Targeting DNA binding for NF-κB as an anticancer approach in hepatocellular carcinoma

Article abstract of DOI:10.3390/cells7100177

Quinoline core has been shown to possess a promising role in the development of anticancer agents. However, the correlation between its broad spectrum of bioactivity and the underlying mechanism of actions is poorly understood. The present study, with the

Full text of DOI:10.3390/cells7100177

cells
Brief Report
Targeting DNA Binding for NF-κB as an Anticancer
Approach in Hepatocellular Carcinoma
Po Yee Chung 1 , Pik Ling Lam 1, Yuanyuan Zhou 1, Jessica Gasparello 2 , Alessia Finotti 2
Adriana Chilin 3, Giovanni Marzaro 3 , Roberto Gambari 2,*, Zhaoxiang Bian 4,
,
Wai Ming Kwok 1, Wai Yeung Wong 1, Xi Wang 1, Alfred King-yin Lam 5
,
Albert Sun-chi Chan 6, Xingshu Li 6, Jessica Yuen Wuen Ma 7, Chung Hin Chui 1,
Kim Hung Lam 1,* and Johnny Cheuk On Tang 1,
*
1
State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and
Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China;
chung.poyee@connect.polyu.hk (P.Y.C.); tcstacey@polyu.edu.hk (P.L.L.);
yuanyuan.09.zhou@connect.polyu.hk (Y.Z.); bcwmkwok@polyu.edu.hk (W.M.K.);
wyrwong@polyu.edu.hk (W.Y.W.); wangxi_1993@126.com (X.W.); chui.ch@gmail.com (C.H.C.)
Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy;
jessica.gasparello@unife.it (J.G.); alessia.finotti@unife.it (A.F.)
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35122 Padova, Italy;
adriana.chilin@unipd.it (A.C.); giovanni.marzaro@unipd.it (G.M.)
Clinical Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China;
bzxiang@hkbu.edu.hk
Griffith Medical School, Griffith University, Gold Coast, QLD 4222, Australia; A.Lam@griffith.edu.au
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China;
chenxz3@mail.sysu.edu.cn (A.S.-c.C.); lixsh@mail.sysu.edu.cn (X.L.)
2
3
4
5
6
7
School of Optometry, The Hong Kong Polytechnic University, Hong Kong, China;
jessicayw.ma@polyu.edu.hk (J.Y.W.M.)
*
Correspondence: gam@unife.it (R.G.); kim.hung.lam@polyu.edu.hk (K.H.L.);
dr-johnny.tang@polyu.edu.hk (J.C.O.T.); Tel.: +39-0532 974443 (R.G.); +852-3400-8727 (J.C.O.T.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 20 September 2018; Accepted: 19 October 2018; Published: 22 October 2018
Abstract: Quinoline core has been shown to possess a promising role in the development of
anticancer agents. However, the correlation between its broad spectrum of bioactivity and the
underlying mechanism of actions is poorly understood. The present study, with the use of
bioinformatics approaches, reported a series of designed molecules which integrated quinoline
core and sulfonyl moiety, with the objective of evaluating the substituent and linker effects on
anticancer activities and associated mechanistic targets. We identified potent compounds (1h
,
2h and ) exhibiting significant anticancer effects towards liver cancer cells (Hep3B) with the
,
5
8
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
relative values of cytotoxicity below 0.40, a value in the range of doxorubicin positive control with
the value of 0.12. Bulky substituents and the presence of bromine atom, as well as the presence
of sulfonamide linkage, are likely the favorable structural components for molecules exerting
a strong anticancer effect. To the best of our knowledge, our findings obtained from chemical
synthesis, in vitro cytotoxicity, bioinformatics-based molecular docking analysis (similarity ensemble
approach, SEA),and electrophoretic mobility shift assay provided the first evidence in correlation
to the anticancer activities of the selected compound
transcription factor NF- B to its target DNA. Accordingly, compound
for the development of quinoline-based NF- B inhibitors and this work added novel information on
5
with the modulation on the binding of
κ
5
represented a lead structure
κ
the understanding of the mechanism of action for bioactive sulfonyl-containing quinoline compounds
against hepatocellular carcinoma.
Cells 2018, 7, 177; doi:10.3390/cells7100177
www.mdpi.com/journal/cells

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Keywords: NF-κB; anticancer; hepatocellular carcinoma; quinolines
1. Introduction
Cancer is one of the most life threatening diseases worldwide [
1]. Thus, continuous development
of effective anticancer drugs with the emergence of drug resistance to existing anticancer agents
remains a big challenge [2]. Research work for isolating and modifying natural alkaloids from plants
and microorganisms can help scientists probe for more biological active leads [3–5]. Natural alkaloids,
such as derivatives of quinolines, are classes of nitrogen-containing heterocycles, are one of the
widest classes of natural products and originate from either natural or artificially synthesized
sources [6]. Some quinoline compounds and their derivatives are available as drugs nowadays [7];
examples include (but are not limited to) antimalarial (e.g., quinine, quinidine, chloroquine), antiviral
(e.g., saquinavir), antibacterial (e.g., ciprofloxacin, sparfoxacin and gatifloxacin), antifungal and
antiprotozoal (e.g., clioquinol), and anticancer (e.g., camptothecin, irinotecan and cabozantinib).
Quinoline scaffold has been recognized as an important source of anticancer drugs as they showed
different effects on cancer biological functions, such as growth inhibition associated with cell cycle
arrest, apoptosis, inhibition of angiogenesis, arrest of cell migration, and modulation of nuclear
receptor responsiveness [
compound which could enhance the antitumor efficiency in human cancer cell lines. Therefore, there
is a continuous research need to isolate and modify naturalalkaloids as biological active leads [ 10].
7]. More recently, Hu et al. [8] reported a tetraisohydroquinoline-based
7,9,
In particular, tropical plants, such as Galipea officinalis and Galipea longiflora, are naturally occurring
sources of several important tetrahydroquinoline and quinoline alkaloids [11]. Extensive studies,
including our reported work, demonstrated that quinoline derivatives have a wide range of biological
activities [12] leading, for instance, to anticancer [13], antimicrobial [14], and anti-inflammatory
effects [15].
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide;
the survival rate remains low (about 20%), even after the introduction of physical treatments, such
as liver transplant surgery and radiation and chemotherapy [16,17]. However, drugs with optimal
efficacy for HCC are still limited, and it has been suggested that the targeted regulation involving the
inhibition on cell migration and epithelia-mesenchymal transition (EMT) processes might be the new
directions for cancer treatment [18]. Thus, development of novel small-molecule drugs with better
potency and limited side effects could help improve survival rate of patients with HCC.
In the present study, we reported a series of designed molecules that integrated quinoline core
and sulfonyl moiety, with the objective of evaluating the substituent and linker effects on anticancer
activities in HCC cells and the associated putative mechanistic target, the transcription factor NF-κB,
with bioinformatic, and molecular analysis. The overall findings provided the first evidence about the
lead structure for the potential development of quinoline-based NF-κB inhibitor.
2. Results and Discussion
We have previously reported a reaction for the preparation of 8-substituted quinoline ethers
under basic condition in organic solvent with the help of phase transfer agent [14]. We now extended
our reported protocol into a facile reaction in water for the substitution of 8-hydroxyquinoline (8HQ)
and extended the protocol for various commercially available quinolines, such as 8-aminoquinoline
(8AQ) and 2-methyl-8-hydroxyquinoline (8QD), to prepare the corresponding substituted quinolines
for the study of substituent and linker (between quinoline core and sulfonyl group) effects on the
biological activities towards cancer cells. Quinoline derivatives 1–8 were synthesized as simple
and straightforward as shown in Scheme 1. In the presence of base and water as solvent, we
diversified the quinoline core by substituting either the hydroxyl or amine group with a variety
of substituted benzenesulfonyl chloride in the product yield of 23–96%. All the synthesized

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compounds were characterized by 1H-NMR and 13C-NMR, and the acquired spectra were shown in
the supplementary information (Figures S1–S23). The molecular structure of our lead compound
5
was further confirmed by the X-ray crystallography; the obtained structure is shown in Figure S24 in
the supplementary information.
Q
Substituted sulfonyl chloride,
R
N
Z
1-8
KOH(aq), r.t.
Y
8HQ: Q, R, Z = H; Y = OH;
8AQ: Q, R, Z = H; Y = NH2;
8BrQ: Q, R = Br, Z = H, Y = OH;
8QD: Q, R = H, Z = CH3, Y = OH;
Scheme 1. Synthesis of 8-substituted sulfonyl-containing quinolines.
Although the product yield of the substituted quinolines was not optimal, its potential for
biomedical application was substantial, as the reactivity of compounds might be governed by both
steric and electronic nature of the substituents on the aromatic ring of substrates. This method
constituted a relatively “green” way to prepare substituted quinolines from commercially available
8-hydroxyquinoline in water. However, it deserved further optimization in the future to achieve
higher yields.
Table 1 shows the biological activities of synthesized sulfonyl-containing quinolines (
study of substituent effect on anticancer activity. These tested compounds were first evaluated
using an in vitro MTS cytotoxicity assay [ ] on hepatocellular carcinoma cells (Figure 1). First,
1–3) for the
2
molecules containing bromine atom showed stronger cytotoxicity on cancer cells than that containing
trifluoromethyl group or unsubstituted. Among them, 1h and 2h exhibited the best anticancer activity
against liver cancer cells (Hep3B), indicating the importance of the presence of bromine atom in the
selected chromophore. Second, regarding the position of bromine atom on quinoline ring (2b–2d),
the bromine atom at ortho-position of the aromatic ring was associated with better activity than those
at m- or p-position. Third, the presence of an additional methyl group at 2 position (i.e., compound
2
) and changing the aromatic ring size (i.e., compound 1 vs. 3 and compound 2 vs. 3) may not
help to improve the anticancer activity on cancer cells for these types of compounds. Accordingly,
the combined effect of the presence of bromine atom and bulky group at the 8-position attributed to
the significant cell death observed in Hep3B cancer cells.
1.0
0.5
*
*
0.0
DMSO1a 1b 1c 1d 1e 1f 1g 1h 2a 2b 2c 2d 2e 2f 2g 2h 3a 3b Dox
Compound
Figure 1. The MTS relative values of compounds
1 to 3 were compared with the vehicle control (DMSO)
and the positive control (doxorubicin, Dox). *: compounds with MTS relative values <0.40.

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Table 1. Bioactivity of the tested compounds (1–3) against liver cancer cell line Hep3B and the related
docking results.
MTS Relative Values a
Nuclear Factor NF-κB
p65 Subunit: E-Value d
Compound
Drug Score c
Log P b
(Mean ± SD)
−20
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
1.05 ± 0.07
0.95 ± 0.03
0.99 ± 0.06
1.06 ± 0.08
1.01 ± 0.04
1.17 ± 0.05
0.71 ± 0.04
0.25 ± 0.03
1.08 ± 0.06
0.82 ± 0.02
1.16 ± 0.03
1.05 ± 0.08
1.05 ± 0.07
1.18 ± 0.005
0.78 ± 0.04
0.30 ± 0.04
1.04 ± 0.02
0.89 ± 0.08
2.76
4.10
3.17
3.19
3.50
3.50
3.21
3.27
3.18
3.59
3.60
3.61
3.91
3.90
3.63
3.66
3.12
3.49
0.15
0.37
0.36
0.38
0.22
0.36
0.21
0.10
0.15
0.33
0.33
0.34
0.20
0.33
0.19
0.09
0.22
0.20
2.59 × 10
−21
4.01 × 10
−16
1.12 × 10
−12
2.38 × 10
−13
7.92 × 10
−13
7.92 × 10
−17
5.39 × 10
−7
8.61 × 10
−17
6.20 × 10
−18
9.52 × 10
−20
1.13 × 10
−15
1.01 × 10
−21
2.74 × 10
−18
1.46 × 10
−16
2g
2h
3a
3b
4.07 × 10
−13
7.92 × 10
−17
1.08 × 10
−15
7.19 × 10
a
Cytotoxicity of all compounds
1
to
3
was determined against Hep3B cells using the cell proliferation assay
M for 48 h. Doxorubicin (Dox) was used as
(MTS assay). Cells were treated with the tested compounds at 15
µ
positive control while dimethyl sulfoxide (DMSO) was used as vehicle control. Each treatment was performed
in three technical and independent replicates, and the average reading to the vehicle control was calculated as
the relative MTS value. MTS relative values of DMSO and positive control (Dox) were determined as 1.00 ± 0.04
b
c
and 0.120 ± 0.004, respectively. Estimated by the ALOGPS 2.1 algorithm [19]. Estimated by the Osiris Property
Explorer (https://www.organic-chemistry.org/prog/peo/).d Estimated by the similarity ensemble approach
(SEA) [20].
To further explore the structural-and-activity relationship of sulfonyl-containing quinolines, we
synthesized some of the dimeric derivatives with different linker groups (
bioactivity of the dimeric quinoline derivatives containing sulfonyl group, while the MTS relative
values are shown in Figure 2. First, compounds containing two quinoline rings ( ) generally exhibited
a much stronger anticancer activity than that of one quinolinyl group (1 ). Second, the anticancer
4–8). Table 2 presents the
4–8
–
3
activity was likely linked to the effect of altering the substituents at 2-position, such as the methyl
group and aldehyde group. Third, we further modified the quinoline ring with bromine atoms at
the 5th and 7th positions with a pronounced anticancer activity result, indicating the presence of a
bromine atom is of certain importance towards anticancer activity. Furthermore, regarding to the linker
effect, the bioactivity of the compound with the oxygen being replaced to nitrogen at the linker part

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(i.e., compound
5) was greatly enhanced. The relative MTS value was reduced to 0.230, which was
comparable to that of the positive control (a standard chemotherapeutic drug, doxorubicin) with an
MTS value of 0.120. Figure 3 displays the morphological changes of Hep3B cells under the exposure
of the lead compound
5. The shrinkage of cancer cells was present after the treatment of compound
5
. In addition, we examined the cell-type specificity of the anticancer activity of compound
5
by
evaluating its anticancer activity against three human cancer cell lines, including human liver cancer
(Hep3B), human esophageal cancer (SLMT-1), and human breast cancer (MCF-7) cells. The determined
MTS50 values in these three cell lines ranged from 1.23 to 6.53 µM (Table 3), suggesting the anticancer
effect of 5 was not limited to the types of cancer.
Table 2. Bioactivity of the tested compounds (
4–
8) against Hep3B cell lines and the related
docking results.
MTS Relative Values a
Nuclear Factor NF-κB
Compound
Drug Score c
Log P b
(Mean ± SD)
p65 Subunit: E-Value d
−24
4
5
6
7
8
0.70 ± 0.07
0.230 ± 0.003
0.868 ± 0.009
0.76 ± 0.03
2.60
2.40
3.04
2.96
3.66
0.45
0.60
0.41
0.37
0.13
4.63 × 10
−71
9.05 × 10
−20
−36
4.45 × 10
1.27 × 10
−9
0.38 ± 0.01
5.18 × 10
a
Cytotoxicity of all compounds were determined against Hep3B cells using the cell proliferation assay (MTS assay)
as described in the legend of Table 1. Each treatment was performed in three technical and independent replicates.
MTS relative values of DMSO and positive control (Dox) were determined as 1.00
±
0.04 and 0.120 ± 0.004
,
b
c
respectively. Estimated by the ALOGPS 2.1 algorithm [19]. Estimated by the Osiris Property Explorer (https:
//www.organic-chemistry.org/prog/peo/). Estimated by the similarity ensemble approach (SEA) [20].
d
1.0
0.5
*
*
0.0
DMSO
4
5
6
7
8
Dox
Compound
Figure 2. The MTS relative values of compounds
4 to 8 were compared with the vehicle control (DMSO)
and the positive control (doxorubicin, Dox). * compounds with MTS relative values <0.40.

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Figure 3. Morphological changes of (
1.8 M), in comparison with ( ) the vehicle control (DMSO) and (
(Dox, at 1.8 M). Cancer cells showed shrinkage after the treatment of compound
A
) Hep3B cells after the 48 h treatment with compound
) the positive control, doxorubicin
and doxorubicin.
5 (at
µ
B
C
µ
5
The cells were stained by crystal violet. Each treatment was repeated in technical triplicates.
Table 3. Cytotoxicity of compound 5 against three human carcinoma cell lines †.
MTS50 Values (µM) (Mean ± SD)
Cell Line
5
Dox
Hep3B
SLMT-1
MCF-7
2.91 ± 0.09
6.53 ± 0.06
1.23 ± 0.03
0.27 ± 0.02
0.41 ± 0.03
0.28 ± 0.04
†
MTS assay was used to evaluate the cytotoxicity of compound
5
on cancer cells. MTS50 is defined as the
concentration of the test agent required to reduce the MTS activity by 50% when compared with the vehicle treated
control. Each treatment was performed in three technical and independent replicates and the corresponding MTS50
values of compound 5 and doxorubicin (Dox) were obtained for the three human carcinoma cell lines, and treated
for 48 h.
The structure-and-anticancer activity relationship of our synthesized compounds was
summarized in Figure 4. Taken together, our findings revealed some key structural requirements,
including the presence of bulky groups (e.g., benzyl ring and quinolyl ring), sulfonamide group, and
bromine atom, for the activity against Hep3B cancer cells.
Figure 4. Structure-and-activity relationship of the designed and prepared sulfonyl-containing
quinolines for studying both the linker and substituent effects on the anticancer activity.
After the lead compound
this series of compounds with some known algorithms [19
5
was identified, we further described the drug-like properties of
21]. Log P estimates the lipophilicity
,
of a chemical compound and drug score reflects the overall potential of a compound to be a drug.
The calculated log P and drug scores are listed in Tables 1 and 2. The data showed that compound
5
had the highest drug score among the test compounds, and had a log P value of 2.40 for drug
permeation across a biological membrane. With the desired in vitro anticancer activity and drug-like
properties, we employed the similarity ensemble approach [20] to identify the potential molecular
targets of our lead compound
5 and other derivatives. The expectation value (E-value) determined
from this approach indicates the chemical similarity between the biological targets and the ligands.

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According to the molecular docking results obtained from the online similarity ensemble approach
search tool [20], the nuclear factor NF- B p65 subunit was identified as the first molecular target
κ
in all the tested compounds, except for 1f, which was at the second priority. The corresponding
E-values are also listed in Tables 1 and 2. These data reflected the class of tested sulfonyl-containing
quinoline derivatives, all potentially participating in the NF-
κ
B pathway. Interestingly,
5
has the
highest priority towards the nuclear factor NF- B p65 subunit with an E-value of 9.05
κ
×
10−71 and
MaxTC of 0.75. Moreover, the derivatives containing two quinolinyl groups had smaller E-values than
those derivatives containing one quinoline ring. This observation was in a good agreement with the
trend obtained from our in vitro experimental results.
A study was also reported that N-(quinolin-8-yl)quinolin-8-sulfonamide (i.e.,compound5 in this
work), used as a ligand in a copper (II) complex, had a good ability in DNA binding and DNA
cleavage [22]. In addition, Kim et al. reported that 2-amino-N-quinoline-8-yl benzenesulfonamide
caused DNA fragmentation and led to subsequent apoptosis in Jurkat T cells [23]. With the support of
these reported studies, we further investigated and revealed the potential binding between compound
5 and DNA and/or DNA binding factors which are crucial for anticancer activities.
Figure 5 shows the potential binding mode of compound
as obtained through molecular docking simulation (AutoDock score =
experiments [24 25] fully confirmed the data depicted in Figure 5. The data obtained (Figure 6)
demonstrated that compound inhibited the interactions between NF-
B and a 32P-labelled target
oligonucleotide mimicking the NF- B binding sites. The activity of compound was similar to
that of a known NF- B inhibitor, trimethylangelicin (TMA); see the representative example shown
in Figure 6B, upper part of the panel [25 27]. Moreover, when quantitative data obtained using
compound , TMA, and another validated NF- B inhibitor (corilagin) [28] were compared, compound
was conclusively demonstrated to exert anti NF- B activity leading to inhibition of the formation of
NF- B/DNA complexes with an efficiency similar to that of other NF- B control inhibitors (Figure 6B,
lower part of the panel). Finally, the inhibitory efficiency of compound was in the range of the
activity of other classes of NF- B inhibitors studied by our group, including psoralene derivatives [25
and a set of low molecular weight compounds identified by virtual screening against p50 NF-
(data not shown) [25 29]. In agreement with the docking results, the inhibition of NF-
interactions was found only when compound
was pre-incubated with NF-kB, with 32P-labelled
target NF- B oligonucleotide added after (Figure 6A,D). On the contrary, no inhibition was obtained
when the compound B was
was pre-incubated with the 32P-labelled EMSA probe and then NF-
added (Figure 6C), supporting the concept of a direct binding of compound to NF- B and not to
5
with the DNA binding region of NF-
κB,
−
5.4 Kcal/mol). The EMSA
,
5
κ
κ
5
κ
–
5
κ
5
κ
κ
κ
5
κ
]
κ
B
,
κB/DNA
5
κ
5
κ
5
κ
DNA. This experiment, together with the docking data, suggested that the possible mechanism of
action of compound 5 likely involved NF-κB binding and regulation.
A
Figure 5. Results of docking simulation between compound
5 and NF-κB. Compound 5 was docked in
NF- B using the previously reported method. ( ) Docking pose of inhibitor (gray carbon colored stick)
κ
A
in NF-κB (pink ribbon). (B) Details of the binding mode. Compound 5 established two H-bonds with
lysine 275 and a face-to-face interaction with arginine 305. The distance and the interaction geometry
between quinoline and arginine are consistent with literature data. (
C) Comparison between the
binding mode, previously reported, for psoralene compounds and the herein considered compound
5.

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Figure 6. Effects of compound
for 20 min at the indicated concentrations with NF-
20 min incubation period was performed after adding the 32P-labelled NF-
complexes were separated by polyacrylamide gel electrophoresis. ( ) Representative data showing the
effects of trimethylangelicin (TMA) on the generation of NF- B/DNA complexes (upper part of the
panel). In the lower part of the panel the comparison of the effects of compound , TMA and corilagin
is shown. ( ) The same protocol described for panel A was followed, however, in this case, compound
was first incubated for 20 min with the 32P-labelled NF-
B probe, after which the NF- B p50 protein
was added. ( ) The effects of compound were not dependent from DMSO, added in the EMSA
5
on NF-
κ
B/DNA interactions. (
B p50 (Active Motif, cat.31101) and then a further
B probe. Then the
A) Compound 5 was pre-incubated
κ
κ
B
κ
5
C
5
κ
κ
D
5
reactions at a concentration corresponding to those of compound 5 incubations.
Heterocycles, such as isoliensinine and coumarin analogs, showed inhibition of NF-κB activation.
The coumarin analog reported by Neelgundmath et al. [30] possessed an ability to induce the apoptosis
in HepG2 cells by suppressing the DNA binding ability of NF- B, while a decrease in NF- B activity
and phosphorylation of NF- B p65 subunit were observed in isoliensinine-treated HepG2 cells [31].
κ
κ
κ
Additionally, a simple quinoline derivative, 8-hydroxyquinoline, was reported to inhibit the NF-
activation in Raw 264.7 cells [32].
κB
Quinoline is a remarkable class of compounds, found in both natural and synthetic sources,
with a broad spectrum of biological activities [33]. We previously reported a series of 8-substituted
quinoline ethers possessing anti-Aspergillus niger activity [14], and more recently, we discovered a
hexahydrofuro[3,2-c]quinoline showed anti-breast cancer activity, as well as its enhanced effect on the
fungistatic activity of micronazole [34]. Simeprevir, an orally-administered quinoline-based specific
protease inhibitor, was approved by the Food and Drug Administration (FDA) for the clinical treatment
of chronic hepatitis C virus infection [35–37]. This molecule contains aquinoline ring and a small
sulfonamide group. Previous studies also reported 2-sulfolmethyl quinolines showing anti-hepatitis B
virus activity [38] and antiproliferative activity [39] against HepG2 cancer cells in vitro.
Sulfonamides are well-known for their medicinal values and coordination properties that are
able to bind to Zinc [40] or formed as metal complex to induce DNA damages by photoradiation [22].
However, studies on the anticancer effect of O-sulfonyl-containing quinolones are still sparse.
The functionality of sulfonyl moiety is presumably advantageous to a chemical structure of
potential drug candidates, because of the availability of hydrogen bonding and the constraint on the
side chains, which allow a specific conformation of molecules. These two important features could
lead to stronger interaction at the active site of the biological targets. Herein, taking the promising

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anticancer effect of NF-
κ
B inhibitors, we aimed to design and synthesize a series of sulfonyl-containing
quinolines with rationale for the evaluation of substituent and linker effects on their in vitro anticancer
activity. All synthesized compounds were screened for their in vitro anticancer activity in Hep3B
hepatocellular carcinoma cells. Subsequently, the lead compound was also tested on esophageal
carcinoma and breast cancer cells. Most importantly, the mechanism of anticancer effect was studied
by the bioinformatics approach with a molecular docking analysis (similarity ensemble approach,
SEA) followed by the associated molecular studies. The overall findings of the present work paved the
new path for developing the sulfonyl-containing quinolines as the leads for future anticancer drug
development which combines the chemical synthesis, genomic, and bioinformatics-based strategies.
The transcription factor NF-κB is a potential therapeutic target. Regulation of NF-κB may
result in a targeted therapy and control of the chemoresistance in cancer cells [41]. This target is
a transcription factor and plays a decisive role in several biological processes, involving cell cycle
regulation [42], cell differentiation, and apoptosis [43
that targeting NF- B could be an effective direction for anticancer treatment, as it suppresses cancer cell
migration and epithelia-mesenchymal transition [45 46]. Previous studies showed that targeting the
NF- B had anticancer effects in breast cancer [47], colon cancer [48], and esophageal cancer cells [49].
Zuo et al. [50] also demonstrated the modulation of NF- B in the regulation of human telomerase
,44]. More importantly, it was strongly suggested
κ
,
κ
κ
reverse transcriptase (hTERT) gene transcription, which was highly correlated to the pathogenesis of
hepatocellular carcinoma.
The results presented support the concept that compound
5 deserves further studies to better
characterize its biological effects on one side, and its mechanism of action on the other. These further
efforts include (but are not limited to) the analysis of the activity on other transcription factors
(in order to further verify the specificity of the treatment), the analysis of transcriptome (in order
to verify the activity on NF-κB regulated genes, including those involved in cell migration and
epithelia-mesenchymal transition process) and chromatin immunoprecipitation assays (in order to
map the lack of binding of NF-κB to gene promoters containing NF-κB binding sites).
In conclusion, the results presented in this study identified a lead compound (compound
a series of 8-substituted sulfonyl-containing quinolines which potentially targets NF- B signaling to
induce cell death in hepatocellular cancer. It also provided the first evidence for the anticancer effect
of quinoline-type compound with the binding to NF- B based on the bioinformatics and proven
5) among
κ
5
κ
by molecular analysis. Further full-scale investigations will involve the anticancer studies on the
suppression on cancer cell migration, epithelia-mesenchymal transition and the use of chromatin
immunoprecipitation (ChIP) assay for probing protein-DNA interactions in HCC and other cancer
types to fully elucidate the underlying anticancer actions for the cell death induced by our lead
compound 5 as well as further development of effective anticancer strategies.
3. Materials and Methods
3.1. Chemistry
3.1.1. Materials
All reagents and chemicals were purchased from commercial sources. The synthesized
compounds were characterized by melting point, mass spectrometry, 1H-, and 13C-NMR. 1H- and
13C-NMR spectra were recorded on a Brucker DPX 400 spectrometer (Bruker, Switzerland) in CDCl3
unless otherwise specified.
3.1.2. General Procedure for the Synthesis of 1–4,6
To get a solution of 8-hydroxyquinoline or 8-hydroxyquinaldine (0.69 mmol, 1.1 equiv) in
water (2 mL), substituted benzenesulfonyl chloride (0.62 mmol, 1.0 equiv) and potassium hydroxide
(0.69 mmol, 1.1 equiv) were added and stirred at room temperature. The reaction progress was

Cells 2018, 7, 177
10 of 14
monitored by thin layer chromatography. After the reaction completed, the aqueous phase was
extracted with dichloromethane and dried over anhydrous sodium sulfate. Solvent was removed by
rotary evaporator (Heidolph, Schwabach, Germany) and the crude product was purified by silica gel
column chromatography.
3.1.3. General Procedure for the Synthesis of 5
A solution of 8-aminoquinoline (0.62 mmol, 1.0 equiv), 8-quinolinesulfonyl chloride (0.62 mmol,
1.0 equiv), and triethylamine (0.62 mmol, 1.0 equiv) in acetonitrile was stirred at room temperature.
The reaction progress was monitored by thin layer chromatography. When the reaction
completed, solvent was removed under reduced pressure. The reaction mixture was extracted with
dichloromethane and washed with water, dried over anhydrous sodium sulfate. Solvent was removed
by rotary evaporator (Heidolph, Schwabach, Germany) and the crude product was purified by silica
gel column chromatography.
3.1.4. General Procedure for the Synthesis of 8
Compound 8 was prepared from the reactant, 5,7-dibromo-8-hydroxyquinoline, using the method
previously reported by our group [14]. The preparation followed the protocol stated in the study,
unless otherwise specified.
3.1.5. General Procedure for the Synthesis of 7
Compound
7 was prepared by the oxidation of compound 6 using selenium dioxide under reflux
in 1,4-dioxane, where the method was previously reported by our group [51].The preparation followed
the protocol stated in the study, unless otherwise specified.
The characterization of the synthesized compounds is described in the supplementary information.
3.2. Biological Analysis
3.2.1. Cell Lines and Cell Culture
Human hepatocellular carcinoma Hep3B, human esophageal squamous cell carcinoma
SLMT-1 [52], and human breast carcinoma MCF-7 cell line were used for the preliminary evaluation
of the possible anticancer activity of the synthesized compounds. Hep3B, and MCF-7 cell lines were
cultured in 90% of DMEM (Dulbecco’s Modified Eagle Medium) culture medium supplemented with
10% fetal bovine serum and 1% penicillin antibiotics while SLMT-1 cell line was cultured in 90% of
MEM (Minimum Essential Medium) Alpha culture medium supplemented with 10% fetal bovine
serum and 1% penicillin antibiotics. The cells were incubated at 37◦C, 5% CO2.
3.2.2. In Vitro MTS Cytotoxicity Assay
The anticancer activity of synthesized compounds was evaluated against three cancer cell lines,
namely Hep3B, SLMT-1, and MCF-7, by standard MTS assay as we reported before [53]. Cells were
seeded in 96 well microtiter plates with a cell density of 5000 for 24 h for anticancer activity screening.
Doxorubicin (Dox) and dimethylsulfoxide (DMSO, molecular biology grade) were used as positive
control and a vehicle control, respectively. Synthesized compounds were added at the desired
concentration and incubated for further 48 h. The relative cell viability in each well was determined by
using MTS assay. Each treatment point was repeated in three technical and independent replicates.
3.2.3. Molecular Docking Analyses
Computational studies were conducted on a 4 CPU (Intel Core2 Quad CPU Q9550, 2.83 GHz,
Ubuntu, London, UK) ACPI x46 Linux workstation with Ubuntu 12.04 operating system (Ubuntu,
London, UK). The NF-κB structure was derived from the Protein Data Bank (PDB code: 1NFK, p50) and
handled with Chimera 5.3.1 software (RBVI, Resource for Biocomputing, Visualization, and Informatics,

Cells 2018, 7, 177
11 of 14
University of California, CA,USA, www.cgl.ucsf.edu/chimera/) [54]. The structure of compound
was prepared with MarvinSketch 5.5 software (Marvin, version 5.5.0.1, Program B; ChemAxon:
5
Budapest, Hungary; www.chemaxon.com/products) and was minimized and protonated (pH = 7.4)
with OpenBabel 2.2.3 software (Version 2.2.3, http://openbabel.org) [55], using the MMFF94s force
field. Docking studies were performed with AutoDock 4.2 software (http://autodock.scripps.edu/)
provided with AutoDock Tools 1.5.4 graphical interface [56]. According to the previous study [25],
docking simulations were performed in a docking box characterized by 0.375 Å spacing and by 54
×
54 54 points dimensions. The box was centered at x = 11.11, y = 18.23, z = 15.33. Docking pose
×
−
was obtained through Lamarckian genetic algorithm searching engine, carrying out 50 runs with a
maximum of 2,500,000 energy evaluations. Cluster analysis was performed on obtained results, with a
root-mean square tolerance of 2.0 Å, finally selecting the lowest energy pose.
3.2.4. Electrophoretic Mobility Shift Assay (EMSA)
Electrophoretic mobility shift assays were performed by using as target DNA double-stranded
32P-labeled oligonucleotides mimicking an NF-
κ
B binding site. Binding reactions were set up as
described elsewhere in binding buffer (5% glycerol, 20 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% TRITON
X-100, 50 mM KCl, 0.2 mM EDTA, 0.05 g/ L dIdC and 1 mM MgCl2) and labeled oligonucleotides
were incubated in the presence of compound at different concentrations. The effects of DMSO
on NF- B/DNA interactions were analyzed at relevant concentrations, demonstrating no inhibitory
effects. After 20 min of binding of compound (resuspended in DMSO) at room temperature to
NF- B oligonucleotides was
B-p50 (Active Motif, cat.31101), incubation with the 32P-labeled NF-
performed in a total final volume of 20 L for additional 20 min, at room temperature. Then,
the samples were electrophoresed at constant voltage (200V for 30 min) through low ionic strength
buffer (0.25 )Tris/borate/EDTA buffer (22.5 mM Tris, 22.5 mM boric acid, 0.5 mM EDTA, pH 8)
µ
µ
5
κ
5
κ
κ
µ
×
on 6% polyacrylamide gels until tracking dye (bromophenol blue) reached the end of a 16 cm slab.
◦
Gels were dried and exposed for autoradiography with intensifying screens at
−
80 C. In these
experiments, DNA/protein complexes migrate through the gel with slower efficiency. The nucleotide
sequences of double-stranded target DNA utilized in these experiments were 50-ATC GGG GGA
AAT CCC AGA AC-30 (sense strand, NF-
κ
B). The synthetic oligonucleotides used in this study were
purchased from IDT (Integrated DNA Technologies, Leuven, Belgium).Control NF- B inhibitors
were trimethylangelicin (TMA) and -1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose (corilagin).
Synthesis of TMA has been reported elsewhere [25 27]; corilagin was obtained from the China
κ
β
–
National Institute for the Control of Pharmaceutical and Biological Products. For quantitative analyses,
the autoradiographs were scanned through a Gel-Doc Instrument (Biorad, Hercules, CA, USA) using
the QuantityOne software (Version 4, Biorad, Hercules, CA, USA).
Supplementary Materials: The supplementary materials are available.
Author Contributions: P.Y.C. and Y.Z. were the PhD students who contributed to the arrangement of all
experiments, analysis of results and writing-up of the paper. R.G. group: J.G., A.F., A.C. and G.M. were responsible
for the overall planning, docking analyses and EMSA experiments. Z.B., C.H.C. and P.L.L. gave directional advices
related to the molecular docking and EMSA analyses. W.Y.W., W.M.K., A.S.-c.C. and X.L. gave advices to the parts
of chemical synthesis. A.K.-y.L., J.Y.W.M. and X.W. contributed to the in vitro cytotoxicity assays and analysis of
results. K.H.L. worked closely with J.C.O.T. to initiate and supervise the present work.
Funding: This work was supported by the Innovation Technology Commission (Hong Kong) to ABCT, HKPU
and the State Key Laboratory of Chirosciences (1-BBX8). R Gambari is supported by AIRC (IG-13575) and by CIB
(Interuniversity Consortium of Biotechnologies, Italy). Lastly, J.C.T. would like to acknowledge the Lo Ka Chung
Foundation Fund (a/c: ZE20 and 954P) and the MOU signed with the Hong Kong Baptist University, Hong Kong
and Griffith University, Australia.
Conflicts of Interest: The authors declare no conflict of interest.

Cells 2018, 7, 177
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