2-Bromo-4,5-Dimethoxy Chalcone Inhibits Cisplatin-induced LLC-PK1 Kidney Cell Death

Full text of DOI:10.1002/bkcs.11454

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DOI: 10.1002/bkcs.11454
BULLETIN OF THE
D. Lee et al.
KOREAN CHEMICAL SOCIETY
2-Bromo-4,5-Dimethoxy Chalcone Inhibits Cisplatin-induced LLC-PK1
Kidney Cell Death
¶,k,**,
Dahae Lee,‡ Heesu Lee,§ Ki Sung Kang,†, and Jae Wook Lee
*
*
†College of Korean Medicine, Gachon University, Seongnam 13120, South Korea.
*E-mail: kkang@gachon.ac.kr
‡School of Pharmacy, Sungkyunkwan University, Suwon 16419, South Korea
§College of Dentisty, Gangneung Wonju National University, Gangneung 25457, South Korea
¶Natural Constituent Research Center, Korea Institute of Science and Technology, Gangneung 25451,
South Korea
kConvergence Research Center of Dementia, Korea Institute of Science and Technology (KIST), Seoul
02792, South Korea
**Department of Biological Chemistry, Korea University of Science and Technology, Daejun 34113,
South Korea. *E-mail: jwlee5@kist.re.kr
Received January 4, 2018, Accepted March 19, 2018
Keywords: Chalcone, Cisplatin, Kidney cells, Renoprotection, MAPK pathway
The kidney has various functions, including modulating
blood pressure, balancing electrolytes, and controlling
blood volume. Among these functions, extracting metabolic
waste from blood plays an important role in kidney.1,2
Additionally, kidney epithelial cells are vulnerable to the
toxic effects of various chemical agents and medications.3,4
Recently, cisplatin, an inorganic platinum-based chemother-
apeutic agent, has been used to inhibit solid malignant
tumors due to its high therapeutic potential.5,6 However,
continuous exposure to cisplatin can cause various signifi-
cant side effects, such as bone marrow suppression, periph-
eral neuropathy, and nephrotoxicity. In particular, a single
dose of cisplatin treatment can cause nephrotoxicity among
one-third of patients.7–9 Therefore, it has been suggested to
develop medication for cisplatin-induced nephrotoxicity.
Chalcone structures, known as α,β-unsaturated ketones,
belong to the naturally occurring flavonoid family and are
abundant in various plants, including fruits, vegetable, tea, and
soy beans.10 Due to its structural simplicity, chalcone has been
used as an effective template in medicinal chemistry for drug
discovery.11 Many studies have shown that chalcones and their
derivatives engage in numerous biological activities, including
anticancer,12 antimalaria,13 antimicrobial,14 anti-inflammatory,15
antiprotoaozal,16 and antiHIV activities.17 Recent studies have
shown the importance of chalcone and its analogues as protec-
tive agents involved in the inhibition of apoptosis mediated
through the mitogen activated protein kinases (MAPK) signal
pathway.18–20 Recent studies have suggested that chalcone and
its analogues have potential in the therapy of drug-associated
chronic or acute kidney diseases.18,19
with 20-bromo-40,50-dimethoxy and 4-bromo group (9) showed
more than 80% of renoprotective effects at 25 μM toward
cisplatin-induced kidney cell death.18 We anticipated that chal-
cone analogues with a 20-bromo-40,50-dimethoxy group may
show renoprotective potential. Therefore, we designed and syn-
thesized chalcone with a 20-bromo-40,50-dimethoxy group.
Chalcones with hydroxy group has been shown from nat-
ural products including isoliquiritigenin,21,22 butein,23,24
cardamonin,25 and etc. The activity of chalcones with
hydroxy group showed potent effects as anticancer, antioxi-
dant, and anti-inflammation. Furthermore, metabolic deriva-
tives of isoliquiritigenin show protective effects to
glutamate-induced neuronal cell death.26 We assumed that
hydroxy chalcone would show more potent effects and
decided to replace substituents of acetophenone moieties in
compound 1 with hydroxyl groups (Scheme 1(a)).
These chalcone analogues with 20-bromo-40,50-dimethoxy
group were prepared through the Claisen–Schmit condensa-
tion reaction using methoxy or MOM (methoxymethyl) pro-
tected hydroxy acetophenones and 20-bromo-40,50-dimethoxy
benzaldehydes under basic conditions (Scheme 1(b)).
MOM protecting group was cleaved under acidic condition.
All crude mixtures were purified by column chromatography
to yield pure chalcones. All synthesized chalcone analogues
were analyzed by 1H NMR and ESI-MS to confirm the chemi-
cal structures (Appendix S1, Supporting information). The
renoprotective potentials of synthesized chalcones were evalu-
ated using LLC-PK1 pig kidney epithelial cells, which are dam-
aged by cisplatin treatment. Renoprotective effects
were
measured
using
3-[4,5-dimethylthiazol-2-yl]-2,5-
In our previous study, chalcone derivatives including halo-
gen, alkyl, and alkoxy groups were synthesized and evaluated
for their protective effects on cisplatin-induced kidney cell tox-
icity. Among the synthesized chalcone analogues, chalcones
diphenyltetrazolium bromide (MTT) reagent. These experi-
ments were repeated three times (Figure 1). In the assay, the
chalcone analogues (1, 3, and 4) showed protective effects at
concentrations of 50 to 200 μM and about 80% of cell
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(a)
(b)
Scheme 1. Design and synthesis of 20-bromo-40,50-dimethoxy
Figure 1. Renoprotective effects of chalcone analogues (1–8).
Control cells were treated with the vehicle only (mean 4 SD,
* p < 0.05 compared to the cisplatin-treated control).
chalcone derivatives.
viability. Renoprotective effects of chalcone analogues (4, 5,
and 6) may not be related with position of hydroxy group in
acetophenone moiety. A compound 6 with mono hydroxyl at
ortho position show no potent effects. A compound 5 with
mono hydroxyl and dimethoxy show no protective effects. A
compound 4 with dihydroxyl at ortho position show protective
effect from 50 μM. Among these eight synthesize chalcones, a
compound 3 was selected based on its renoprotective potential.
To evaluate the protective effects of compound 3 on apo-
ptotic cell death, we analyzed the population of live and
dead cells using image-based cytometric assays. Annexin V
conjugated with alexa fluor 488 fluorescent probe and propi-
dium iodide were used to stain dead or apoptotic cells. As
shown in Figure 2(a) and (b), cisplatin treatment increased
the number of red and green fluorescent spots, which is cor-
related with the number of apoptotic and dead cells. In
Figure 2(b), the ratio of apoptotic cells increased to 34%
with 25 μM cisplatin treatment. On the other hand, following
treatment with cisplatin (25 μM), compound 3 (25 and
50 μM) on LLC-PK1 cells gradually decreased the popula-
tion of apoptotic and dead cells (Figure 2(b)). We selected
compound 3 for further mechanism studies of its renoprotec-
tive effects on kidney cells. Cell viability after compound
3 treatment was almost 100% at a concentration of 50 μM.
In our previous studies, the MAPKs signal pathway and
apoptotic signal cascades were shown to be involved in
cisplatin-induced kidney cell death. Additionally, it is
known that cisplatin treatment induced p53-mediated acti-
vation of caspases in LLC-PK1 kidney cells. Cisplatin
(b)
(a)
Cisplatin (25 μM)
Control
Compound 3 (μM)
40
35
30
25
20
15
10
5
*
Cisplatin (25 μM)
+ compound 3 (25 μM)
Cisplatin (25 μM)
+ compound 3 (50 μM)
*
0
C
0
25 50
Cisplatin (25 μM)
Figure 2. Protective effects of compound 3 on cisplatin-induced
LLC-PK1 cell death. (a) LLC-PK1 cells were treated with 25 μM
and 50 μM of compound 3 followed by 25 μM of cisplatin and incu-
bated for 24 h. The control group (labeled as C) was treated with cul-
ture media with 0.5% DMSO. Apoptotic and dead cells were stained
with green and red fluorescence, respectively. (b) Percentages of apo-
ptotic (annexin V+) and dead cells were determined by counting the
number of green and red fluorescence spots using a Tali image-based
cytometer. Control cells were treated with the vehicle only (mean
4 SD, * p < 0.05 compared to the cisplatin-treated control).
treatment typically increases reactive oxygen species and
the activities of p38, c-JNK, and ERK kinase.18,19
Therefore, we investigated the mechanism of protective
effect of compound 3 by observing protein levels of p38,
JNK, ERK, and these phosphorylated proteins. In Figure 3,
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KOREAN CHEMICAL SOCIETY
-
-
-
+
+
-
+
-
+
-
cisplatin treatment induced the MAPKs signal pathway by
increasing protein levels of p-p38, p-JNK, and p-ERK, but
not of p38, JNK, and ERK, whereas these levels dose-
dependently decreased after treatment with compound
3 (25 and 50 μM) (Figure 3). Mitogen-activated protein
kinases (MAPKs) are important enzymes that respond to a
variety of extracellular stimuli and regulate cell differentiation,
proliferation, and survival. MAPKs comprise ERK, JNK, and
p38. All three principal MAPKs can be detected in various
renal cells involved in ischemia–reperfusion, toxic renal
injury, and acute renal injury.
Cisplatin (25 μM)
Compound 3 (25 μM)
Compound 3 (50 μM)
+
-
P-JNK
JNK
P-P38
p38
JNK and p38, which signal the cell response to stress
and inflammation, are activated in ischemia–reperfusion.
ERK growth factor-related signaling is activated during
toxic renal injury, whereas JNK and ERK are activated dur-
ing acute renal injury.27–29 During cisplatin-induced kidney
cell death, programmed apoptosis mainly occurs in proxi-
mal tubule epithelial cells.
P-ERK
ERK
Cleaved caspase -8
The ischemia or toxic injury activates initiator caspases
(caspase-8, −10, −2, and − 9), executioner caspases (cas-
pase-3, −7, and −6), and PARP, which is cleaved by cas-
pases. Additionally, the BCL family plays a role during
kidney failure. This cascade activates Bax (pro-apoptotic
family member) and decreases Bcl-2 (antiapoptotic family
member), which significantly changes the Bax/Bcl-2 ratio
in BCL family members from the mitochondria.30–32 In our
study, we explored the protective effect by observing the
protein level of apoptotic signal cascades. In agreement
with previous studies, as shown in Figure 3, cisplatin treat-
ment induced the apoptotic signal pathway by increasing
the protein level of caspase-8, −9, −3, bad, bcl-2, and frag-
ments of PARP. However, it dose-dependently decreased
after treatment with compound 3 (25 and 50 μM). Thus,
the protection effect of compound 3 on injured proximal
tubule cells after treatment with cisplatin was mediated by
deactivation of MAPKs and apoptotic signaling pathways.
To evaluate the effect of compound 3 to cisplatin anticancer
activity, A2780 human ovarian cancer cells were treated with
various concentration of compound 3 and cisplatin (Figure 4).
Cotreatment of compound 3 up to 200 μM did not diminish
cytotoxic effect of cisplatin. This result indicated that com-
pound 3 with cisplatin can alleviate neprotoxicity without
decreasing anticancer effect of cisplatin. As anticancer agent,
cisplatin-induced cancer cell death by activating several signal
pathways, including ATR, p53, p73, and MAPK, and apopto-
sis pathway.33 In A2780 human ovarian cancer, compound
3 may inhibits MAPK pathway which is activated by cis-
platin. Therefore, cotreatment of compound 3 and cisplatin
does not diminish anticancer activity.34
BID
Bax
Bad
Bcl-2
Cleaved caspase -9
Cleaved caspase -3
PARP
GAPDH
Figure 3. Effect of compound 3 on the expression of p-JNK, p-
p38, p-ERK, bad, Bcl-2, cleaved caspase-9, cleaved caspase-3,
and cleaved PARP. LLC-PK1 cells were treated with 25 and
50 μM of compound 3 followed by 25 μM of cisplatin and incu-
bated for 24 h. After cell lysis, the levels of p-JNK, p-p38, p-
ERK, bad, Bcl-2, cleaved caspase-9, cleaved caspase-3, and
cleaved PARP were analyzed by Western blotting.
In conclusion, these results suggested that some of the syn-
thesized chalcone analogues has therapeutic potential by pre-
venting cisplatin-induced nephrotoxicity. Among these
analogues, compound 3 is the most potent analogue as it
inhibits the activation of the MAPK-apoptosis signal pathway.
Experimental
MAPKs are a family of serine/threonine kinase enzymes
which are consisted of extracellular-receptor kinases
(ERK1/2), the c-Jun N-terminal kinases (JNK), and the p38
MAPKinases.35 Activation of MAPK pathway is implicated
with cisplatin-induced nephrotoxicity.34 Therefore, inhibition
of MAPK pathway by a pharmacological inhibitor shows pro-
tective effect on kidney cells.
1.1.. Synthesis of (E)-3-(2-bromo-4,5-dimethoxyphenyl)-
1-(2-hydroxy-5-methylphenyl)prop-2-en-1-one (3). The
solution of 2-hydroxy-5-methylacetophenone (30 mg,
0.2 mmol) in ethanol (4 mL) was treated with 2-bromo-4,5-
dimethoxybenzaldehyde (48.8 mg, 0.2 mmol) and followed
by 0.5 mL of 8N NaOH solution. The reaction was stirred
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Note
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Figure 4. The effects of co-treatment of compound 3 and cisplatin
on the A2780 human ovarian cancer cell viability was determined
after 24 h culture by MTT assay, and the results were expressed as
percentage of viable cells. The data shows co-treatment of com-
pound 3 and cisplatin does not decrease antiproliferative activity of
cisplatin on ovarian cancer cell line. Control cells were treated with
the vehicle only (mean 4 SD, * p < 0.05 compared to the control).
at room temperature for 24 h. The reaction mixture was
extracted with ethyl acetate (30 mL × 3) and H2O after
adjusted pH 7. Solvent was dried with Na2SO4 and evapo-
rated under reduced pressure. The crude was purified by
column chromatography (hexane/ethyl acetate, 5:1) to
afford compound 3 (42.1 mg, 56.0%).
1H NMR (CDCl3): 12.60 ppm (s, 1H), 12.6 ppm (s, 1H),
8.20 ppm (d, J = 15.2 Hz, CH CH, 1H), 7.66 ppm (d,
J = 1.2 Hz, 1H), 7.44 ppm (d, J = 15.2 Hz, 1H), 7.32 ppm
(dd, J = 8.4 ppm, 2 Hz, 1H), 7.21 ppm (s, 1H), 7. 11 ppm
(s, 1H), 6.94 ppm (d, J = 8.4 Hz, 1H), 3.98 ppm (s, 3H),
3.93 ppm (s, 3H). ESI-MS: m/z calcd for C18H18BrO4 [M +
H] = 377.04 found 377.0.
Acknowledgments. This research was supported by Basic
Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Sci-
ence, ICT & Future Planning (2015R1C1A1A02037383)
and Korea Institute of Science and Technology Institutional
Program (2Z04690), and National Research Council of Sci-
ence & Technology (CRC-15-04-KIST).
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
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