
accumulation in vitro[12, 14a, 15b]. To examine whether 1a induced
DNA damage in HUVECs and MCF-7 cells, western blotting
assay was exploited to detect the protein expression of p53 (Ser
15) and histone (Ser 139). As can be seen in Figure 7A and
S21, there is an obvious elevation of p53 (Ser 15) and histone
(Ser 139) protein expression in 1a treatment, which indicated
that 1a induced DNA damage-mediate signaling pathway
contributed to cells apoptosis and antiangiogenesis.
Organoselenium 1a suppresses the VEGF-VEGFR2-
ERK/AKT signaling axis.
Angiogenesis is strictly regulated by various angiogenesis
stimulators and antiangiogenesis cytokines.VEGF, the most
accepted pro-angiogenic growth factor, palys a critical role in
regulating angiogenesis through binding to VEGF receptors
(VEGFR1, 2 and 3)[20], and VEGFR2 is considered to exhibit the
major role. After activation of VEGFR2, several downstream
protein kinase pathways can be activated and thus modulated
1a (μM )
A
B
a
0
2.5
5
10
a
a
a
100
75
50
25
0
VEGF
c
1.0 0.8 0.3
0
VEGFR2
AKT
e
1.0 0.8 0.4 0.1
b
d
f
1.0 1.0 1.0 0.9
1.0 1.1 0.4 0.1
p-AKT
ERK
ROS has been considered to play an essential role in
modulating apoptosis pathways[23]. Overproduction of ROS
result in intracellular oxidative products accumulation, such as
DNA strand breaks (DSBs). Increasing evidences demonstrate
that ROS generation is involved in seleno complexes induce cell
apoptosis[16, 24]. Therefore, DCFH-DA, a fluorescein-labeled
probe, was used to detect 1a-induced ROS accumulation. As
shown in Figure 7B, treatment of HUVECs with indicated
concentration of 1a resulted in conspicuousness ROS
accumulation in a time- and dose-dependent manner, which is
further confirmed by the increasing fluorescence in Figure S22.
Based on the importance of ROS, thiol-reducing antioxidants,
glutathione (GSH), was introduced to examine the role of
1a (μM ) - 2.5 5 2.5 5 2.5 5
-
-
+
-
U0126 (10 μM)
-
-
-
-
-
-
-
-
+
-
+
-
-
1.0 1.0 1.0 1.0
1.0 0.9 0.8 0.6
LY2294002 (10 μM)
+
+
+
p-ERK
β-actin
Figure 6. 1a suppresses VEGF–VEGFR2–ERK/AKT signaling axis. (A). 1a
inhibits the protein expression of VEGF, VEGFR2, ERK and AKT. (B). ERK
and AKT inhibitor enhances 1a-induced cell growth inhibition against HUVECs.
Cells were pre-treated with 10 μM LY2294002 (Akt-upstream inhibitor) or 10
μM U0126 (ERK inhibitor) for 1 h and co-treated with 1a for 72 h. Cell viability
was detected by MTT assay. All data here are expressed as means±SD of
triplicates. Bars with different characters are statistically different at P<0.05
level.
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the antibodies used in this study were purchased from Cell
Signaling Technology (Beverly, MA). KoAc, 5-bromo-2-(4-
methylphenyl) benzimidazole, NaH, DMF, (BOC)2O, PdCl2,
B
D
A
1a (μM)
0
175
150
125
100
2.5 M
5.0 M
10 M
0
2.5
5
10
Ser15-p53
p-histone
0
0.2
0.8
1.0
andbis(pinacolato)diboron,
1,4-dioxane,
Pd(PPh3)4
were
obtained from sigma. All of the solvents used were of high-
performance liquid chromatgraphy (HPLC) grade. SeDs were
well solute in dimethyl sulphoxide (DMSO).The stock solution of
SeDs were prepared in DMSO and kept at -20℃. SeDs were
diluted in culture medium to obtain the desired concentration as
needed.
0
0.4
0.9
1.0
β-actin
0
10 20 30 60 90 120
Time (min)
C
120
a
a
90
60
30
d
e
p-histone
b
0.1 1.0 0.2 0.5
1.0 0.1 0.9 0.7
c
p-ERK
Synthesis of selenadiazole derivatives
0
β-actin
Generally, the 1a-1e and 2a-2c were synthesized as
1a (μM)
-
-
5
10
-
5
+
10
+
-
GSH (5 mM)
-
+
previously described[16, 24-25]
.
Figure 7. 1a triggered DNA damage in HUVEC cells through ROS
accumulation. (A). 1a-induced DNA damage in HUVEC cells. (B). 1a induced
Synthesis of compound B
intracellular ROS generation in
a time- and dose-dependent manner in
3
mmol [1,1'-biphenyl]-3,3',4,4'-tetraamine (A) was
HUVECs. (C). ROS scavengers prevents 1a-induced cell growth inhibition. (D)
DNA damage and ERK activation of HUVECs. All data here are expressed as
means±SD of triplicates. All images shown here are representative of three
independent experiments with similar results. Bars with different characters (a-
e) are statistically different at P<0.05 level.
dissolved in 250 mL hydrochloric acid solution (HCl: H2O = 1:5)
in a 500 mL flask. Then 3 mmol SeO2 which was dissolved in 20
mL hot distilled water was added into the flask drop by drop
through a constant pressure funnel. The mixture was stirred for
2 h at r.t. After the 2 h, sodium hydroxide solution was used to
change the pH to about 7.0, filtered, got the products, 4-
intracellular ROS in 1a-induced apoptotic cell death. As shown
in Figure 7C, pretreatment with 5 mM GSH for 2 h effectively
protected HUVECs against 1a-induced growth inhibition. The
result indicated that 1a inhibited HUVECs growth was depended
on ROS generation. Furthermore, GSH addition significantly
attenuated 1a-induced DNA damage and ERK inactivation,
indicating that ROS acts as upstream mediator of DAN damage
and ERK pathway (Figure 7D). Taken together, these results
demonstrated that 1a inhibited angiogenesis-mediated human
breast tumor growth by induction of HUVECs apoptosis through
ROS-mediated DNA damage.
(benzo[c][1,2,5]selenadiazol-5-yl)benzene-1,2-diamine
Yield: 90%.
(B).
General procedure for the synthesis of 1a-1f
1 mmol B dissolved in 25 mL DMF in a 50 mL flask. 1 mmol
corresponding aromatic aldehyde was added into the flask with
a catalyst, p-Methylbenzene sulfonic acid. The mixture stirred for
40 min at 80°C. After the reaction completed, the mixture was
put into Na2CO3 (a.q.) stirring for 30 min, filtered to give the
crude products, which was chromatographed over silica gel
column (petroleum ether: ethyl acetate = 5:1) to afford pure
compound 1a-1f.
Conclusions
General procedure for the synthesis of 1g-1h
In this study, we have demonstrated the antiangiogenesis
activities of SeDs. 1a could effectively inhibit human breast
tumor growth in xenograft mice through suppressing tumor
angiogenesis by targeting VEGFR2 mediated AKT and ERK
signaling pathway and then reinforced the apoptosis effect
induced by 1a through triggering DNA damage in endothelial
cells. These results clearly demonstrate the antiangiogenic
potency of SeDs and the underlying molecular mechanisms.
0.5 mmol B, 0.5 mmol 4-aminobenzoic acid and 10 g poly
phosphoric acid (PPA) were added and mixed in DMSO (50 mL)
homogeneously. The mixture was heated thrice at 20 % for 90 s;
then the mixture was cooled down to room temperature, then
water was added, crude product was filtered off and dried at
room temperature. Solid product obtained by flash silicon
column chromatography. （ Petroleum ether ： ethyl acetate =
20:1）.
General procedure for the synthesis of 2a-2d
Experimental Section
1 mmol B dissolved in 25 mL DMF in a 50 mL flask. 2 mmol
corresponding aromatic aldehyde was add into the flask with a
catalyst, p-Methylbenzene sulfonic acid. The mixture stirred for 2
h at 80 °C. After the reaction completed, the mixture was put into
Na2CO3 (a.q.) stirring for 30 min, filtered to give the crude
products, which was chromatographed over silica gel column
(petroleum ether: ethyl acetate = 7:1) to afford pure compound
2a-2d.
Materials
VEGF was purchased from BD PharMingen (Bedford,MA).
ECGM and M199 medium were purchased from Life
Technologies, Invitrogen), Propidium iodide, DCF-DA, MTT,
BCA kit for protein determination were purchased from Sigma.
DMEM, FBS and penicillin-streptomycin were purchased from
Invitrogen (Carlsbad, CA). Caspases substrate (Caspase-3/8/9),
U0126 and LY2294002 were acquired from Calbiochem. All of
Characterization of 1a-1h
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ESI mass spectrometry was used to charecterized the final
products of these complexes. 1a Yield: 50 %; ESI-MS: m/z
391.1 [M+H+]+. 1b Yield: 45 %; ESI-MS: m/z 409.4 [M-H+]-. 1c:
Yield: 40 %; ESI-MS: m/z 377.2 [M+H+]+. 1d: Yield: 40 %; ESI-
MS: m/z 393.3 [M+H+]+. 1e: Yield: 47 %; ESI-MS: m/z 420.4 [M-
H+]-.
was concentrated under vacuo and the residue was purified by
alumina column chromatography with Ethyl acetate and
Petroleum ether as eluants to give the title compound as a light
yellow solid. Yield: 80 %. ESI-HRMS: m/z 327.12415
1
[C20H14N4O]+. HNMR (600 MHz, DMSO-d6, δ): 13.07 (s, 1H),
8.30 (s, 1H), 8.14 (d, J = 28.2 Hz, 5H), 7.72 (s, 2H), 7.39 (d, J =
7.2 Hz, 2H), 2.40 (s, 3H). 13CNMR (151 MHz, DMSO-d6, δ):
150.18, 148.79, 145.10, 140.48, 134.56, 130.07, 127.60, 127.06,
116.85, 21.48.
+
1f: Yield: 38 %; ESI-MS: m/z 419.8291 [M+H+] . Mp 331-
332 °C. Elemental analysis calcd (%) for C21H17N5Se: C, 60.29;
H, 4.10; N, 16.74; found (%): C, 60.22; H, 4.05; N, 16.51. IR
(KBr): ν 750 (Se-N-Se), ν 1365 (C-N) ν 1610, 1550, 803 (C=C
Cell culture
1
arom) cm-1. H-NMR: 400 MHz, DMSO-d6, δ, ppm) 12.78-12.74
Primary human umbilical vascular endothelial cells
(HUVEC) were a kind gift from Dr. Liu (Guangzhou Jinan
Biomedicine Research and Development Center, Guangdong
Provincial Key Laboratory of Bioengineering Medicine, Jinan
University, Guangzhou 510632, PR China). A375, A549, MCF-7,
Hela, HepG2 and HK-2 were purchased from the American Type
Culture Collection. HUVECs were cultivated in endothelial cell
growth medium (ECGM):M199 medium (Life Technologies,
Invitrogen) supplemented with 20% fetal bovine serum (FBS,
Gibco) at 37℃ in a humidified (5% CO2, 95% air) atmosphere.
HK-2 and several human cancer cells (MCF-7, A375, HepG2,
A549 and Hela) were maintained in DMEM medium
supplemented with 10 % FBS, 100 units/mL penicillin and 50
units/mL streptomycin at 37 ℃ in a humidified incubator with 5 %
CO2 atmosphere.
(d, 1H), 8.09-7.94(m, 5H), 7.94-7.85 (m, 1H), 7.71-7.57 (m, 2H),
6.87-6.85 (d, 2H), 3.01 (s, 6H) (Figure S5-S6).
1g: Yield: 43 %; TOF MS: m/z 392.0428 [M+H+]+. Mp 324-
325 °C. Elemental analysis calcd (%) for C19H13N5Se: C, 58.47;
H, 3.36; N, 17.94; found (%): C, 58.42; H, 3.35; N, 17.91. IR
(KBr): ν 3419 (N-H), ν 750 (Se-N-Se), ν 1365 (C-N) ν 1608,
1
1480, (C=C arom) cm-1. H-NMR: (400 MHz, DMSO-d6, δ, ppm)
δ 8.10 (s, 1H), 8.04 (m, 1H), 8.01 (d, 2H), 7.95-7.89 (t, 2H),
7.71-7.63 (m, 2H) , 6.72-6.69 (d, 2H). 13C NMR (400 MHz,
DMSO-d6, δ): 160.75, 159.18, 153.52, 151.48, 141.75, 132.49,
130.45, 128.25, 123.23, 121.81, 118.99, 113.81. (Figure S7-
S9).
1h: Yield: 35 %; TOF MS: m/z 434.0510 [M+H+]+. Mp 319-
320 °C. Elemental analysis calcd (%) for C19H13N5Se: C, 58.47;
H, 3.36; N, 17.94; found (%): C, 58.42; H, 3.35; N, 17.91. IR
(KBr): ν 3427 (N-H), ν 750 (Se-N-Se), ν 1355 (C-N) ν 1615,
Tumor xenograft study
MCF-7 cells (about 1×106) in 100 μL serum-free medium
were subcutaneously injected into the right oxter of male nude
mice. After the average tumor volume reached about 50-70 mm3
after 7 days, mice were randomly divided into 3 groups (8
mice/group) for control group, 2.5 mg/kg group and 5 mg/kg
group (in this present study, we have carried out preliminary
experiment to detect the LD50 (Lethal Dose, 50 %) of 1a before
the Tumor xenograft study. We found that the LD50 of 1a was
about 10 mg/kg, therefore, we choose 1/2 LD50 and 1/4 LD50
dosage of 1a for the cancer treatment model.). Drugs were
injected every other day, caudal vein, from the first day until the
sixteenth day (8 times). At the termination of the experiments,
tumors were harvested, photographed and weighed. Tumor
dimensions were measured with calipers and the volume was
calculated using the formula: volume = l×w2/2, with l being the
maximal length and w being the width. All animal experiments
were approved by the Animal Experimentation Ethics Committee.
1483, (C=C arom) cm-1. H-NMR: (400 MHz, DMSO-d6, δ, ppm)
1
10.23 (s, 1H), 8.17-8.11 (t, 3H), 8.05-7.99 (m, 2H), 7.95 – 7.92
(m, 1H), 7.79 – 7.70 (m, 4H), 2.10 (s, 3H). 13C NMR (400 MHz,
DMSO-d6, δ): 168.79, 160.51, 159.21, 141.08, 130.40, 129.73,
127.32, 126.71, 124.47, 123.23, 119.01, 118.84, 24.19 (Figure
S10-S12).
Characterization of 2a-2d
2a: Yield: 31 %; ESI-MS: m/z 467.3 [M+H+]+. 2b: Yield:
28 %; ESI-MS: m/z 535.4 [M+H+]+. 2c: Yield: 25 %; ESI-MS: m/z
495.3 [M+H+] +.
2d: Yield: 30 %; ESI-MS: m/z 495.2 [M-H+]-. Mp 268-267 °C.
Elemental analysis calcd (%) for C26H18N4O2Se: C, 62.78; H,
3.65; N, 11.26; found (%): C, 62.42; H, 2.61; N, 11.31. IR (KBr):
ν 748 (Se-N-Se), ν 2965 (C-H), ν 3920 (C-OH), ν 1370 (C-N) ν
1570, 1420, 814 (C=C arom) cm-1. 1H-NMR: (500 MHz, CDCl3, δ, Hematology Analysis
ppm) 8.11 (t, 2H), 8.02 (dd, 1H), 7.92 (d, 1H), 7.73 (dt, 2H), 7.54
(dd, 2H), 6.89 (dd, 4H) 6.79-6.59 (m, 2H) 5.56 (s, 1H), 5.47 (s,
1H) (Figure S13-S14).
The mice were intravenous administration with 1a at a dosage of
2.5 mg/kg and 5.0 mg/ kg of mouse body weight (n = 3, per
group) and then sacrificed at 72 h.Then the the blood sample
(72 h) was used for hematology analysis at Guangzhou
Overseas Chinese Hospital.
Synthesis of compound 3a
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(p-tolyl)-
1H-benzo[d]imidazole
(0.27
g,
0.81
mmol),
5-
bromobenzo[c][1,2,5]oxadiazole (0.185 g, 0.93 mmol ) and 25
mg Pd(PPh3)4 were dissolved in 1,4-dioxane (10 mL) under N2 at
Measurement of cytotoxicity
Cytotoxicity of organoselenium towards HUVECs, HK-2 and
human cancer cell lines was measured as previously
described[26]. Briefly, cells at a density of 2×103 per well were
rt.. After Na
2CO3 (0.26 g, 2.45 mmol) being added to the mixure
solution, the reaction was stirred for 16 h at 100 °C. The reaction
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allowed to grow for 24 h and then treated with different
concentrations of complexes for 72 h. After that cell viability was
measured by MTT assay. Cell viability in the presence of GSH
or NAC (with powerful reducing capacity) was measured by
trypan blue staining as previously reported[27]. The cell viability
was expressed as % of control (as 100 %). The control groups
were treated with 0.5 % DMSO.
This work was supported by National High-level personnel of
special support program (W02070191), YangFan Innovative &
Entepreneurial
Research
Team
Project
(201312H05),
Fundamental Research Funds for the Central Universities,
National Key Scientific Instrument and Equipment Development
Project. 2017YFF0104904 and Natural Science Foundation of
Guangdong Province (2017A030313091).
Cell cycle distribution assay
Flow cytometric assay was exploited to detect the cell cycle
distribution after 1a incubation for the indicated times[28]. Briefly,
1a treated cells were harvested and stained with PI after fixed
with 70 % ethanol at -20 ℃ overnight, and the cell cycle
distribution of the labeled cells were analyzed by Flow cytometer.
The proportion of sub-G1 peaks were used to represent as
apoptosis cells.
Keywords: Selenadiazole • Anticancer •VEGF • Apoptosis •
ROS
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Herein we demonstrate that the
introduction of –CH3 group into SeDs
(1a) potently inhibits MCF-7 tumor
growth in nude mice without severe
systematic cytotoxicity through
suppressing tumor angiogenesis.
Further mechanistic studies indicate
that 1a-induced antiangiogenesis
effect was achieved by abolishing
VEGF-VEGFR2-ERK/AKT signal axis
and enhanced the apoptosis effect
through triggering ROS-mediated
DNA damage.
Haoqiang Lai #,a, Xiaoyan Fu#,a,
Chengcheng Sang,a Liyuan Hou,a
Pengju Feng,a Xiaoling Li,*b and
Tianfeng Chen*a
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