Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells

Article abstract of DOI:10.1016/j.bmcl.2015.10.093

Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in contro

Full text of DOI:10.1016/j.bmcl.2015.10.093

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5705–5707
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of apoptotic chalcone analogues in HepG2 human
hepatocellular carcinoma cells
Cheon-Soo Park a,y, Yongchel Ahn b,y, Dahae Lee c, Sung Won Moon d,e, Ki Hyun Kim f, Noriko Yamabe c,
Gwi Seo Hwang c, Hyuk Jai Jang a, Heesu Lee g, Ki Sung Kang c, , Jae Wook Lee e,h,
⇑
⇑
a Department of Surgery, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung 210-711, Republic of Korea
b Department of Hematology and Oncology, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung 210-711, Republic of Korea
c College of Korean Medicine, Gachon University, Seongnam 461-701, Republic of Korea
d Department of Chemistry, Gangneung Wonju National University, Gangneung 210-340, Republic of Korea
e Natural Product Research Center, Korea Institute of Science and Technology, Gangneung 210-340, Republic of Korea
f Natural Product Research Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
g Department of Oral Anatomy, College of Dentistry, Gangneung Wonju National University, 201-340, Republic of Korea
h Department of Biological Chemistry, University of Science and Technology (UST), Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma
HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was signifi-
cantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24 h induced
cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that com-
pound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic
apoptotic pathway.
Received 1 October 2015
Revised 28 October 2015
Accepted 30 October 2015
Available online 31 October 2015
Keywords:
Chalcone
Anticancer
HepG2
Ó 2015 Elsevier Ltd. All rights reserved.
Apoptosis
Hepatocellular carcinoma (HCC) is a highly lethal tumor that
commonly occurs in patients with chronic liver disease and cirrho-
sis.1 HCC has a poor prognosis due to its high recurrence rate and
resistance to chemotherapy.2 To date, liver transplantation is the
best form of treatment because it removes the tumor as well as
damaged hepatic tissues that may provoke chronic liver disorders.1
Several experimental and clinical investigations have occurred,
with the goal of preventing recurrence and secondary tumors
and improve the clinical outcome of HCC patients.3
Chalcone is an aromatic ketone or enone structure that forms
the central core and exhibits a wide range of biological activities,
including anticancer effects.4 Isoliquiritigenin is a natural pigment
with the simple chalcone structure 4,20,40-trihydroxychalcone.
Isoliquiritigenin inhibits the proliferation of human hepatoma cells
(HepG2) by G2/M phase arrest and programmed cell death.5
Xanthohumol, the major prenylated chalcone found in hops,
induces apoptosis in two HCC cell lines (HepG2 and Huh7).6 In
addition, chalcone derivatives from the fern Cyclosorus parasiticus
exhibit potent cytotoxicity against six cancer cell lines (e.g., lung
cancer A549, HepG2, breast cancer MCF-7 and MDA-MB-231,
leukemia ALL-SIL, and pancreatic cancer SW1990).7 Based on these
findings, in this study we synthesized eight chalcone analogues
and evaluated their cytotoxic effects on human hepatoma HepG2
cells (see Fig. 1).
We synthesized chalcone analogues (1–8) with methoxy or
halogenated groups that have electron-donating or electron-
accepting properties. For synthesis, we used Claisen–Schmidt
condensation between substituted acetophenone and benzalde-
hyde in the presence of aqueous NaOH solution.8 A reaction
mixture of benzaldehyde and acetophenone in EtOH treated with
NaOH solution was refluxed overnight. The synthesized chalcone
derivatives were purified by either silica gel column chromatogra-
phy or recrystallization with EtOH. All products were characterized
using 1H nuclear magnetic resonance (NMR), 13C NMR, and liquid
chromatography–mass spectrometry (LC–MS).
Cytotoxic effects of the chalcone analogues were assessed in
HepG2 cells using a MTT assay.9 As shown in Figure 2, two
compounds (3 and 5), each possessing a 3,5-dimethoxy-4-bromo
phenyl group, inhibited HepG2 cell growth by more than 50%. Of
⇑
Corresponding authors. Tel.: +82 33 750 5402; fax: +82 33 750 5416 (K.K.S.);
tel./fax: +82 33 650 3502 (L.J.W.).
E-mail addresses: kkang@gachon.ac.kr (K.S. Kang), jwlee5@kist.re.kr (J.W. Lee).
These authors contributed equally to this work.
y
http://dx.doi.org/10.1016/j.bmcl.2015.10.093
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

5706
C.-S. Park et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5705–5707
O
O
O
aq. NaOH
EtOH
+
H
R2
R1
R2
R1
O
O
O
O
O
O
O
O
N
Br
Cl
Br
O
3
1
2
O
O
O
O
O
N
Br
Br
O
4
5
6
O
O
Cl
Cl
8
7
Figure 1. Synthesis of chalcone analogues and their structures.
these, compound 5 inhibited HepG2 cell growth up to 91% and 98%
of the control growth at 50 and 100 M, respectively. However, for
l
the other chalcone analogues (1, 2, 4, 6–8), we observed weak to no
inhibition in cell growth when compared to the control. Conse-
quently, we concluded that, of the eight synthesized chalcone
derivatives, compound 5 was the most cytotoxic to HepG2 cells
Figure 3. Effects of compound 5 on HepG2 cell apoptosis. (A) Representative
images for the detection of apoptosis and (B) percentage of annexin V+ apoptotic
cells. Dead cells and apoptotic cells were stained red and green, respectively.
Apoptosis was determined using a Tali image-based cytometer. *p < 0.05 compared
to the control.
(IC50: 26
lM) and less cytotoxic activity against normal porcine
kidney proximal tubular epithelial LLC-PK1 cells (IC50: 83
lM).
Therefore, further mechanistic studies were conducted with com-
Representative data of the annexin V/PI flow cytometry results
pound 5.
are shown in Figure 3. Exposure to compound 5 resulted in signif-
To determine whether compound 5 induced apoptosis in HepG2
cells, we performed flow cytometry using annexin V as a fluores-
cein isothiocyanate (FITC) conjugate in combination with propid-
ium iodide (PI) as an exclusion dye to determine cell viability.10
icant apoptosis. Compound 5, at 25 and 50 lM, resulted in
8.5 2.2% and 31.2 2.9% early apoptotic cell populations, respec-
tively, compared to the control (1.9 0.5%) (Fig. 3).
The control HepG2 cells had a normal appearing morphology
(Fig. 4(A)). However,
a large proportion cells shrank, and
Figure 4. Compound 5 activates the extrinsic apoptotic pathway in HepG2 cells. (A)
Morphological changes were confirmed using phase-contrast microscopy. (B)
Protein expression levels of cleaved caspase-8, BID, Bax, Bcl-2, cleaved caspase-9,
cleaved caspase-3, PARP, and GAPDH in HepG2 cells. Cells were seeded in 96-well
plates at a density of 1 ꢀ 104 cells/well and incubated for 24 h at 37 °C. The cells
were treated with different concentrations of compound 5. Results from Western
blot analysis shows the levels of cleaved caspase-8, BID, Bax, Bcl-2, cleaved caspase-
9, cleaved caspase-3, PARP, and GAPDH in HepG2 cells treated with compound 5 for
Figure 2. Comparison of the cytotoxic effects of all chalcone derivatives on HepG2
cells. HepG2 cells were pretreated with various concentrations (up to 100
lM) of
24 h. Whole-cell lysates (20 lg) were separated by SDS–PAGE, transferred onto
chalcone derivatives for 24 h. Cell viability was assessed using
a
MTT assay.
PVDF membranes, and probed with the indicated antibodies. Proteins were
visualized using an ECL detection system.
*p < 0.05 compared to the control.

C.-S. Park et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5705–5707
5707
progressive condensation and break up (karyorrhexis) of the
Supplementary data
nucleus were detected in most cells after co-treatment with the
compound 5 (Fig. 4(A)). Western blotting was performed to evalu-
ate the expression of proteins involved in the apoptotic response to
determine if apoptosis occurs via the intrinsic (mitochondrial) or
extrinsic pathway.11 Results from Western blot analysis are shown
in Figure 4. Exposure to compound 5 for 24 h induced the cleavage
of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). In
addition, we observed a significant increase in the ratio of Bax/
Bcl-2 (Fig. 4(B)). Caspase-9 is activated through the mitochon-
dria-dependent intrinsic pathway.12 Based on no changes in
cleaved capase-9, compound 5 induced apoptosis via the extrinsic
apoptotic pathway.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
093.
References and notes
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Cesar et al. reported a relationship between the structural
characteristics of synthetic chalcones and their anti-tumor
activities.4 Treatment of HepG2 cells with synthetic 20-hydroxy-
chalcones for 24 h induced apoptosis, which was mediated via
the activation of caspase-9 and subsequently nuclear apoptosis.4
5. Hsua, Y. L.; Kuob, P. L.; Lina, C. C. Life Sci. 2005, 77, 279.
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Culture Collection (ATCC, Rockville, USA). HepG2 cells were maintained in
DMEM (Gibco, Gaithersburg, USA) supplemented with 10% fetal bovine serum
Zhang et al. reported that chalcone with
a p-substituted
methyl group at the B ring and thiosemicarbazide had the greatest
inhibitory effect on HepG2 cells compared to other synthesized
chalcone-type thiosemicarbazide compounds.13 In the present
study, we determined that 3,5-dimethoxy-4-bromo phenyl
structure of compounds (3 and 5) is important for inducing
apoptosis in HepG2 cells via the extrinsic pathway.
In summary, compound 5 showed strong cytotoxicity and
promoted apoptosis in human hepatoma HepG2 cells. Exposure
to compound 5 for 24 h induced the cleavage of caspase-8 and
-3, and PARP. The percentage of apoptotic cells was significantly
higher in compound 5-treated cells than in control cells. These
(Gibco), 100 units/mL penicillin, and 100 lg/mL streptomycin, and incubated
at 37 °C in a humidified incubator with 5% CO2. The MTT assay was conducted
according to a previously published study: Eom et al BMB Rep. 2015, 48, 461.
10. Apoptotic cells were analyzed by annexin V and PI staining using an Annexin-
V-FLUOS Staining Kit (Roche, Indianapolis, USA) according to the
manufacturer’s recommendations. Briefly, whole cells were collected, rinsed
with DPBS, incubated with 100
lL annexin-V binding buffer containing 2
l
a
L
annexin-V and PI at rt for 15 min in the dark, and analyzed by
2 lL
FACSCalibur flow cytometer. At least 20,000 events were evaluated in these
experiments: Lee et al Bioorg. Med. Chem. Lett. 2015, 25, 1929.
11. Human hepatoma cells were grown in 60 mm dishes and treated with 25 and
50 lM compound 5 for 24 h. Whole-cell extracts were prepared as reported
previously using RIPA buffer (Cell Signaling, Beverly, USA) supplemented with
1ꢀ protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride: Park
et al J. Ginseng Res. 2014, 38, 22. For Western blot analysis, proteins (whole-cell
findings suggest that compound
analogue that contributes to apoptotic cell death in human
hepatoma cells. Future studies will focus on the effect of
5 is the active chalcone
extracts: 30
lg/lane) were separated by electrophoresis through a NuPAGE 4–
12% Bis-tris gel (Invitrogen, Carlsbad, USA), transferred onto
a
PVDF
compound
metastasis.
5 on unexplored areas like tumor invasion and
membrane, and analyzed with the epitope-specific primary and secondary
antibodies. Bound antibodies were visualized using ECL Advance Western
Blotting Detection Reagents (GE Healthcare, Waukesha, USA) and an
ImageQuant LAS-4000 Imager (Fujifilm, Tokyo, Japan).
Acknowledgments
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Lee, S. I.; Lee, S. C.; Chung, J. W. BMB Rep. 2015, 48, 531.
13. Zhang, H. J.; Qian, Y.; Zhu, D. D.; Yang, X. G.; Zhu, H. L. Eur. J. Med. Chem. 2011,
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This research was supported by the Gangneung Asan Hospital
Biomedical Research Center Promotion Fund and a grant from
the Korea Institute of Science and Technology (2Z04371).