Novel synthetic coumarins that targets NF-κB in Hepatocellular carcinoma

Article abstract of DOI:10.1016/j.bmcl.2014.12.065

Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor worldwide, and is the third most common cause of cancer related death. Constitutive activation of NF-κB is the underlying mechanism behind tumorigenesis and this protein regulates the expression of genes involved in proliferation, survival, drug resistance, angiogenesis and metastasis. The design of inhibitors which suppress NF-κB activation is therefore of great therapeutic importance in the treatment of HCC. In this study, we investigated the effect of newly synthesized coumarin derivatives against HCC cells, and identified (7-Carbethoxyamino-2-oxo-2H-chromen-4-yl)methylpyrrolidine-1 carbodithioate (CPP) as lead compound. Further, we evaluated the effect of CPP on the DNA binding ability of NF-κB, CXCL12-induced cell migration and invasion, and the regulated gene products in HCC cells. We found that CPP induced cytotoxicity in three HCC cells in a time and dose dependent manner, and suppressed the DNA binding ability of NF-κB. CPP significantly decreased the CXCL12-induced cell migration and invasion. More evidently, CPP inhibits the expression of NF-κB targeted genes such as cyclin D1, Bcl-2, survivin, MMP12 and C-Myc. Furthermore, the molecular docking analysis suggested that CPP interacts with the p50 binding domain of the p65 subunit, scoring best among the 26 docked coumarin derivatives of this study. Thus, we are reporting CPP as a potent inhibitor of the pro-inflammatory pathway in Hepatocellular carcinoma.

Full text of DOI:10.1016/j.bmcl.2014.12.065

Bioorganic & Medicinal Chemistry Letters 25 (2015) 893–897
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel synthetic coumarins that targets NF-
jB in Hepatocellular
carcinoma
a,b,
c,
Mahabaleshwaraiah Neelgundmath
, Koragere Rajashekar Dinesh
,
d,
e
e
e
Chakrabhavi Dhananjaya Mohan , Feng Li , Xiaoyun Dai , Kodappully Sivaraman Siveen ,
f,g
f
f
e
f
Shardul Paricharak , Daniel J. Mason , Julian E. Fuchs , Gautam Sethi , Andreas Bender ,
d
b,
⇑
c,
⇑
Kanchugarakoppal S. Rangappa , Obelannavar Kotresh , Basappa
a
Department of Chemistry, P.C. Jabin Science College, Hubli, India
Department of Chemistry, Karnataka Science College, Dharwad, India
Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Central College Campus, Palace Road, Bangalore 560001, India
Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore 570005, India
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore, Singapore
b
c
d
e
f
Centre for Molecular Science Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, United Kingdom
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
g
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor worldwide, and is the third
Received 31 August 2014
Revised 27 November 2014
Accepted 19 December 2014
Available online 30 December 2014
most common cause of cancer related death. Constitutive activation of NF-
nism behind tumorigenesis and this protein regulates the expression of genes involved in proliferation,
survival, drug resistance, angiogenesis and metastasis. The design of inhibitors which suppress NF-
activation is therefore of great therapeutic importance in the treatment of HCC. In this study, we inves-
tigated the effect of newly synthesized coumarin derivatives against HCC cells, and identified (7-Carbeth-
oxyamino-2-oxo-2H-chromen-4-yl)methylpyrrolidine-1 carbodithioate (CPP) as lead compound. Further,
jB is the underlying mecha-
j
B
Keywords:
Hepatocellular carcinoma
Coumarin
NF-jB
Bioinformatics
we evaluated the effect of CPP on the DNA binding ability of NF-
invasion, and the regulated gene products in HCC cells. We found that CPP induced cytotoxicity in three
HCC cells in a time and dose dependent manner, and suppressed the DNA binding ability of NF- B. CPP
significantly decreased the CXCL12-induced cell migration and invasion. More evidently, CPP inhibits the
expression of NF- B targeted genes such as cyclin D1, Bcl-2, survivin, MMP12 and C-Myc. Furthermore,
jB, CXCL12-induced cell migration and
j
Inflammation
j
the molecular docking analysis suggested that CPP interacts with the p50 binding domain of the p65 sub-
unit, scoring best among the 26 docked coumarin derivatives of this study. Thus, we are reporting CPP as
a potent inhibitor of the pro-inflammatory pathway in Hepatocellular carcinoma.
Ó 2014 Elsevier Ltd. All rights reserved.
Hepatocellular carcinoma (HCC) is the most common form of
kappa B (NF-jB) is an inducible transcription factor present ubiqui-
liver cancer, and is the third most common cause of cancer related
mortality. Obesity, hepatitis B or hepatitis C virus infection, con-
tously in the cytoplasm of almost all mammalian cells, and was
identified by Baltimore and colleagues in 1986. In an unstimulated
1
5
sumption of aflatoxin B1, and alcoholic hepatitis are the major risk
factors of HCC. The majority of patients with HCC are diagnosed at
cell, NF-jB is associated with IjB (inhibitory jB), and resides in the
cytoplasm. Different ligands acting upon the receptor result in a
2
6
a late stage of the disease, with therapy limited to only liver trans-
plantation and surgical liver sectioning. Early detection of HCC may
contribute to the strengthening of prognosis.3 Nuclear factor
cascade of upstream kinases, finally leading to phosphorylation
and ubiquitylation of I
jB, which is targeted for degradation. NF-
,4
j
B translocates into nucleus and regulates transcription of the
7
genes involved in inflammation. NF-jB is referred to as a dou-
ble-edged sword by researchers because of its critical role in proper
Abbreviation: CPP, (7-Carbethoxyamino-2-oxo-2H-chromen-4-yl)methylpyrrol-
idine-1 carbodithioate.
functioning of the immune system, and the possibility that onco-
8
⇑
Corresponding authors. Tel.: +91 9686449876 (Basappa).
E-mail address: salundibasappa@gmail.com (Basappa).
These authors contributed equally.
genesis may arise from the slightest change in regulation. Several
anti-apoptotic, angiogenic, inflammatory, metastasis-related, and
http://dx.doi.org/10.1016/j.bmcl.2014.12.065
960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
0

8
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M. Neelgundmath et al. / Bioorg. Med. Chem. Lett. 25 (2015) 893–897
Figure 1. Synthesis of coumarin-based small molecules libraries.
3
Figure 3. (A) CPP suppresses NF-jB DNA binding ability in HepG2 cells. HepG2 cells
Figure 2. (A) HepG2 cells (5 Â 10 /mL) were plated in triplicate, treated with
were treated with 50
lM CPP for indicated time points; nuclear extracts were
indicated concentrations of CPP, and then subjected to MTT assay after 24, 48 and
2 h to analyze proliferation of cells. Standard deviations between the triplicates
are indicated. Indicates p value <0.05. (B) CPP potentiates PARP cleavage. HepG2
prepared, and 5 g of the nuclear extract protein was used for ELISA-based DNA
l
7
⁄
binding assay. The results shown are representative of two independent experi-
⁄
6
ments P <0.05. (B) CPP suppresses NF-
j
B regulated gene products involved in
cells (2 Â 10 /mL) were treated with CPP for time intervals, after which whole-cell
6
proliferation, survival and angiogenesis. HepG2 cells (2 Â 10 /mL) were treated
with 50 M CPP for indicated time intervals, after which whole-cell extracts were
prepared and 30 g proteins of those extracts were resolved on 10% SDS–PAGE,
membrane sliced according to molecular weight and probed against cyclin D1, Bcl-
, survivin and C-Myc antibodies. The same blots were stripped and reprobed with
extracts were prepared and 30 lg protein of those extracts were resolved on 10%
l
SDS–PAGE, membrane sliced according to molecular weight and probed against
PARP antibodies. The same blots used to detect b-actin to verify equal protein
loading.
l
2
b-actin antibody to verify equal protein loading. The results of cyclin D1, Bcl-2,
survivin and C-Myc shown are representative of two independent experiments. (C)
6
CPP induces MMP12 cleavage. HepG2 cells (2 Â 10 /mL) were treated with CPP for
proliferative proteins, including cIAP, Bcl-2, c-Myc, MMP 12, and
B signaling.8
,9
indicated concentrations, after which whole-cell extracts were prepared and 30 lg
COX2, are regulated by NF-
j
proteins of those extracts were resolved on 10% SDS–PAGE, membrane sliced
according to molecular weight and probed against MMP12 antibodies. The same
blots used to detect b-actin to verify equal protein loading.
Coumarins were originally isolated in early 1820 from tonka
beans, and were later used for various domestic purposes.10 Since

M. Neelgundmath et al. / Bioorg. Med. Chem. Lett. 25 (2015) 893–897
895
Figure 4. CPP suppresses migration in HCC cells. An IBIDI culture insert (IBIDI GmbH) consists of two reservoirs separated by a 500
l
m thick wall created by a culture insert
5
in a 35 mm petri dish. For migration assay, an equal number of HepG2 cell (70
l
L; 5 Â 10 cells/mL) were added into the two reservoirs of the same insert and incubated at
3
7 °C. After 12 h, the insert was gently removed creating a gap of ꢀ500
lm. The cells were treated with CPP (50 lM) for 8 h before being exposed to 100 ng/mL CXCL12 for
2
4 h. Width of wound was measured at time zero and 24 h of incubation in the absence or presence of CXCL12. The representative photographs showed the same area at time
zero and after 24 h of incubation and columns represent mean distance of migrated cells in the presence or absence of CPP and CXCL12.
their first identification, researchers have discovered an enormous
number of coumarin based natural compounds with different phar-
macological properties. Fraxetin, esculetin, and daphnetin are some
of the coumarin related natural compounds with very good
agent,20 which results in the formation of substituted-4-bro-
momethylcoumarins 1(a–m). A one pot reaction of carbon disul-
fide and dimethylamine/pyrrolidine in ethanol was prepared for
compounds 1(a–m), stirring at room temperature for 24 h, which
resulted in the formation of coumarinyldithiocarbamates (Fig. 1).
Organic materials were extracted with dichloromethane, before
1
1
inhibitory activity against cyclooxygenase and lipoxygenase.
Researchers have exploited the anti-tumor, appetite-suppressor,
anti-inflammatory and antioxidant activities of many natural and
2 4
being separated and dried over anhydrous Na SO . The solvent
1
2
synthetic coumarin based compounds both in vitro and in vivo.
was removed under reduced pressure to obtain the desired prod-
ucts, and the crude product was further purified by flash column
chromatography. In some cases, it was necessary to obtain
crystallized structures from the mixture of ethanol and chloroform
solvents. The structures of all the target compounds were charac-
As part of our continued research into identification of new inhibi-
tors against various signaling cascades in cancer and inflammation,
herein, we investigated whether coumarins may exert their anti-
3–19
cancer effect by disruption of NF-
j
B signaling.1
We synthesized
1
13
a library of coumarin derivatives, and evaluated their effect on
Hepatocellular carcinoma cells. We found (7-Carbethoxyamino-
terized by elemental analysis, GCMS, IR, H NMR, and C NMR
spectrometry.
2
-oxo-2H-chromen-4-yl)methylpyrrolidine-1 carbodithioate (CPP)
to be a potent cytotoxic molecule, causing significant reduction in
NF- B targeted gene products, increasing apoptosis, and suppress-
ing the DNA binding ability of NF- B.
We investigated cytotoxic effect of a newly synthesized series
of compounds on HepG2 cells using an MTT assay as described pre-
2
1,22
j
viously (Supplementary Table 3).
CPP was found to be most
j
effective compound among the tested compounds, and MTT assay
was used to further investigate whether CPP can inhibit the prolif-
eration of HepG2, PLC/PRF5, and Huh7. Our results showed that
CPP inhibits the proliferation of all the three cell lines (data not
shown for PLC/PRF5 and Huh7) in both a dose and time dependent
manner (Fig. 2A). However CPP did not show high cytotoxic effect
The synthetic route for coumarin-based small molecule libraries
is outlined in Figure 1. 4-Bromoethylacetoacetate is obtained from
the bromination of ethyl-acetoacetate, and subsequently treated
with various substituted phenols under Pechmann cyclization
conditions using concentrated sulfuric acid as the dehydrating

8
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M. Neelgundmath et al. / Bioorg. Med. Chem. Lett. 25 (2015) 893–897
on LO2 cells up to 72 h at 50
have cytotoxic effect on normal cells.
l
M indicating that the CPP does not
can result in suppression of its gene products. Therefore, we inves-
tigated whether the NF- B signaling in HepG2 cells can be modu-
j
We also investigated whether suppression of constitutively
lated by newly synthesized coumarins. Among the new series of
coumarins, CPP proved to be a potent anti-proliferative agent
against all the three HCC cells and did not show toxicity towards
non-diseased cells. Further evaluation showed that CPP decrease
active NF-
time-dependent cleavage of a 116 kDa Poly ADP-ribose polymerase
PARP) protein into an 85 kDa fragment (Fig. 2B). These results
jB in HepG2 cells by CPP leads to apoptosis. CPP induced
(
clearly suggest that CPP induces substantial apoptosis in HepG2
cells.
the DNA binding ability of NF-
in corresponding gene products. This reduction was apparent dur-
ing evaluation of the levels of NF- B regulated gene products such
jB, indicating the possible reduction
NF-
cell proliferation and survival. Therefore, we investigated whether
CPP can modulate the DNA binding activity of NF- B in HCC cells.
Analysis of nuclear extracts prepared from HepG2 cells, using an
ELISA based TransAM NF- B assay kit, showed that CPP inhibited
the NF- B-DNA binding activities in a time dependent manner
Fig. 3A). These results suggest that CPP can suppress the DNA
binding ability of NF- B.
NF- B activation has been shown to regulate the expression of
various gene products involved in cell survival, proliferation, angi-
jB is a transcription factor that plays an important role in
j
as cyclin D1, Bcl-2, Survivin, C-Myc, and MMP12. PARP is an inte-
j
gral part of cellular DNA repair machinery and a target of cas-
3
0
pase-3 and caspase-7.
Our western blotting results clearly
j
suggest the cleavage of PARP from a 116 kDA protein into an
89 kDA fragment, evidencing that CPP induces apoptosis. We also
found that CPP exert anticancer effects by inhibiting migration
and invasion of HCC cells. Thus, our results strongly demonstrate
that the anti-oncogenic property of CPP in HCC is mediated
j
(
j
j
through suppression of NF-jB signaling pathway.
ogenesis and chemoresistance.5
,23,24
We found that expression of
the cell cycle regulator cyclin D1, and the antiapoptotic proteins
Bcl-2, survivin, and, C-Myc, and MMP12 which are reported to be
regulated by NF-
jB, were modulated by CPP treatment. Their
expression decreased across different dose and time intervals
(Fig. 3B and C).
We also determined the effect of CPP on HCC cell invasion, a key
hallmark of cancer.2
5,26
Using an in vitro migration assay, we found
that HepG2 cells migrated faster under the influence of CXCL12,
and this effect was abolished upon treatment with CPP (Fig. 4).
To elucidate further the effect of CPP on CXCL12-induced cell inva-
sion, we used the BD BioCoat Matrigel invasion assay system and
found that treatment with CPP suppressed CXCL12-induced inva-
sion of HepG2 cells (Fig. 5).
We simulated molecular docking of coumarins with a I
B crystal structure (PDB: 1IKN). For details of the docking routine,
please see Supplementary information. Docked coumarins were
observed at the interface of p65 (a subunit of NF- B) and I
an inhibitory protein of NF- B), thereby replacing the p50
jBa/NF-
j
j
jBa
(
j
sub-unit in the complex (Fig. 6). A hydrophobic cavity formed by
His-245 and Arg-246 of p65 and Tyr-248, Tyr-251, and Tyr-289 is
targeted. These regions are partially occupied by Val-251 and
Leu-269 in the native complex with p50. Lead compound CPP
scored best amongst the 26 docked coumarin compounds. In
addition to the hydrophobic interactions predicted for most
members of the series, this compound extends further into a more
polar region. Thereby, we observe hydrogen bonding between
CPP’s ester moiety and the side-chain of Tyr-251 and hydrogen
bonding to the backbone of Ser-288. The coumarin scaffold is
tightly bound via Van der Waals interactions and a hydrogen bond
to the backbone of Asp-290.
In conclusion, the over activation of NF-
jB in cancer cells pro-
vides a unique target for the design of therapeutic agents. NF-
jB
is reported to regulate more than 200 genes connected to cell pro-
liferation (cyclin D1 and C-Myc), survival (survivin), anti-apoptosis
(
(
Bcl-2), inflammation (COX2), angiogenesis (VEGF), and metastasis
8
,27
MMPs).
Of these gene products, cyclin D1 and C-Myc are cru-
cial cell cycle regulators involved in proliferation, Bcl-2 and survi-
vin are anti-apoptotic proteins, and survivin is reported to
associate with drug resistance in breast cancer.2 MMPs are a large
class of proteins with more than 20 types, and generally noted as
extracellular matrix degrading proteases. Many MMPs contribute
to the dislodging and migration of tumor cells, and have been
implicated in cancer progression. MMP1, MMP3 and MMP12 are
8
5
Figure 5. CPP suppresses invasion in HCC cells. A, HepG2 (1 Â 10 cells) were
seeded in the top-chamber of the BD Matrigel invasion chamber. After pre-
incubation with or without 50 lM CPP for 8 h, transwell chambers were then
placed into the wells of a 24-well plate into which we added either the basal
medium only or basal medium containing 100 ng/mL CXCL12 in a predetermined
arrangement. After incubation, for 24 h cell invasion was analyzed and columns
represent mean percentage of invaded cells.
the major NF-jB regulated matrix metalloproteinase genes. The
role of MMP12 in cancer development is not articulate, but
researchers correlated the elevated levels of MMP12 with poor
prognosis of HCC in the past.29 Blockade of NF-
jB DNA binding

M. Neelgundmath et al. / Bioorg. Med. Chem. Lett. 25 (2015) 893–897
897
Figure 6. Predicted molecules interactions between CPP and the I
jB
a/NF-
jB complex. (A) Ribbon diagram of the native I
j
B
a/NF-jB complex. The p50 sub-unit is show in
green, p65 in cyan, and IjBa in pink. (B) Ensemble of docking solution for the 26 coumarin compounds (in stick representation, and atomic coloring with green carbons)
targeting the p50/p65 interface. (C) Close-up view of the binding mode of lead compound CPP. CPP shows hydrogen bonding (yellow dots) to the backbone of Ser-288, the
backbone of Asp-290, and the side-chain of Tyr-251.
1
1
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Acknowledgments
This research was supported by University Grants Commission
63.
(
41-257-2012-SR), Vision Group Science and Technology, Depart-
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to K.S.R. This work was supported by grants from NUS Academic
Research Fund [Grants R-184-000-207-112] to G.S. Basappa thanks
Karnataka University, INDIA for PAVATE fellowship. C.D.M. thanks
DST for INSPIRE fellowship. S.P. thanks the Netherlands Organisa-
tion for Scientific Research (NWO, grant number NWO-017.009-
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ONE 2012, 7, e39444.
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Supplementary data (detailed experimental procedures for the
synthesis and the pharmacological investigations) associated with
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