Synthesis and evaluation of the anticancer activity of bischalcone analogs in human lung carcinoma (A549) cell line

Article abstract of DOI:10.1016/j.ejphar.2020.173396

Bischalcone has gained much attention because of its wide range of application in pharmaceutical chemistry. This work aims to evaluate the antiproliferation effects and explore the anticancer mechanism of bischalcone analogs on human lung cancer A549 cell

Full text of DOI:10.1016/j.ejphar.2020.173396

European Journal of Pharmacology 888 (2020) 173396
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Full length article
Synthesis and evaluation of the anticancer activity of bischalcone analogs
in human lung carcinoma (A549) cell line
Jie Yang**, Wen-Wen Mu, Guo-Yun Liu*
School of Pharmacy, Liaocheng University, 1 Hunan Street, Liaocheng, Shandong, 252000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bischalcone has gained much attention because of its wide range of application in pharmaceutical chemistry.
This work aims to evaluate the antiproliferation effects and explore the anticancer mechanism of bischalcone
analogs on human lung cancer A549 cells. In this study, we synthesized a series of bischalcone analogs via Aldol
condensation reaction; MTT method was used to evaluate the antiproliferation effects; the 2′,7′-dichloro-
fluorescein fluorescence assay was used to determine the intracellular reactive oxygen species levels; the
glutathione reductase-DTNB recycling assay was used to detect the redox imbalance; determination of thio-
barbituric acid-reactive substance was used to evaluate the lipid peroxidation; Rhodamine 123 was used to test
the mitochondrial membrane potential (MMP); the FITC/PI kit was used to detect the apoptosis; Western blotting
was used to detect the expression of Bax and Caspase 3. After treatment with curcumin and bischalcone analogs,
compounds 1d and 1g, the more stabilities compounds than curcumin, exhibited much higher potency in A549
cells than curcumin and other bischalcone analogs. Further mechanism of action studies revealed that 1d and 1g
exhibited more stronger reactive oxygen species production abilities than curcumin and accompanied by the
redox imbalance, lipid peroxidation, the loss of MMP, the activition of Bax and Caspase 3, and ultimately resulted
in apoptosis of A549 cell. These data suggest that enhancing the reactive oxygen species generation ability of
bischalcone analogs may be a promising strategy for the treatment of human lung cancer.
Bischalcone
Curcumin analogs
Reactive oxygen species
Apoptosis
Human lung carcinoma (A549) cell line
Bax
1. Introduction
A1nchez+JC&cauthor_id=30068667Santos et al., 2017). One of the
simplest natural compounds is chalcone, a curcumin analog and it is a
Lung cancer is one of the most devastating cancers with high
morbidity and mortality in the worldwide (Malvezzi et al., 2019). The
mortality rate for lung cancer will increase in the world, especically in
women by 40% in 2030 (https://pubmed.ncbi.nlm.nih.gov/?term=Mar
t%C3%ADn-S%C3%A1nchez+JC&cauthor
bioactive component of many edible plants (https://pubmed.ncbi.nlm.
nih.gov/?term=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor
_id=30068667Wang et al., 2019). Chalcone, bearing an α,β-unsaturated
carbonyl moiety, has been shown to be a potential chemopreventive and
anticancer agents (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%
C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Wang et al.,
2019). Chalcone could enhance intracellular reactive oxygen species
levels in human liver hepatocellular carcinoma (HepG2) cells. The bis-
chalcone, containing two chalcone units in a single structure, also
exhibts spectral biological activity including anti-inflammatory and
anticancer activity (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%
C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Reddy et al.,
´
_id=30068667Martín-Sanchez et al., 2018). The common strategies,
containing surgery, chemotherapy, radiotherapy and targeted therapy,
are used to treat the lung cancer. While the side-effects of gastrointes-
tinal response and renal dysfunction have been reported to be associated
with chemotherapy (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%
C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Liang et al.,
2019). Therefore, it is essential to search for new anticncer agents to
treat lung cancer.
2012). Similar compounds featuring two α,β-unsaturated carbonyl units
Natural products and their derivatives have contributed significantly
to drug discovery, especially in the development of anticancer drugs
(https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%C3%
have been described in the literature, while information of their anti-
cancer activity and mechanism against lung cancer is relatively
inadequate.
* Corresponding author.
** Corresponding author.
E-mail address: guoyunliu@126.com (J. Yang).
https://doi.org/10.1016/j.ejphar.2020.173396
Received 7 June 2020; Received in revised form 17 July 2020; Accepted 22 July 2020
Available online 14 August 2020
0014-2999/© 2020 Elsevier B.V. All rights reserved.

J. Yang et al.
European Journal of Pharmacology 888 (2020) 173396
In this study, a series of bischalcone analogs are synthesized ac-
MHz, (CD3)2CO), δ 8.12 (d, J = 9.0 Hz, 4H), 7.97 (d, J = 15.9 Hz, 2H),
7.92 (s, 4H), 7.77 (d, J = 15.9 Hz, 2H), 6.99 (d, J = 9.0 Hz, 4H); 13C NMR
(75 MHz, CDCl3), δ 187.0, 162.2, 157.8, 141.8, 136.6, 131.2, 129.1,
123.0, 115.4.
cording to the Aldol condensation protocol (Scheme 1). Thus we focus
on the cytotoxicity and explore the anticancer mechanism against
human lung cancer A549 cells.
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(2-methoxyphenyl)prop-2-en-1-
one (1d). Yield: 42.5%, light yellow solid; mp 150-152 ◦C, 1H NMR (400
MHz, CDCl3), δ 7.65-7.60 (m, 8H), 7.51-7.47 (m, 2H), 7.45 (d, J = 16 Hz,
2H), 7.07 (t, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0, 2.4 Hz, 2H), 3.92 (s, 6H);
13C NMR (100 MHz, CDCl3), δ 192.6, 158.2, 141.9, 136.9, 133.1, 130.4,
129.1, 128.8, 127.8, 120.8, 111.6, 55.8, 55.7.
2. Materials and methods
2.1. Materials
Roswell Park Memorial Institute (RPMI)-1640 was from Hyclone. 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
rhodamine 123, 2´,7´-dichlorofluorescein diacetate, the reduced (GSH)
and oxidized (GSSG) glutathione, 2-vinylpyridine (97%) and thio-
barbituric acid were obtained from Beyotime. Radio Immunoprecipita-
tion Assay (RIPA) buffer, 50*Cooktail proteinase inhibitor, GAPDH, the
primary antibodies and HRP-labeled secondary antibodies were pur-
chased from Servicebio. Annexin V-FITC/PI apoptosis detection kit was
purchased from BD Biosciences. Substituted acetophenone, 1,4-phtha-
laldehyde and curcumin were from EnergyChemical. All other chem-
icals were of the highest quality available.
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(3-methoxyphenyl)prop-2-en-1-
one (1e). Yield: 50.6%, yellow powder; mp 180-182 ◦C, 1H NMR (400
MHz, CDCl3), δ 7.84 (d, J = 16 Hz, 2H), 7.70 (s, 4H), 7.63-7.55 (m, 6H),
7.45 (t, J = 8.0 Hz, 2H), 7.17 (dd, J = 8.0, 2.4 Hz, 2H), 3.90 (s, 6H); 13
C
NMR (100 MHz, CDCl3), δ 189.9, 159.9, 143.5, 139.4, 136.9, 129.6,
129.0, 123.1, 121.1, 119.4, 112.9, 55.5.
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(4-methoxyphenyl)prop-2-en-1-
one (1f). Yield: 55.8%, yellow powder; mp 245-248 ◦C; 1H NMR (300
MHz, (CDCl3), δ 8.05 (d, J = 8.4 Hz, 4H), 7.80 (d, J = 15.9 Hz, 2H), 7.68
(s, 4H), 7.59 (d, J = 15.9 Hz, 2H), 7.00 (d, J = 8.4 Hz, 4H), 3.90 (s, 6H);
13C NMR (75 MHz, CDCl3), δ 188.4, 163.5, 142.7, 136.9, 130.9, 130.8,
128.8, 122.8, 114.0, 113.9, 55.5.
2.2. Synthesis of the curcumin analogs
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(2-fluorinephenyl)prop-2-en-1-
one (1g).
2.2.1. General procedure for the synthesis of 1a-1i
Yield: 56.2%, yellow powder; mp 135-138 ◦C; 1H NMR (500 MHz,
(CDCl3), δ 7.82 (t, J = 8.0 Hz, 2H), 7.73 (d, J = 16.0 Hz, 2H), 7.67 (s,
4H), 7.53-7.56 (m, 2H), 7.44 (d, J = 16.0 Hz, 2H); 7.26 (d, J = 8.0 Hz,
2H); 7.17 (t, J = 8.0 Hz, 2H).
The bischalcone analogs were synthesized according to the published
procedure (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S
%C3%A1nchez+JC&cauthor_id=30068667Winter et al., 2014). Briefly,
aqueous NaOH (60%, w/v, 8 ml) was added dropwise to a vigorously
stirred solution of 1,4-phthalaldehyde (5 mmol) and relevant aceto-
phenone (10 mmol) in ethanol (20 ml) at 0 ◦C. After 24 h stirring at room
temperature, distilled water and 10% hydrochloric acid solution were
added for total precipitation of the compounds. Then the crude products
were purified with a silica gel column. Their structures were confirmed
by 1H and 13C NMR spectroscopy.
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(3-fluorinephenyl)prop-2-en-1-
one (1h).
Yield: 61.5%, yellow powder; mp 141-144 ◦C; 1H NMR (500 MHz,
(CDCl3), δ 8.07-8.10 (m, 4H), 7.81 (d, J = 16.0 Hz, 2H), 7.71 (s, 4H),
7.55 (d, J = 16.0 Hz, 2H); 7.18 (d, J = 8.0 Hz, 4H).
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(4-fluorinephenyl)prop-2-en-1-
one (1i).
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(2-hydroxylphenyl)prop-2-en-1-
one (1a). Yield: 44.2%; Yellow solid; m.p.: 228-231 ◦C, 1H NMR (400
MHz, d6-DMSO), δ 12.48 (s, 2H), 8.30 (dd, J = 8.0, 1.6 Hz, 2H), 8.17 (d,
J = 16 Hz, 2H), 8.03 (d, J = 8.0 Hz, 4H), 7.90 (d, J = 16 Hz, 2H), 7.61-
7.57 (m, 2H), 7.05-7.03 (m, 4H); 13C NMR (100 MHz, CDCl3), δ 193.9,
162.2, 144.0, 137.1, 136.8, 131.4, 130.1, 123.6, 121.4, 119.6, 118.2.
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(3-hydroxylphenyl)prop-2-en-1-
one (1b). Yield: 51.6%, light yellow solid; m.p.: 244-246 ◦C, 1H NMR
(400 MHz, d6-DMSO), δ 9.83 (s, 2H), 7.98-7.94 (m, 6H), 7.77 (d, J = 16
Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H), 7.48 (s, 2H), 7.41 (t, J = 8.0 Hz, 2H),
7.09 (dd, J = 8.0, 2.4 Hz, 2H); 13C NMR (100 MHz, CDCl3), δ 189.5,
158.2, 143.3, 139.4, 137.1, 130.3, 129.8, 123.6, 120.8, 120.1, 115.1.
(2E, 2′E)-3,3′-(1,4-phenylene)bis(1-(4-hydroxylphenyl)prop-2-en-1-
one (1c). Yield: 55.8%, yellow powder; mp 219-222 ◦C; 1H NMR (300
Yield: 63.8%, yellow powder; mp 235-237 ◦C; 1H NMR (500 MHz,
(CDCl3), δ 8.06 (dd, J = 8.0 Hz, 4H), 7.81 (d, J = 16.0 Hz, 2H), 7.70 (s,
4H), 7.55 (d, J = 16.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 4H).
2.3. MTT assay
Human lung carcinoma cells (A549) were from the Shanghai Insti-
tute of Biochemistry and Cell Biology, Chinese Academy of Sciences.
Cells were cultured with RPMI-1640 medium at 37 ◦C in a humidified
atmosphere with 5% CO2. A549 cells were seeded in 96-well plates at a
density of 3 × 103/well and incubated for 24 h. Then the cells were
treated for another 48 h with compounds at the selected concentration.
The next steps were processed according to the reported reference (https
Scheme 1. Molecular structures and synthetics routs of bischalcone analogs.
2

J. Yang et al.
European Journal of Pharmacology 888 (2020) 173396
://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%C3%A1nchez
+JC&cauthor_id=30068667Liu et al., 2016).
of the proteins with a Bicinchoninic Acid (BCA) protein assay kit. After
separation in the gel and transfer, the polyvinylidene fluoride (PVDF)
membranes were blocked and incubated with the primary antibodies
before incubation with the corresponding Horseradish Peroxidase
(HRP)-labeled secondary antibody. Subsequently, the blots were washed
and visualized by the enhanced chemiluminescence (ECL) detection.
2.4. Stability assay
Stability of curcumin and bischalcone analogs was monitored as
described previously (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%
C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Liu et al., 2016).
Briefly, curcumin or bischalcone analogs (1d and 1g) was dissolved in
RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf
serum, then their maximun absorbance were monitored by using a
micro-plate reader for 120 min at 10-min intervals.
2.11. Statistical analysis
The data are expressed as the mean ± S.D. of at least three inde-
pendent experiments.
Data were analyzed by use of SPSS software 17.0 (SPSS, Inc., Chi-
cago, IL, USA). The difference between two groups was analyzed by one-
way analysis of variance (ANOVA) followed by Dunnett test (compare
all drug treatment groups vs. control). The values were considered sig-
nificant at P < 0.05. *: P < 0.05; **: P < 0.01; ***: P < 0.001 compared
with control.
2.5. Cell apoptosis analysis
The apoptosis induction activity of curcumin and 1d were detected
as previously described (https://pubmed.ncbi.nlm.nih.gov/?term=Mart
%C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Liu et al.,
2016). 3 × 105 A549 cells were treated with the tested compounds in
6-well plates for 24 h. After that, the cells were collected and determined
by FITC/PI kit using a flow cytometry.
3. Results
3.1. Synthesis
2.6. Intracellular reactive oxygen species measurement
Compounds proposed in this work were synthesized according to
previously described procedure (https://pubmed.ncbi.nlm.nih.gov/?
term=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor
The 2′,7′-dichlorofluorescein fluorescence assay was used to deter-
mine the intracellular reactive oxygen species levels as described pre-
viously (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%
C3%A1nchez+JC&cauthor_id=30068667Liu et al., 2018). 3 × 105
A549 cells were treated with the tested compounds in 6-well plates for 6
_id=30068667Winter et al., 2014) (Scheme 1). 1a-1i were synthesized
via Aldol condensation from 1,4-phthalaldehyde and relevant aceto-
phenon at moderate yields after purification by silica gel column chro-
matography. All compounds were characterized by 1H NMR and 13C
NMR.
h. Next, the harvested cells were incubated with 3 μM DCFH-DA for 30
min at 37 ◦C and analyzed immediately by flow cytometry.
3.2. Antiproliferative activity of bischalcone analogs in vitro
2.7. Measurement of GSH and GSSG levels
It has been reported that bischalcone showed a spectral anti-cancer
A549 (3 × 105 cells/well) cells were seeded in 6-well plates in
growth medium and treated with the tested compound for 6 h. Intra-
cellular GSH and GSSG contents were performed by the glutathione
reductase-DTNB recycling assay as described previously (https://pubm
ed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%C3%A1nchez+JC&ca
uthor_id=30068667Zhang et al., 2018).
properties
(https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%
ADn-S%C3%A1nchez+JC&cauthor_id=30068667Reddy et al., 2012).
Therefore, MTT assays were used to evaluated the cytotoxicity of cur-
cumin and bischalcone analogs on A549 cancer cells after a 48 h treat-
ment in vitro, and the IC50 values are listeded in Table 1. Compounds 1d
and 1g displayed more excellent cytotoxicity than curcumin and other
bischalcone analogs. This result may be due to the “ortho effect” of
substituents which could enhance the antiproliferation activity of
mono-carbonyl curcimin analogs against cancer cells (https://pubmed.
ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%C3%A1nchez+JC&cauth
or_id=30068667Liu et al., 2016). Especially, 1d and 1g surfaced as
potent lead compounds exhibiting about 9-time and 5-time higher po-
tency than curcumin, respectively. This result may be relation with the
more stabilities of 1d and 1g in RPMI 1640 supplemented with 10%
(v/v) heat-inactivated fetal calf serum than curcumin (Fig. 1). There-
fore, 1d and 1g were selected for further underlying their anti-cancer
mechanisms on A549 cells.
2.8. Determination of thiobarbituric acid-reactive substance (TBARS)
3 × 105 A549 cells were incubated with the tested compound for 18 h
in 6-well plates. Then, the cells were harvested and lysed. Lipid perox-
idation was determined by the protocol as previously described (https
://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%C3%A1nchez
+JC&cauthor_id=30068667Mu et al., 2020).
2.9. Analysis of mitochondrial membrane potential
3 × 105 A549 cells were treated with the tested compound for 18 h in
6-well plates. Next, the cells were collected and incubated with
3.3. Apoptosis induction in A549 cells after treatment with 1d and 1g
Rhodamine 123 (5 μ
M) for 30 min at 37 ◦C and analyzed using a flow
cytometry as previously described (https://pubmed.ncbi.nlm.nih.gov/?
term=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor
_id=30068667Yang et al., 2019).
Apoptosis, known as type I programmed cell death, is a common cell
death pathway in chemotherapeutic agent (https://pubmed.ncbi.nlm.
nih.gov/?term=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor
_id=30068667Benedetti et al., 2018). To investigate the surveyed
reduction in cell viability brought by 1d and 1g was rely on induction of
apoptosis, an annexinV-FITC/PI double staining was executed and fol-
lowed by flow cytometry analysis. As shown in Fig. 2, 1d and 1g
exhibited significant apoptosis of A549 cells in a dose-dependent
2.10. Western blotting analysis
After treatment with the tested compounds, the expression of Cas-
pase 3 and Bax proteins of A549 cells were analyzed by Western blot as
previously descripted (https://pubmed.ncbi.nlm.nih.gov/?term=Mart
manner (P < 0.05). In particular, treatment with 60
μ
M 1d and 60
μ
M
M
%C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Ma et
al.,
1g, which exhibited stronger apoptosis inducing activity than 60
μ
2020). Briefly, the harvested A549 cells were lysed with ice-cold RIPA
lysis buffer containing proteinase inhibitors, followed by quantification
curcumin, caused 66.4% and 52.4% late apoptotic cells, respectively.
The sequence of apoptotic activity of these compounds was in
3

J. Yang et al.
European Journal of Pharmacology 888 (2020) 173396
Table 1
Cytotoxicity of curcumin and its analogs against A549 cells.
Comps.
IC50
(μ
M)
Comps.
IC50
(μ
M)
>200
>200
19.2 ± 1.4
9.7 ± 0.8
>200
>200
>200
5.5 ± 0.7
180.7 ± 9.9
52.3 ± 1.1
The IC50 value is the concentration of a compound tested to cause 50% inhibition of cell viability after 48 h of treatment, and is expressed as the mean ± S.D. for three
determinations.
factor in the apoptosis induced by bischalcones.
3.5. Effects of 1d and 1g on the redox balance
Given that intracellular redox imbalance, which was evaluated by
the ratio of GSH and its disulfide GSSG, usually occurred when a sus-
tained higher reactive oxygen species productionDiebold). The gluta-
thione reductase-DTNB recycling assay was used to evaluate the ratio of
GSH/GSSG of A549 cells after 1d and 1g treatment. It was found that 1d
and 1g sharply decreased the ratio of GSH/GSSG in a dose-dependent
manner (P < 0.05. Fig. 4). In addition, at a concentration of 60 μM,
1d caused a 3-fold decline in the ratio of GSH/GSSG relative to the
control (P < 0.001. Fig. 4), which was in line with its more powerful
reactive oxygen species production abilities. These results indicated that
the intracellular extensive reactive oxygen species resulted in the redox
imbalance of A549 cells.
3.6. Effects of 1d and 1g on the lipid peroxidation of A549 cells
Fig. 1. Stability assessment on curcumin (50 μM), 1d (50 μM) and 1g (50 μM)
in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf serum at
25 ◦C by monitoring the decrease in their maximun absorbance.
The sustained higher reactive oxygen species production of cells also
could target to the lipids of the plasma membrane and result in lipid
peroxidation
(https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%
accordance with their cytotoxicity in A549 cells, which suggest that the
cytotoxicity of the bischalcones was rely on their induction of apoptosis.
ADn-S%C3%A1nchez+JC&cauthor_id=30068667Serrano et al., 2020),
which was raised as determination of the amounts of malondialdehyde
(MDA). As seen in Fig. 5, exposure of the cells to 1d and 1g resulted in a
remarkable increase of the levels of MDA and exhibited a prime
3.4. Effects of 1d and 1g on the intracellular reactive oxygen species
generation
dose-dependent manner. Additionally, treatment with 60 μM 1d and 60
μ
M 1g, the amounts of MDA with about 2-fold and 1.9-fold increase
respectively compared with the control (P < 0.01. Fig. 5).
Intracellular reactive oxygen species can modulate cell proliferation,
apoptosis and other physiological reactions (https://pubmed.ncbi.nlm.
nih.gov/?term=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor
3.7. Effects of 1d and 1g on the loss of mitochondrial membrane potential
(MMP)
_id=30068667Zhang et al., 2019). Therefore, we decided to determined
whether 1d and 1g could increase reactive oxygen species generation in
A549 cells using 2′,7′-dichlorofluorescin diacetate. As expected,
remarkable increase in the generation of reactive oxygen species was
investigated in A549 cells exposed to different concentrations of 1d and
As demonstrated in the literature, the excess production of reactive
oxygen species is often accompanied with the disruption of mitochon-
drial membrane, a critical event in early cell apoptosis Kroemer). For the
sake of investigating whether the disruption of the MMP was related to
induction of cell apoptosis, the MMP in A549 cells was determined with
Rhodamine 123 by flow cytometry. Fig. 6 shows that 1d and 1g caused a
significant decrease of MMP in a perfect dose-dependent fashion (P <
0.01). Moreover, 1d and 1g were more active than the leading curcumin
1g (15, 30 and 60 μM) relative to that of control (P < 0.01. Fig. 3). It was
observed that 1d and 1g showed an excellent dose-dependent fashion in
reactive oxygen species production and exhibited more potent ability
than curcumin. Specifically, cells treated with 60
μM 1d showed a
5-time increase compared to the control (P < 0.001. Fig. 3). These re-
sults were relationship with the apoptotic activity of these compounds
and revealed that reactive oxygen species generation ability is a critical
in the loss of MMP. In particular, treatment with 60
μM 1d and 60 μM 1g
resulted in about 3-time and 2-time decrease of the MMP respectively
4

J. Yang et al.
European Journal of Pharmacology 888 (2020) 173396
Fig. 2. Flow cytometric analysis for apoptotic induction of A549 cells treated with curcumin and bischalcone analogs (1d and 1g) for 24 h *P < 0.05, **P < 0.01,
***P < 0.001 compared with control.
relative to the control (P < 0.001. Fig. 6). These results were in line with
the reactive oxygen species production abilities and apoptosis inducing
activities of 1d and 1g on A549 cell.
4. Discussion
Lung cancer is one of the most common life-threatening malig-
nancies in the worldwide (Malvezzi et al., 2019), accounting for almost
13% of all cancer diagnoses and nearly 19% of total cancer deaths (Zhou
et al., 2020). Approximately 85% of lung cancer events are non-small
cell lung cancer (NSCLC) (Wu et al., 2020). Currently, all kinds of
various of strategies, specially the chemtherapy technique, are applied
in lung cancer diagnosis and treatment (https://pubmed.ncbi.nlm.nih.
gov/?term=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor
3.8. 1d and 1g promoted the Bax and Caspase 3 expression
Bax and Caspase 3 proteins play integral roles in the initiation and
execution of apoptosisBoice). Therefore, we investigated for the Bax and
Caspase 3 activities by western blotting. As expected, both the expres-
sion levels of Bax and Caspase 3 proteins were remarkably increased
after compounds 1d and 1g treatment compared with control (Fig. 7). In
particular, compound 1d could increase the expression of Caspase 3 in a
dose-dependent manner, and 1g enhanced the levels of Bax protein in a
dose-dependent fashion. These results indicated that the bischalcone
analogs 1d and 1g could promote A549 cell apoptosis through activating
the expression of Bax and Caspase 3.
_id=30068667Liang et al., 2019). The well-known chemotherapeutic
agents, containing paclitaxel (https://pubmed.ncbi.nlm.nih.gov/?term
=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Zhang
et al., 2020), cisplatin (https://pubmed.ncbi.nlm.nih.gov/?term=Mart
%C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Zhu et al.,
2020) and gemcitabine (https://pubmed.ncbi.nlm.nih.gov/?term=Mart
%C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Cao et al.,
2020), are used in lung cancer therapy. Whereas, patients with NSCLC
had a poor prognosis, which attributed to the resistance of cancer cells to
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J. Yang et al.
European Journal of Pharmacology 888 (2020) 173396
Fig. 6. Effects of curcumin and bischalcone analogs (1d and 1g) on the loss of
mitochondrial membrane potential at the indicated concentrations in A549
cells for 12 h *P < 0.05, **P < 0.01, ***P < 0.001 compared with control.
Fig. 3. The reactive oxygen species generation induced by curcumin and bis-
chalcone analogs (1d and 1g) at the indicated concentrations on A549 cells. *P
< 0.05, **P < 0.01, ***P < 0.001 compared with control.
Fig. 7. The expression levels of Bax and Caspase 3 proteins in A549 cells
promoted by bischalcone analogs 1d and 1g.
chemotherapy and metastasis (Ashrafizadeh et al., 2020). Extensive ef-
forts need to be done to discovery new potential and impactful agents to
fight against this disease.
Natural products have contributed notably to antitumor agents and it
is estimated that most of the antitumor agents are of natural origin or
inspired by natural compounds-. Curcumin, the naturally active ingre-
dient found in turmeric, could affect various molecular pathway, such as
nuclear factor-κB (NF-κB) (Liu et al., 2019), vascular endothelial growth
factors (Qin et al., 2020) and PI3/Akt (Song et al., 2020) in treatment of
lung cancer. Curcumin can induce apoptosis to reduce the viability and
proliferation of lung cancer cells (Chen et al., 2020). Our previous work
also illustrated that curcumin and its analogs could induce apoptosis by
enhancing reactive oxygen species production in lung cancer (Liu et al.,
2016). Bischalcones, the curcumin analogs with omitting the active
methylene group, are promising candidates as these are important in-
termediates and raw materials widely used as precursors to drugs
(Sharma et al., 2018). Curcumin-inspired bischalcone could accelerate
cancer cell death through the inhibition of ATP-binding cassette super-
family G member 2 (ABCG2) protein (Winter et al., 2014) and promoting
endoplasmic reticulum stress (Sansalone et al., 2019).
Fig. 4. The changes of GSH/GSSG ratios in A549 cells after treatment with 1d
at the indicated concentrations for 6 h. Each experiment was performed in
triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control.
In this text, our data found that the bischalcone analogs 1d and 1g,
the fluorine or methoxyl attached to the ortho-position of the aromatic
ring(s), showed more potent cytotoxicity than that of curcumin and
other bischalcone analogs (Table 1). This phenomenon may be attrib-
uted to the “ortho effect” of substituents (https://pubmed.ncbi.nlm.nih.
gov/?term=Mart%C3%ADn-S%C3%A1nchez+JC&cauthor
_id=30068667Dai et al., 2015). Previously, the ortho hydroxyl group
was affirmed to enhance remarkably the antiproliferation activity of
Fig. 5. The effects of bischalcone analogs (1d and 1g) at the indicated con-
centrations on the lipid peroxidation of A549 cells. Values are expressed as
MDA equivalents (pmol)/mg protein. Each experiment was performed in trip-
licate. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control.
cinnamaldehyde
(https://pubmed.ncbi.nlm.nih.gov/?term=Mart%
C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Chew et al.,
2010) and chalcones (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%
C3%ADn-S%C3%A1nchez+JC&cauthor_id=30068667Gan
et
al.,
2013). Our previous work also proven that the ortho trifluoromethyl
6

J. Yang et al.
European Journal of Pharmacology 888 (2020) 173396
(https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%C3%
A1nchez+JC&cauthor_id=30068667Dai et al., 2015) and ortho fluorine
group (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%
C3%A1nchez+JC&cauthor_id=30068667Liu et al., 2016) could
strengthen the cytotoxicity of mono-carbonyl curcumin analogs. Intro-
duction of ortho groups may reveal a conformational change of a ligand
by stereoelectronic effects, to impel its binding with the redox-sensitive
target proteins (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%
ADn-S%C3%A1nchez+JC&cauthor_id=30068667Dai et al., 2015). The
stereoelectronic effects could be proclaimed by calculating the geome-
tries of 1d and 1g in the lowest energy conformation using Gaussian g09
program. Other factors including stabilities and cellular uptake of 1d
and 1g should devote their cytotoxicity. Our results exhibited that
compounds 1d and 1g showed the more stabilities than curcumin in
RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf
serum (Fig. 1). Curcumin analogs possessing “ortho effect” of substituents
showed excellent cellular uptake in lung cancer in our previous work
(https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S%C3%
A1nchez+JC&cauthor_id=30068667Liu et al., 2016).
A sustained higher reactive oxygen species production could result in
the intracellular redox imbalance. This study indicated that the ratios of
GSH/GSSG were present as an outstanding dose-dependent fashion after
treatment with compounds 1d and 1g. The result was consistent with the
reactive oxygen species generation abilities of 1d and 1g. Compounds
1d and 1g will go a step further to result in lipid peroxidation and the
loss of mitochondrial membrane potential with an excellent dose-
dependent manner. Moreover, 1d and 1g could increase the expres-
sion of Bax and Caspase 3 proteins, which play potential roles in the
initiation and execution of apoptosisBoice). The mechanisms of
bischalcone-mediated apoptosis in A549 cells are still not fully clarified.
The stereoelectronic effects of bischalcones and the specific mechanism
of reactive oxygen species and apoptosis still requires further research.
5. Conclusions
In conclusion, we had synthesized a panel of bischalcone analogs
through the Aldol condensation reaction, and confirmed their structures
by the 1H NMR and 13C NMR. Then, we found that 1d and 1g exhibited
more potent cytotoxicity against A549 cells than curcumin and other
bischalcone analogs. Furthermore, the results indicated that reactive
oxygen species exerted a key role in 1d and 1g-triggered apoptosis and
act as an upstream signal to regulate the redox balance, lipid perox-
idatin, the disruption of MMP, the expression levels of Bax and Caspase 3
(Scheme 2). Therefore, the present study supported that enhancing the
reactive oxygen species production may be a potential strategy to
improve the anticancer activity of bischalcone analogs.
Apoptosis, a common cell death pathway in chemotherapeutic agent,
play a potent role in the development and maintenance of tissue ho-
meostasis (https://pubmed.ncbi.nlm.nih.gov/?term=Mart%C3%ADn-S
%C3%A1nchez+JC&cauthor_id=30068667Benedetti et al., 2018).
This study found that compounds 1d and 1g exhibited significant
apoptosis of A549 cells with 66.4% and 52.4% late apoptotic cells,
respectively. Increasing evidence has supported that reactive oxygen
species overproduction, as an upstream apoptotic factor, is associated
with apoptotic cell death (Pelicano et al., 2004). This study has shown
that reactive oxygen species levels increased with an excellent
dose-dependent manner induced by compounds 1d and 1g (P < 0.01.
Fig. 3). The reactive oxygen species generation by curcumin analogs
may be due to its ability to covalently modify TrxR (Fang et al., 2005).
Compounds 1d and 1g could irreversibly inhibit TrxR and the modified
enzyme converted into a prooxidant that triggered reactive oxygen
species generation by an acquired NADPH oxidase.
Ethics approval and consent to participate
No human or animal related experiments in the present study.
Consent for publication
All authors give the consent for the publish of this study.
Scheme 2. 1d induce reactive oxygen species-Mediated apoptosis in A549 cells.
7

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European Journal of Pharmacology 888 (2020) 173396
Liu, G.Y., Jia, C.C., Han, P.R., Yang, J., 2018. 3,5-Bis(2-fluorobenzylidene)-4-piperidone
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Availability of data and material
Data made available to all interested researchers upon request.
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CRediT authorship contribution statement
Jie Yang: Methodology, Formal analysis, Investigation, Resources,
Data curation, Writing - original draft, Writing - review & editing,
Funding acquisition. Wen-Wen Mu: Methodology, Formal analysis,
Investigation, Resources, Data curation. Guo-Yun Liu: Writing - original
draft, Writing - review & editing, Supervision, Project administration,
Funding acquisition.
´
´
´
´
Martín-Sanchez, J.C., Lunet, N., Gonzalez-Marron, A., Lidon-Moyano, C., Matilla-
`
Santander, N., Cleries, R., Malvezzi, M., Morais, S., Costa, A.R., Ferro, A., Lopes-
´
Conceicao, L., La Vecchia, C., Martínez-Sanchez, J.M., 2018. Projections in breast
and lung cancer mortality among women: a bayesian analysis of 52 countries
worldwide. Canc. Res. 78, 4436–4442. https://doi.org/10.1158/0008-5472.CAN-
18-0187.
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgements
Mu, W.W., Cao, Y.X., Tie, X.R., Li, T., Zhu, Z.Z., Yang, J., Liu, G.Y., 2020. Fluorined
mono-carbonyl curcumin analogues induce reactive oxygen species to enhance
apoptosis inhuman colon cancer HCT116 cells. Lat. Am. J. Pharm. 39, 361–365.
Pelicano, H., Carney, D., Huang, P., 2004. ROS stress in cancer cells and therapeutic
implications. Drug Resist. Updates 7, 97–110. https://doi.org/10.1016/j.
drup.2004.01.004.
Qin, L.M., Zhong, M.L., Adah, D., Qin, L., Chen, X.P., Ma, C.Q., Fu, Q., Zhu, X.P., Li, Z.L.,
Wang, N.N., Chen, Y.F., 2020. A novel tumour suppressor lncRNA F630028O10Rik
inhibits lung cancer angiogenesis by regulating miR-223-3p. J. Cell Mol. Med. 24,
3549–3559. https://doi.org/10.1111/jcmm.15044.
This work was supported by the National Natural Science Foundation
of China (Grant No. 81901420), Shandong Provincial Natural Science
Foundation (Grant No. ZR2018LH022), the Open Project of Shandong
Collaborative Innovation Centre for Antibody Drugs (Grant No. CIC-
AD1822 and CIC-AD1824).
Reddy, M.V.B., Shen, Y., Ohkoshi, E., Bastow, K.F., Lee, K., Wu, T., 2012. Bis-chalcone
analogues as potent NO production inhibitors and as cytotoxic agents. Eur. J. Med.
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Sansalone, L., Veliz, E.A., Myrthil, N., Stathias, V., Walters, W., Torrens, I.I., Schürer, S.
C., Vanni, S., Leblanc, R.M., Graham, R.M., 2019. Novel curcumin inspired bis-
chalcone promotes endoplasmic reticulum stress and glioblastoma neurosphere cell
death, 11, p. 357. https://doi.org/10.3390/cancers11030357.
Appendix A. Supplementary data
Santos, C.C.F., Paradela, L.S., Novaes, L.F.T., Dias, S.M.G., Pastre, J.C., 2017. Design and
synthesis of cenocladamide analogues and their evaluation against breast cancer cell
lines. MedChemComm 8, 755–766. https://doi.org/10.1039/c6md00577b.
Serrano, J.J., Delgado, B., Medina, M.A., 2020. Control of tumor angiogenesis and
metastasis through modulation of cell redox state. BBA-Reviews on Cancer. https://
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