Discovery and anticancer evaluation of a formononetin derivative against gastric cancer SGC7901 cells

Article abstract of DOI:10.1007/s10637-019-00767-7

Background Gastric cancer (GC) is the second most common cause of cancer-related death worldwide. Novel anticancer drugs against gastric cancer are urgently needed. Methods Compound 10 was designed and synthesized via a molecular hybridization strategy ba

Full text of DOI:10.1007/s10637-019-00767-7

Investigational New Drugs
https://doi.org/10.1007/s10637-019-00767-7
SHORT REPORT
Discovery and anticancer evaluation of a formononetin derivative
against gastric cancer SGC7901 cells
Jian-Ning Yao1 & Xue-Xiu Zhang1 & Yan-Zhen Zhang1 & Jia-Heng Li2 & Dong-Yao Zhao1 & Bing Gao1 & Hai-Ning Zhou1
Shi-Lin Gao1 & Lian-Feng Zhang1
&
Received: 4 January 2019 /Accepted: 21 March 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Summary
Background Gastric cancer (GC) is the second most common cause of cancer-related death worldwide. Novel anticancer drugs
against gastric cancer are urgently needed. Methods Compound 10 was designed and synthesized via a molecular hybridization
strategy based on the natural product formononetin. It was evaluated for their antiproliferative activity against three gastric cancer
cell lines (SGC7901, MKN45 and MGC803). Results Derivative 10 displayed potently antiproliferative activity with an IC50
value of 1.07 μM against SGC7901 cells. Derivative 10 could inhibit the growth and migration against gastric cancer SGC7901
cells through the Wnt/β-Catenin and AKT/mTOR pathways. From the in vivo expremints, it could effectively inhibited
SGC7901 xenograft tumor growth in vivo without significant loss of the body weight. Conclusion Derivative 10 is an novel
antitumor agent with potential for further clinical applications to treat gastric cancer.
.
.
.
.
Keywords Gastric cancer Formononetin SGC7901 Growth Migration
Introduction
anticancer, antiinflammatory and neuroprotective activies by
regulating the mitogen-activated protein kinase signal path-
way or estrogen receptor [4–6]. Formononetin could induce
cell cycle arrest by decreasing cyclin D1 mRNA and protein
expression in human breast cancer in vitro and in vivo [7].
Formononetin in combination with temozolomide displayed a
synergistic role on Glioma C6 cells through inducing tumor
cells apoptosis [8]. However, formononetin as a natural prod-
uct displayed weak or no activity (Fig. 1) with IC50 values
>30 μM against MGC803 cells, PC3 cells and MCF7 cells
[9–11]. In order to discovery more potent antitumor com-
pounds based on the natural formononetin, the structural mod-
ification of formononetin was very necessary.
Gastric cancer (GC) as the second leading cause of cancer-
related death worldwide is relatively high in eastern Asia [1].
In China, as the dominant types of cancer in the age group 60
to 74 years, gastric cancer is the leading cause of death and a
major digestive system public health problem, with 0.50 mil-
lion deaths and 0.68 million new cases in 2015 [2]. Although
targeted chemotherapy has increased long-term survival of
patients with gastric cancer, the clinical application has been
limited owing to the severe adverse effects [3]. Therefore,
novel drugs with high anticancer efficiency against gastric
cancer are urgently needed.
Formononetin isolated from the red clover displayed a lot
of pharmacological effects such as vasorelaxant, antioxidant,
Coumarins exhibited a wide range of pharmacological ac-
tivities in drug discovery [12–16]. The promising biological
profile and easy synthetic modification have stimulated the
design and development of coumarin-based derivatives as po-
tential antitumor agents [17]. Molecular hybridization is a
useful strategy in drug design and development based on the
combination of pharmacophoric moieties of different bioac-
tive units to produce a new hybrid with improved affinity and
efficacy, when compared to the parent drugs [18, 19]. From
these interesting findings, we introduced the coumarin unit to
design a potent anticancer molecule based on the natural
formononetin via a molecular hybridization strategy (Fig. 2).
Jian-Ning Yao and Xue-Xiu Zhang contributed equally to this work.
* Lian-Feng Zhang
lianfengzhangzzu@163.com
1
Department of Gastroenterology, the First Affiliated Hospital of
Zhengzhou University, Zhengzhou 450052, China
2
Reproductive Medicine Department, the Third Affiliated Hospital of
Zhengzhou University, Zhengzhou 450052, China

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Fig. 1 The main problem of formononetin as a natural and anticancer product
In this work, the synthesis and anticancer mechanisms of the
formononetin-coumarin hybrid were investigated.
76.32, 76.00, 75.68, 64.86, 30.95, 28.50. Found, m/z:
246.0873 [M + H]+. C12H12N3O3. Calculated: 246.0879.
1H and 13C NMR spectra of compound 10
Materials and methods
o
1
White solid, m.p.: 156~158 c, yield:32.89%. H NMR
(400 MHz, DMSO-d6) δ 8.43 (s, 1H), 8.36 (s, 1H), 8.01 (dd,
J = 21.3, 9.2 Hz, 2H), 7.62 (d, J = 8.6 Hz, 1H), 7.53 (d, J =
8.6 Hz, 2H), 7.35 (d, J = 2.1 Hz, 1H), 7.13 (dd, J = 8.9, 2.1 Hz,
1H), 6.99 (dd, J = 13.6, 5.4 Hz, 3H), 6.91 (dd, J = 8.6, 2.2 Hz,
1H), 6.29 (d, J = 9.5 Hz, 1H), 5.33 (s, 2H), 4.59 (t, J = 6.8 Hz,
2H), 4.11 (t, J = 5.9 Hz, 2H), 3.80 (s, 3H), 2.44–2.23 (m, 2H).
13C NMR (100 MHz, DMSO-d6) δ 175.09, 162.83, 161.92,
160.74, 159.49, 157.79, 155.81, 153.99, 144.77, 142.33,
130.55, 129.97, 127.47, 125.59, 124.51, 123.87, 118.27,
115.67, 114.10, 113.12, 113.06, 112.95, 101.72, 65.91,
62.37, 55.63, 47.14, 29.67. Found, m/z: 552.1779 [M + H]+.
C31H26N3O7. Calculated: 552.1771.
1H and 13C NMR spectra of compound 8
o
1
White solid, m.p.: 104~106 c, yield:50.66%. H NMR
(400 MHz, DMSO-d6) δ 7.99 (d, J = 9.5 Hz, 1H), 7.63 (d,
J = 8.6 Hz, 1H), 7.10–6.82 (m, 2H), 6.29 (d, J = 9.5 Hz,
1H), 4.19 (t, J = 6.0 Hz, 2H), 3.67 (t, J = 6.6 Hz, 2H), 2.28
(p, J = 6.3 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
161.95, 160.74, 155.82, 144.76, 130.01, 113.42–112.82 (m),
101.73, 66.56, 40.60, 40.39, 40.18, 39.97, 39.77, 39.56,
39.35, 32.06, 31.47. Found, m/z: 282.9978 [M + H]+.
C12H12BrO3. Calculated: 282.9970.
1H and 13C NMR spectra of compound 9
MTT assay
o
1
White solid, m.p.: 100~103 c, yield:41.34%. H NMR
(400 MHz, CDCl3) δ 7.57 (d, J = 9.5 Hz, 1H), 7.31 (d, J =
8.3 Hz, 1H), 6.86–6.67 (m, 2H), 6.19 (d, J = 9.5 Hz, 1H),
4.21–4.04 (m, 2H), 3.51 (dt, J = 27.8, 6.4 Hz, 2H), 2.29 (p,
J = 6.1 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 160.84,
160.11, 154.85, 142.32, 127.80, 112.29, 111.73, 100.55,
Three gastric cancer cell lines (SGC7901, MKN45 and
MGC803) were bought from the first affiliated hospital
of zhengzhou university and shanghai institute of bio-
chemistry and cell biology. 1000 Cells were seeded in
96-well plates per well. For each well, 20 μl 3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
Fig. 2 Molecular hybridization
strategy to design the targeted
compound

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Scheme 1 Synthesis of the formononetin-coumarin hybrid 10. Reagents and conditions: a propargyl bromide, NaOH, acetone, reflux; b 1,3-
.
dibromopropane, K2CO3, CH2Cl2, reflux; c NaN3, CH3CN, reflux; d intermediate 7, CuSO4 5H2O, sodium ascorbate, DMSO:H2O (1:1), r.t
(MTT, 5 mg/ml) was added and plates were incubated at
37 °C for 1 h. After removing the supernatant, 100 μl
DMSO per well was added [20].
throughput screening, and the assay was performed in the 96-
well microplate by reported references [22, 23].
Western blot analysis
Wound healing
SGC7901 cells were lysed in a RIPA lysis buffer and centri-
fuged at 15000 rpm for 30 min. The protein samples were
electrophoresed on SDS-PAGE and transferred to PVDF
membrane. Then, PVDF membrane was incubated with cor-
responding primary and secondary antibodies. The proteins
were visualized using chemiluminescence (ECL) detection
reagents [24].
SGC7901 cells were seeded in 6-well plates and the
formononetin-coumarin hybrid 10 was added for 48 h.
Wounds were created using a pipette tip and medium was added
for 24 h. Each experiment was repeated at least three times [21].
SIRT 1 inhibition assay in vitro
The peptide substrate is incubated with human recombinant
SIRT1 along with its cofactor NAD+. Fluorophore was de-
tected using an excitation wavelength of 360 nm and an emis-
sion wavelength of 460 nm. The reactions were suited to high-
Animals study in vivo
Animals experiments were processed according to protocols
and guidelines established by the ethics committee of
Fig. 3 Gastric cancer cells
(SGC7901, MKN45 and
MGC803) were treated with the
hybrid 10 for 48 h

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zhengzhou university. Mice were subcutaneously implanted
with SGC7901 cells (1 X 107 cells per mouse) on the right
flank of nude mice. Once tumor volumes reached to 100 mm3,
the mice were randomly divided into two groups (n = 5 mice
for each group). The treatment group received intragastric
administration of formononetin-coumarin hybrid 10 per day
for a period of 21 days [25].
lines (SGC7901, MKN45 and MGC803) were selected to test
the cell viability and the formononetin-coumarin hybrid 10
(0.1 μM, 0.4 μM, 0.8 μM, 1 μM, 2.5 μM and 3 μM) was
added for 48 h. As shown in Fig. 3, the formononetin-
coumarin hybrid 10 could inhibit gastric cancer cells growth
in a concentration-dependent manner. Especially, the
formononetin-coumarin hybrid 10 displayed potently antipro-
liferative activity with an IC50 value of 1.07 μM against
SGC7901 cells. However, formononetin displayed the weak
activity against three cell lines (SGC7901, MKN45 and
MGC803) with IC50 values >20 μM. This result suggests that
coumarin moiety may play a synergistic role for inhibitory
activity against cancer cells.
Results
Synthesis of analogue 10
The novel formononetin hybrid 10 was synthesized as illus-
trated in Scheme 1. Natural formononetin was coupled with
propargyl bromide in acetone in the presence of sodium hy-
droxide to obtain formononetin intermediate 7. In addition,
coumarin intermediate 9 was obtained via the nucleophilic
substitution reaction of commercially available coumarin with
1,3-dibromopropane. The target formononetin-coumarin hy-
brid 10 was synthesized from the alkyne intermediate 7 and
azide intermediate 9 using a click reaction in the presence of
copper(II) sulfate pentahydrate.
Analogue 10 inhibited SIRT1 expression
against SGC7901 cells
Sirtuin 1 (SIRT1) as a member of SIRT family is up-regulated
in several types of tumors [27]. As a nicotinamide adenine
dinucleotide-(NAD+-) dependent histone deacetylase, SIRT1
functions as a master regulator of ageing, apoptosis, and stress
response [28]. Recently, SIRT1 has been an important target to
discovery SIRT1 inhibitors and anticancer agents [29].
To explore the SIRT1 enzyme inhibitory activity of hybrid
10, the reported SIRT1 inhibition assay in vitro was used [30].
As shown in Fig. 4, formononetin-coumarin hybrid 10 displayed
a high degree of inhibitory activity toward SIRT1 with an IC50
of 2.52 μM. The tumor suppressor p53 was identified as the
critical target of the SIRT1 inhibitor–induced cell death in cancer
cells [31]. From the western blot results, the expression of
Analogue 10 inhibited gastric cancer cells growth
Gastric cancer (GC) has been the fourth most common cancer
and the second leading cause of cancer-related death in the
world [26]. It is very necessary to discovery anticancer drugs
to treat gastric cancer. In this work, three gastric cancer cell
a
c
b
d
Fig. 4 a In vitro SIRT1 inhibition assay; b The expression of SIRT1 and p53 treated with hybrid 10 (control, 0.5 μM, 1 μM, and 2 μM) in SGC7901
cells; c Fold of control for SIRT1 protein; d: Fold of control for P53 protein. * P < 0.05 was considered significant

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SIRT1 protein was decreased and total p53 expression level was
upregulated after treated with formononetin-coumarin hybrid
10. All these expreiments illustrated that formononetin-
coumarin hybrid 10 might be a novel SIRT1 inhibitor.
suggests that hybrid 10 could inhibit the migration process
in SGC7901 tumor cells.
Analogue 10 regulated the Wnt/β-catenin pathway
in SGC7901 cells
Analogue 10 inhibited SGC7901 cells migration
Wnt/β-Catenin pathway displayed an important role in various
cellular process including cell proliferation and migration [33].
In Wnt/β-Catenin signaling pathway, intracellular levels of β-
catenin are regulated by a multiprotein complex encompassing
kinases such as glycogen synthase kinase-3β (GSK-3β). In
the nucleus, β-catenin binds to members of the TCF family
of transcription factors, such as TCF-4 protein [34].
Based on the migration results and western bolt analysis
above, we also explore whether formononetin-coumarin hybrid
10 could regulate Wnt/β-Catenin pathway in SGC7901 cells.
Western blot assay was performed to study the expression
levels of β-catenin, Wnt5α, phospho β-catenin, GSK-3β,
SGC7901 cell migration was evaluated by wound healing and
western blot experiments. Microphotographs showed that un-
treated gastric cancer SGC7901 cells filled most of the
wounded area for 48 h after scratching the cell monolayer,
whereas treatment with hybrid 10 (1 μM) markedly sup-
pressed repairment of the wound (Fig. 5). In addition, we also
examined the protein expression of migration-related makers
such as E-cadherenin and N-cadherenin [32]. Based on the
results from Fig. 5, the expression of E-cadherin was upregu-
lated and the expression of N-cadherence was downregulated
by formononetin-coumarin hybrid 10. All these result
a
b
c
d
Fig. 5 a Effect of hybrid 10 on SGC7901 cells migration; b The expression of migration related markers in SGC7901 cells; c Fold of control for E-
cadherin protein; d Fold of control for N-cadherin protein

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a
b
c
d
e
f
Fig. 6 Derivative 10 regulated Wnt/β-Catenin pathway in SGC7901 cells. **: p < 0.01 verse control. ****: p < 0.0001 verse control
and TCF4 in HepG2 cells. As shown in Fig. 6, the expression
levels of β-catenin, Wnt5α, phospho β-catenin, and TCF-4
were all decreased and the expression level of GSK-3β was
increased in treated SGC7901 cells. All these results indicated
that hybrid 10 inhibited SGC7901 cells growth and migration
via regulating the Wnt/β-Catenin pathway.
[36]. In addition, mammalian target of rapamycin (mTOR)
as an atypical serine/threonine protein kinase played an impor-
tant role in the regulation of cellular growth and motility [37].
From Fig. 7, formononetin-coumarin hybrid 10 could
downregulate the phosphorylation levels of AKT and mTOR
in SGC7901 cells in a concentration-dependent manner. The
tests on AKT/mTOR signaling pathway illustrated that
formononetin-coumarin hybrid 10 might also regulate AKT/
mTOR signaling pathway to inhibit growth and migration of
SGC7901 cells.
Analogue 10 regulated the AKT/mTOR pathway
in SGC7901 cells
The activation of AKT/mTOR pathway resulted in hyperac-
tive signaling cascades related to cellular growth, proliferation,
and migration [35]. In AKT/mTOR pathway, protein kinase B
(also known as AKT) activation initiated a signal transduction
cascade that promoted the cellular growth and proliferation
Analogue 10 inhibited SGC7901 cells growth in vivo
To evaluate the antitumor effects of formononetin-coumarin
hybrid 10 in vivo, a SGC7901 xenograft model was
a
b
c
Fig. 7 Derivative 10 regulated AKT/mTOR pathway in SGC7901 cells. *: p < 0.05 verse control, **: p < 0.01 verse control

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Fig. 8 a Average tumor volume
(mm3); b Tumor weight; c weight
of mice. **P < 0.01, significantly
different compared with the
control by test
established in nude mice by subcutaneously injecting
SGC7901 cells. Mice were then randomly assigned to two
groups (control and 100 mg/kg 10) with 5 mice per group.
The results in Fig. 8 showed that formononetin-coumarin
hybrid 10 (100 mg/kg) caused a considerable suppression
of tumor growth. The average tumor weights of control
and formononetin-coumarin hybrid 10 groups were 1.45
0.26 g and 0.41 0.21 g (inhibitory rate: 71.72%), re-
spectively. Importantly, the in vivo antitumor efficacy of
formononetin-coumarin hybrid 10 was achieved without
causing any obvious loss of body weight. These results
suggested that formononetin-coumarin hybrid 10 has a
low toxicity toward mice.
Discussion
We designed and synthesized a novel formononetin-coumarin
hybrid by the molecular hybridization strategy based on the
natural product formononetin. From this rational modifica-
tion, formononetin-coumarin hybrid 10 displayed the more
potent antiproliferative activity than formononetin against

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Inflamm 2018:3048532–3048532. https://doi.org/10.1155/2018/
3048532
MGC803 cells. Hybrid 10 was an novel SIRT1 inhibitor with
an IC50 value of 2.52 μM by decreasing the expression of
Sirt1 and increasing the expression of P53. In addition,
formononetin-coumarin hybrid 10 could inhibit the migration
against SGC7901 tumor cells via Wnt/β-Catenin and AKT/
mTOR pathways. Importantly, formononetin-coumarin hy-
brid 10 potently inhibited tumor growth in vivo with a low
toxicity toward mice.
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Author contributions Jian-Ning Yao, Xue-Xiu Zhang and Lian-Feng
Zhang designed the research. Jian-Ning Yao, Xue-Xiu Zhang, Yan-
Zhen Zhang, Jia-Heng Li, Dong-Yao Zhao, Bing Gao, Hai-Ning Zhou,
Shi-Lin Gao, and Lian-Feng Zhang performed all the experiments. All
authors read and approved the final manuscript.
Funding This work was supported by the fund of The First Affiliated
Hospital of Zhengzhou University.
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Compliance with ethical standards
Conflict of interest All the authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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