Synthesis of renoprotective chalcone analogues that protect against cisplatin-induced cytotoxicity in LLC-PK1 cells

Full text of DOI:10.1002/bkcs.10984

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DOI: 10.1002/bkcs.10984
BULLETIN OF THE
N. Yamabe et al.
KOREAN CHEMICAL SOCIETY
Synthesis of Renoprotective Chalcone Analogues That Protect Against
Cisplatin-induced Cytotoxicity in LLC-PK1 Cells
Noriko Yamabe,†,†† Dahae Lee,†,†† Heesu Lee,‡ Myung Sook Shin,§ Gwi Seo Hwang,†
§,#,¶,
Ki Sung Kang,†, and Jae Wook Lee
*
*
†College of Korean Medicine, Gachon University, Seongnam 461-701, South Korea.
*E-mail: kkang@gachon.ac.kr
‡Department of Oral Anatomy, College of Dentistry, Gangneung Wonju National University, Gangneung
210-340, South Korea
§Natural Constituent Research Center, Korea Institute of Science and Technology, Gangnung 210-340,
South Korea. *E-mail: jwlee5@kist.re.kr
#Convergence Research Center for Dementia, Korea Institute of Science and Technology, Seoul, South
Korea
¶Department of Biological Chemistry, Korea University of Science and Technology (UST),
Daejun 305-333, South Korea
Received July 21, 2016, Accepted September 13, 2016, Published online October 25, 2016
Keywords: Chalcone, Cisplatin, Mitogen-activated protein kinases, Nephrotoxicity
cells.19 In our previous research on the synthesis of bioac-
Kidneys have major roles for balancing electrolytes, main-
taining pH homeostasis, and controlling blood pressure. In
particular, kidneys also remove excess of waste products,
medication, and endogenous toxic waste. Therefore, kid-
neys are major targets for toxic effects of various chemical
agents or medications. In a recent survey performed on crit-
ical patients, nephrotoxicity caused by medication is
responsible for about 25% of hospital admissions for acute
kidney injury (AKI).1–4
Cisplatin (cis-dichlorodiammine platinum(II), CDDP) is
one of the most potent anticancer drugs for the treatment of
epithelial malignancies including stomach, neck, head, blad-
der, testicular, ovarian, and lung cancers.5 However, the util-
ity of cisplatin is often limited, because of its toxicities
inducing cytotoxicity, gastrotoxicity, myelosuppression, and
allergic reactions.6 Among toxicities of cisplatin, the main
dose-limiting side effect is its severe nephrotoxicity.7–10
Cisplatin accumulates in the kidney to a greater degree
via transport than in other organs.10 Accumulated cisplatin
in kidney cells activates the mitogen-activated protein
kinases (MAPK) cascade and molecular responses that are
involved in the typical stress response.11,12 The mechan-
isms that lead to cisplatin-induced AKI involve oxidative
stress, inflammation, and renal cell apoptosis.3,13–15
tive constituents from natural resources, we found that chal-
cone derivatives had significant anti-inflammatory effects
and renoprotective effect against cisplatin-induced damage
in kidney cells.20
In our previous research, potent chalcone derivatives
exhibited slight cytotoxicity at high concentration in LLC-
PK1 cells.20 Therefore, we tried to develop chalcone deri-
vatives with no adversary effects at high concentration. We
synthesized novel chalcone derivatives to evaluate their
potential renoprotective effects. Chalcone analogues (1–15)
were prepared by Claisen-Schmidt condensation reaction
using various acetophenone and benzaldehyde in the pres-
ence of 20% NaOH and EtOH solutions (Scheme 1).20–23
A reaction mixture of benzaldehyde and acetophenone in
EtOH treated with NaOH solution was refluxed overnight.
The crude chalcone derivatives were purified by either silica
gel column chromatography or recrystallization with EtOH.
The synthesized chalcone derivatives were analyzed
using 1H-nuclear magnetic resonance (NMR), 13C-NMR,
and liquid chromatography-mass spectrometry (LC-MS).
The newly synthesized chalcone derivatives have methoxy-,
halogenated-, or nitro-substitution groups that have electron-
donating or electron-withdrawing properties. We assumed
that the properties of these substitution groups could
affect on the renoprotective and cytotoxic effects in
LLC-PK1 cells.
Several naturally occurring compounds that protect
against cisplatin-induced kidney damage have been identi-
fied.16,17 The kidney cell protection effects of sulforaphanes
are mediated by inhibition of the apoptosis pathway via the
c-Jun N-terminal kinase (JNK)-p53-caspase apoptotic cas-
cade.18 Ginsenosides from Panax ginseng protect against
cisplatin-induced nephropathy in renal tubular epithelial
To compare with potency chalcone derivatives, the reno-
protective potential of the 15 synthesized chalcone deriva-
tives (1–15) was evaluated using 25 μM of cisplatin-treated
LLC-PK1 cells and followed by 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As
shown in Figure 1, compounds (1, 4, 8, 11, 14, and 15)
†† These authors contributed equally to this work.
Bull. Korean Chem. Soc. 2016
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
1

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BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
O
O
O
(A)
20% NaOH
EtOH
+
H
1 (μM)
2 (μM)
3 (μM)
R2
120
120
100
80
60
40
20
0
120
100
80
60
40
20
0
100
80
60
40
20
0
R1
R2
R1
O
O
O
OCH3
OCH3
N
N
O
F
O
N
F
F
OCH3
3
2
Cisplatin (25 μM)
4 (μM)
Cisplatin (25 μM)
5 (μM)
Cisplatin (25 μM)
6 (μM)
1
O
O
O
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
H3CO
O2N
F
4
5
6
O
O
O
H3CO
OCH3
Br
OCH3
Br
Cl
H3CO
Cisplatin (25 μM)
8 (μM)
Cisplatin (25 μM)
9 (μM)
Cisplatin (25 μM)
7 (μM)
9
8
7
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
O
O
O
OCH3
Br
Cl
Br
Cl
N
O
OCH3
12
O
11
10
O
O
Cisplatin (25μM)
12 (μM)
Cisplatin (25μM)
11 (μM)
Cisplatin (25μM)
10 (μM)
Cl
O
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Cl
14
15
13
Scheme 1. Synthesis of chalcone analogues and their structures.
ameliorated the reduced cell viability in a dose-dependent
manner (Figure 1). However, chalcone derivatives (7 and
10) with 3-bromo-2,4-dimethoxy group showed no reno-
protective effects and cytotoxicity in high concentration.
Compounds (1 and 8) with 3,4,5-trimethoxy or naphthalene
group showed relatively high potency in renoprotective
effects. Chalcone derivatives (11, 14, and 15) with chloro-
substitution group showed potent renoprotective effects
among other substitution group. Notably, among 4-allyloxy
phenyl derivative (14 and 15), except from bromo-
substitution (7) which show lower potency, the derivatives
with meta and para chloro-substitution exhibited relatively
high potency in renoprotective effects compared to other
derivatives with cabazolyl group (11) (Figure 1(a)).
Cisplatin (25 μM)
13 (μM)
Cisplatin (25 μM)
14 (μM)
Cisplatin (25 μM)
15 (μM)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Cisplatin (25 μM)
Cisplatin (25 μM)
Cisplatin (25 μM)
(B)
Cisplatin +
#15 (10 μM)
Cisplatin
Cisplatin +
#15 (50 μM)
Control
Of these chalcone derivatives, compound 15 ameliorated
cisplatin-induced nephrotoxicity to 92.0 and 98.3% of the
control value at 50 and 125 μM, respectively. In particular,
compound 15 exhibited no cytotoxicity at higher than
125 μM. In addition, microscopic image analysis shows
that compound 15 treated cells have high renoprotective
effects compared with cisplatin-treated cells (Figure 1(b)).
Consequently, we conclude that of the 15 synthesized
chalcone derivatives, compound 15 is the most potent com-
pound that protects against cisplatin-induced nephrotoxicity
(Figure 1).
The renoprotective potential of synthetic or naturally
occurring compounds is related to their antioxidant, antia-
poptotic, and anti-inflammatory effects.16,18,20 An image-
based cytometric assay was performed to verify the antia-
poptotic potential of compound 15; Figure 2(a) presents
representative images. Compound 15 significantly inhibited
the percentage of apoptotic cells: compared with the
cisplatin-treated group, significant inhibition of 30 and 85%
Figure 1. Comparison in the protective effect of synthesized
15 chalcones derivatives on cisplatin-induced renal cell damage.
(a) Protection assay with compounds (1–15) in cisplatin-induced
(25 μM) LLC-PK1 cells. (b) Representative microscopic image of
the renoprotective effect of compound 15 following treatment with
25 μM cisplatin.
was observed with 10 and 50 μM compound 15, respec-
tively (Figure 2(b)). Next, we used Western blot analysis to
measure the expression of inflammatory signal proteins,
including p-JNK, p-p38, p-ERK, and cleaved caspase-3, in
LLC-PK1 cells. Upregulated phosphorylation of JNK, p38,
and ERK by cisplatin treatment was decreased markedly
after co-treatment with compound 15 (Figure 3). The results
demonstrate that blocking the MAPKs-caspase-3 signaling
cascade plays a critical role in mediating the protective
effect of compound 15 against cisplatin-induced
nephrotoxicity.
Bull. Korean Chem. Soc. 2016
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
Control
Cisplatin
(A)
(B)
(A)
(B)
60
50
40
30
20
10
0
30
25
20
15
10
#15 (μM)
0
0
10
50
Cisplatin (25 μM)
-
+
+
+
Cisplatin + #15 (50 μM)
Cisplatin + #15 (10 μM)
p-p38
5
0
3
p38
2.5
2
1.5
1
p-JNK
Figure 2. Effects of compound 15 on apoptosis in LLC-PK1 cells.
(a) Representative images of apoptosis detection. (b) Percentage
of annexin V-positive-stained apoptotic cells. Dead and apoptotic
cells were stained with red and green fluorescence. Apoptosis was
determined using a Tali image-based cytometer.
0.5
0
JNK
1.4
1.2
1
0.8
0.6
0.4
0.2
0
p-ERK
ERK
To evaluate the effect of compound 15 on the anticancer
activity of cisplatin, the human cervical cancer HeLa cells
were treated with the compound 15 and cisplatin alone or in
combination. Co-treatment with compound 15 at the dose up
to 50 μM did not attenuate the cytotoxic properties of cis-
platin (Figure 4). No obvious anticancer effect of compound
15 alone was observed. Our findings suggest that compound
15 with cisplatin may alleviate the nephrotoxicity without
compromising therapeutic efficiency of cisplatin.
Cleaved Caspase-3
GAPDH
10
9
8
7
6
5
4
3
2
1
0
Chalcone derivatives 1, 4, 8, 11, 14, and 15 have thera-
peutic potential for protecting against anticancer drug-
induced nephrotoxicity. The chalcone analogues with meta
or para-chloro phenyl and 4-allyloxy phenyl group (14 and
15) had highly potent effects in the kidney cell protection
assay, while the chalcone derivatives (7 and 10) with 3-
bromo-2,4-dimethoxy phenyl group had no protective
effects. Compound 15 had no cytotoxic effects on LLC-
PK1 cells, even at high concentrations (250 μM). Com-
pound 15 exhibited the strongest renoprotective effects by
blocking the MAPKs-caspase-3 signaling cascade.
Figure 3. The MAPKs-caspase-3 signaling pathway mediates the
protective effect of compound 15 against cytotoxicity in cultured
LLC-PK1 cells. The Western blot analysis shows the levels of
phosphorylated (p)-p38, p38, p-ERK, ERK, p-JNK, JNK, cleaved
caspase-3, and GAPDH in LLC-PK1 cells treated with compound
9 with or without cisplatin at different concentrations for 24 h.
120
100
80
60
40
20
0
In anticancer activity, cisplatin stimulated cancer cell sig-
nal pathways, including ATR, p53, p73, and MAPK, and
culminate the activation of apoptosis.24 Compound 15 may
selectively block MAPK signal pathway activated by cis-
platin. Therefore, co-treatment of cisplatin with compound
15 exhibited no attenuating anticancer effects in the human
cervical cancer cells.
MAPK signal pathways are all known to play roles in
modulating nephrotoxicity, caused by cisplatin. Stimulation
of these signal pathways induced apoptosis in kidney cells.
(E)-3-(2-bromo-4,5-dimethoxyphenyl)-1-(4-bromophenyl)
prop-2-en-1-one19 showed potent renoprotective effects at
25 μM by blocking MAPK-p53-caspase-3 signal pathway.
However, high dose of this compound showed decreasing
renoprotective effects.
-
-
2.5
-
5
-
10 25 50
-
-
-
-
-
2.5
5
5
10 25 50
10 25 50
#15 (μM)
Cisplain (μM)
-
-
-
2.5
5
10 25 50 2.5
Compound 15 is therefore a relatively potent chemical
agent that has a significant effect on kidney protection in
cell culture. Compound 15 might ultimately lead to the
development of chemotherapeutic agents.
Figure 4. The effect of the combination treatment of compound
15 and cisplatin on the cell viability in HeLa cells. Cell viability
was determined after 24 h culture by cell counting assay kit, and
the results were expressed as percentage of viable cells.
Bull. Korean Chem. Soc. 2016
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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KOREAN CHEMICAL SOCIETY
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The Synthesis of (E)-3-(4-(allyloxy)phenyl)-1-(4-chloro-
phenyl)prop-2-en-1-one (15). The reaction condition was
followed the same procedure. 4-Chloroacetophenone
(77.5 mg,
0.5 mmol)
and
4-allyloxybenzaldehyde
(81.5 mg, 0.5 mmol) were afforded to compound 15
(86 mg, 57.7%). 1H-NMR (CDCl3, 400 MHz) δ: 7.95
(dd, J = 6.8, 2 Hz, 2H), 7.78 (d, J = 15.6 Hz, 1H), 7.59
(d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.36 (d,
J = 15.6 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.10–6.01
(m, 1H), 5.42 (dd, J = 14.0, 1.6 Hz, 1H), 5.32 (dd,
J = 10.4, 1.2 Hz, 1H), 4.59 (dt, J = 5.2, 1.6 Hz, 2H).
13C-NMR (CDCl3, 100 MHz) δ: 189.2, 160.9, 145.2,
139.0, 136.8, 132.7, 132.0, 130.3, 129.8, 129.7, 128.9,
127.9, 127.6, 119.2, 118.1, 115.2, 115.0, 68.9. ESI-MS
299.1 [M + H].
Acknowledgments. This research was funded by the
Korea Institute of Science and Technology Institutional
Program (2Z4610, 2Z04690) and the National Research
Council of Science & Technology (NST) grant by the
Korea government (MSIP) (No. CRC-15-04-KIST).
Supporting Information. Additional supporting informa-
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© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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