3,6-disubstituted 1,2,4-triazolo[3,4-b] thiadiazoles with anticancer activity targeting topoisomerase II alpha

Article abstract of DOI:10.2147/OTT.S254856

Background: Topoisomerase IIα (topIIα) maintains the topology of DNA in order to ensure the proper functioning of numerous DNA processes. Inhibition of topIIα leads to the killing of cancer cells thus constituting such inhibitors as useful tools in cancer therapeutics. Triazolo[3,4-b]thiadiazole derivatives are known for their wide range of pharmacological activities while previous studies have documented their in vitro anticancer activity. The purpose of the current study was to investigate if these chemical compounds can act as topIIα inhibitors in cell-free and cell-based systems. Materials and Methods: The MTT assay was performed in DLD-1, HT-29, and LoVo cancer cells so as to evaluate the antiproliferative activity of KA25, KA26, and KA39 triazolo[3,4-b]thiadiazole derivatives. The KA39 compound was tested as a potential topIIα inhibitor using the plasmid-based topoisomerase II drug screening kit. The inhibitory effect of the three derivatives on topIIα phosphorylation was studied in HT-29 and LoVo cancer cells according to Human Phospho-TOP2A/Topoisomerase II Alpha Cell-Based Phosphorylation ELISA Kit. Moreover, flow cytometry was utilized in order to explore apoptotic induction and cell cycle growth arrest, upon treatment with KA39, in DLD-1 and HT-29 cells, respectively. In silico studies were also carried out for further investigation. Results: All three triazolo[3,4-b]thiadiazole derivatives showed an in vitro antiproliferative effect with the KA39 compound being the most potent one. Our results indicated that KA39 induced both early and late apoptosis as well as cell cycle growth arrest in S phase. In addition, the compound blocked the relaxation of supercoiled DNA while it also inhibited topIIα phosphorylation (upon treatment; P<0.001). Conclusion: Among the three triazolo[3,4-b]thiadiazole derivatives, KA39 was shown to be the most potent anticancer agent and catalytic inhibitor of topIIα phosphorylation as well.

Full text of DOI:10.2147/OTT.S254856

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O R I G I N A L R E S E A R C H
3,6-Disubstituted 1,2,4-Triazolo[3,4-b]
Thiadiazoles with Anticancer Activity Targeting
Topoisomerase II Alpha
This article was published in the following Dove Press journal:
OncoTargets and Therapy
Sofia Sagredou1
Background: Topoisomerase IIα (topIIα) maintains the topology of DNA in order to ensure
1
the proper functioning of numerous DNA processes. Inhibition of topIIα leads to the killing
Panagiotis Dalezis
Nikolaos Nikoleousakos1
of cancer cells thus constituting such inhibitors as useful tools in cancer therapeutics.
1
Triazolo[3,4-b]thiadiazole derivatives are known for their wide range of pharmacological
activities while previous studies have documented their in vitro anticancer activity. The
purpose of the current study was to investigate if these chemical compounds can act as topIIα
inhibitors in cell-free and cell-based systems.
Michail Nikolaou
2
Maria Voura
Konstantinos Almpanakis2
3,4
Mihalis I Panayiotidis
Materials and Methods: The MTT assay was performed in DLD-1, HT-29, and LoVo
cancer cells so as to evaluate the antiproliferative activity of KA25, KA26, and KA39
triazolo[3,4-b]thiadiazole derivatives. The KA39 compound was tested as a potential
topIIα inhibitor using the plasmid-based topoisomerase II drug screening kit. The inhibitory
effect of the three derivatives on topIIα phosphorylation was studied in HT-29 and LoVo
cancer cells according to Human Phospho-TOP2A/Topoisomerase II Alpha Cell-Based
Phosphorylation ELISA Kit. Moreover, flow cytometry was utilized in order to explore
apoptotic induction and cell cycle growth arrest, upon treatment with KA39, in DLD-1
and HT-29 cells, respectively. In silico studies were also carried out for further investigation.
Results: All three triazolo[3,4-b]thiadiazole derivatives showed an in vitro antiproliferative
effect with the KA39 compound being the most potent one. Our results indicated that KA39
induced both early and late apoptosis as well as cell cycle growth arrest in S phase. In
addition, the compound blocked the relaxation of supercoiled DNA while it also inhibited
topIIα phosphorylation (upon treatment; P<0.001).
2
Vasiliki Sarli
Dimitrios T Trafalis1
1Laboratory of Pharmacology, Faculty of
Medicine, National and Kapodistrian
University of Athens, Athens 11527,
Greece; 2Department of Chemistry,
Aristotle University of Thessaloniki ,
Thessaloniki, 54124, Greece;
3Department of Electron Microscopy &
Molecular Pathology, The Cyprus
Institute of Neurology & Genetics,
Nicosia 2371, Cyprus; 4The Cyprus
School of Molecular Medicine, Nicosia
1683, Cyprus
Conclusion: Among the three triazolo[3,4-b]thiadiazole derivatives, KA39 was shown to be
the most potent anticancer agent and catalytic inhibitor of topIIα phosphorylation as well.
Keywords: triazoles, thiadiazoles, topoisomerase IIα, catalytic cycle, Ser-1106,
phosphorylation
Introduction
DNA topology is crucial for a wide range of cellular processes including cell
viability, replication, transcription, as well as chromosomal segregation, recombi-
nation, and condensation/decondensation.1,2 More specifically, cells recruit
a specific category of enzymes known as topoisomerases which carry out the
required alterations in the topological state of DNA.3 Two types of topoisomerases,
I and II, are ubiquitously expressed, both of which can regulate DNA supercoiling
by i) generating transient strand breaks, ii) removing any knots and tangles, and iii)
Correspondence: Sofia Sagredou;
Dimitrios T Trafalis
Email ssagredou@med.uoa.gr;
dtrafal@med.uoa.gr
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sealing the broken strands in the duplex molecule.4 respectively. Moreover, modifications at different positions
The major difference between topoisomerase I and II is of heterocyclic compounds as well as the fusion of the two
the generation of single and double strand breaks in the
double helix, respectively.5 With regard to topoisomerase
II, higher mammals express two isoforms of the enzyme,
topoisomerase IIα and IIβ, which share similar amino acid
sequence and catalytic activities but their cellular func-
tions are quite distinct.6 For instance, topoisomerase IIα
(topIIα) is a cell-cycle dependent enzyme (specifically at
the G2/M phase) while topoisomerase IIβ’s (topIIβ) func-
tion is not in line with cell-cycle progression.7 With
respect to topIIα, various posttranslational modifications
(phosphorylation, ubiquitination, and SUMOylation) can
modify the functionality of the enzyme in different ways.8
Among them, phosphorylation (specifically at Ser-1106) is
considered to be a key mechanism capable of altering the
catalytic activity of the enzyme.9,10 Topoisomerase II oper-
ates a catalytic cycle, known as a double-strand passage
reaction, with two DNA duplexes (termed G- and
T-segments) being formed. The enzyme binds and cleaves
the G-segment which then leads to an intermediate and
transient cleavage complex consisting of DNA and topoi-
somerase II. In the meantime, the T-segment is translo-
core structures of triazoles and thiadiazoles rings they all
constitute alternative synthetic possibilities capable of
leading to novel chemical entities.17 For instance, accord-
ing to current findings, fusion of 1,2,4-triazole and
1,3,4-thiadiazole rings displayed significant cytotoxic
activity against several cancer cell lines implying that
these newly synthesized compounds may act as potent
anticancer agents.17–24 The aim of the present study was
to investigate whether topIIα constitutes a potential target
of triazolo[3,4-b]thiadiazole derivatives KA25, KA26, and
KA39 including an evaluation of their biological activity
against three human colorectal cancer cell lines and one
normal. To determine the inhibitory effect of the three
heterocyclic compounds, in topIIα’s activity, our study
was carried out as follows: The KA39 agent, by being
the most active, was tested in the DNA relaxation reaction
performed by topoisomerase II whereas all three tested
compounds were assayed in the inhibition of the phosphor-
ylation of the topIIα isoform. The experimental study
regarding the potential inhibition of topIIα phosphoryla-
tion was conducted in two human colorectal cancer cell
lines, HT-29 and LoVo, both of which express the target
protein in sufficient levels.25 Finally, other and more
detailed studies were conducted about the potency of
KA39 compound to induce apoptosis as well as cell
cycle growth arrest.
cated through
a “gate” provided by the cleaved
G-segment. Then, topoisomerase II seals the broken
strands in the duple helix of the G-segment, releases the
molecule and then the “gate” closes again so that the
enzyme retrieves its enzymatic activity in order to start
a new catalytic cycle.5,11 The double-strand passage
requires two cofactors, magnesium and adenosine tripho-
sphate (ATP), so that the catalytic cycle can be transacted.
More specifically, magnesium is required for all steps of
the catalytic cycle whereas ATP is necessary for i) trans-
portation of the T-segment through the “open gate”, and ii)
conformational changes necessary for the next round of
catalysis to occur.12,13
Materials and Methods
Chemistry
The synthesis of the 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles
was performed according to the synthetic protocol previously
described by our group.17 Commercially available
2-(3,4-dimethoxyphenyl)acetic acid reacted with oxalyl chlor-
ide and esterified to yield the corresponding methyl ester,
which was subsequently converted to sulfonyl chloride 2
after treatment with chlorosulfonic acid in CHCl3
(Scheme 1). Then, sulfonyl chloride 2 reacted with Me2NH
to give sulfonamide 3 in excellent yield. Hydrazinolysis of this
sulfonamide gave the carboxylic acid hydrazide 4, which
reacted with KOH, CS2 in EtOH to give the intermediate
potassium thiocarbamate that was cyclized in the presence of
hydrazine hydrate to triazole 5.26 Condensation of triazole 5
with 2.5-dinitro-benzoic acid, 4-chloro-, and 4-fluoro-
cinnamic acid in POCl3 produced the desired 1,2,4-triazolo
On the other hand, triazoles and thiadiazoles are het-
erocyclic compounds consisting of a five-membered ring
where triazole contains three nitrogen atoms and thiadia-
zole two nitrogen and one sulfur atoms. Both classes of
heterocycles are known for processing a wide spectrum of
biological activities like anti-microbial, anti-inflammatory,
anti-convulsant, antioxidant, anti-leishmanial, anti-viral,
anti-hypertensive and anti-depressant.14–16 Of great inter-
est is the potent anticancer activity which is attributable to
reaction sites that both heterocyclic compounds carry
including the toxophoric N-C-S moiety and the acidic
proton at C-2 in the thiadiazole and triazole rings, [3,4-b]-1,2,4-thiadiazoles KA39, KA26, and KA25, as
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Scheme 1 Synthesis of 3,6-disubstituted 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles.
illustrated in Scheme 1. All reactions were carried out under an with H2O (20 mL) and the mixture was extracted with
dichloromethane (3 x 20 mL). The organic extracts were
dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel
column chromatography (eluent; hexane/ethyl acetate, 4/
1), to afford S1 as a yellowish oil in 99% yield. The
spectral data were accordance with those reported in the
atmosphere of argon unless otherwise specified. High purity
commercial reagents and solvents were purchased and used
without further purification. Reactions were monitored by thin
layer chromatography and using UV light as a visualizing
agent and ethanolic p-anisaldehyde solution, aqueous ceric
sulfate/phosphomolybdic acid, and heat as developing agents.
The 1H and 13C NMR spectra were recorded at 500 and 126
MHz, and tetramethylsilane was used as an internal standard.
Chemical shifts are indicated in δ values (ppm) from internal
1
literature.27 S1: H NMR (500 MHz, CDCl3) δ 6.81 (s,
3H), 3.88 (s, 3H), 3.86 (s, 3H), 3.69 (s, 3H), 3.57 (s, 2H).
Methyl 2-(2-(Chlorosulfonyl)-4,5-Dimethoxyphenyl)
Acetate, 2
1
reference peaks (TMS H 0.00; CDCl3 1H 7.26, 13C 77.00;
DMSO-d6 1H 2.50, 13C 39.51) and coupling constants (J) in
Hz. Melting points (mp) are uncorrected. The LC-MS spectra
were recorded on a LC20AD Shimadzu connected to
Shimadzu LCMS-2010EV equipped with C18 analytical col-
umn SUPELCO Discovery (C18, 25 cmx4.6 mm, 5 μm).
To a solution of S1 (3.7 g, 17.64 mmol) in CHCl3 (35 mL)
chlorosulfonic acid (5.28 mL, 79.4 mmol) was added drop-
wise at 0°C and the reaction was stirred at room temperature
for 1 hour. The reaction was quenched with H2O (35 mL) and
the mixture was extracted with dichloromethane (x3). The
organic layers were combined, dried over Na2SO4, filtered
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (eluent; hex-
ane/ethyl acetate = 2/1), to yield compound 2 as a white solid
Methyl 2-(3,4-Dimethoxyphenyl)Acetate, S1
2-(3,4-dimethoxyphenyl)acetic acid (1 g, 5.1 mmol) was
dissolved in 24 mL dry DCM, a catalytic amount of dry
DMF was added and the mixture was cooled at 0°C. Then,
oxalyl chloride (1.32 mL, 15.31 mmol) was added drop-
wise and the reaction left stirring at room temperature for
0.5 hours. The addition of MeOH (20 mL) in portions at 0°
C was followed and the mixture was stirred at room
1
(88% yield). 2: mp = 107–110°C; H NMR (500 MHz,
CDCl3) δ 7.52 (s, 1H), 6.88 (s, 1H), 4.11 (s, 2H), 3.97 (s,
3H), 3.95 (s, 3H), 3.73 (s, 3H); 13C NMR (126 MHz, CDCl3)
δ 170.5, 154.1, 148.0, 134.6, 127.8, 115.3, 111.3, 56.4, 56.4,
52.4, 38.0; ESI-MS m/z for C11H13ClNaO6S [M+Na]+ calcd
temperature for 10 minutes. The reaction was quenched 331.0, found 330.85.
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mp = 113–116°C; 1H NMR (500 MHz, DMSO-d6) δ 13.41
(s, 1H), 7.26 (s, 1H), 7.03 (s, 1H), 5.58 (s, 2H), 4.31 (s, 2H),
3.82 (s, 3H), 3.79 (s, 3H), 2.61 (s, 6H); 13C NMR (126 MHz,
DMSO-d6) δ 165.8, 151.8, 151.5, 147.1, 127.7, 126.3, 115.6,
112.7, 55.9, 36.8, 28.1 (1 carbon is missing due to over-
lapping); ESI-MS m/z for C13H19N5NaO4S2 [M+Na]+
calcd 396.08, found 396.00.
Methyl 2-(2-(N,N-Dimethylsulfamoyl)-
4,5-Dimethoxyphenyl)Acetate, 3
To the solution of 2 (450 mg, 1.46 mmol) in THF (1.8 mL)
2.92 mmol of dimethylamine (1.47 mL, solution 2 M in
THF) were added at 0°C. The reaction mixture was stirred
at room temperature for 1 hour. Then, it was concentrated
under reduced pressure to afford compound 3 as a pale
1
yellow solid in 99% yield. 3: mp = 143–146°C; H NMR
(500 MHz, CDCl3) δ 7.43 (s, 1H), 6.81 (s, 1H), 3.99 (s,
2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.68 (s, 3H), 2.68 (d, 6H);
13C NMR (126 MHz, CDCl3) δ 171.5, 152.2, 147.5, 127.5,
127.3, 115.4, 113.1, 56.3, 56.1, 52.0, 37.9, 36.6; ESI-MS
m/z for C13H19NNaO6S [M+Na]+ calcd 340.08, found
339.90.
General Procedure for the Preparation of
1,2,4-Triazolo-[3,4-b]-1,3,4-Thiadiazoles
The equimolar mixture of 5 (0.268 mmol) and selected
aromatic carboxylic acids (0.268 mmol) in phosphorous
oxychloride (0.4 mL) was stirred while heated under reflux
for 2 hours. The reaction mixture was cooled to room
temperature and then poured into ice. Then, the pH was
adjusted to 8 with solid K2CO3. The mixture was extracted
with dichloromethane and the organic extracts were dried
over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (eluent; ethyl acetate) to give the corre-
sponding 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles.
2-(2-Hydrazinyl-2-Oxoethyl)-N,N-Dimethyl-
4,5-Dimethoxybenzenesulfonamide,4
The mixture of 3 (300 mg, 0.95 mmol) in aqueous hydrazide
80% (0.47 mL, 7.79 mmol) was refluxed for 0.5 hour. The
reaction mixture was extracted with dichloromethane
(3x10 mL) and the combined organic extracts were dried
over Na2SO4, filtered and concentrated under reduced pres-
sure to afford compound 4 (yellow solid) in 66% yield. The
spectral data were accordance with those reported in the
(E)-2-((6-(4-Fluorostyryl)-[1,2,4]Triazolo[3,4-b]
[1,3,4]Thiadiazol-3-Yl)Methyl)-4,5-Dimethoxy-N,
N-Dimethylbenzenesulfonamide, KA25
1
literature.28 4: H NMR (500 MHz, CDCl3) δ 7.70 (s, 1H),
7.32 (s, 1H), 7.01 (s, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.79 (s,
2H), 2.71 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 170.6,
152.4, 147.6, 128.2, 125.9, 114.5, 112.8, 56.2, 38.4, 37.0;
ESI-MS m/z for C12H19N3NaO5S [M+Na]+ calcd 340.09,
found 339.85.
1
Yellow solid, 73% yield, mp = 210–212°C; H NMR (500
MHz, DMSO-d6) δ 7.70 (dd, J = 8.6, 5.6 Hz, 2H), 7.65 (d,
J = 16.4 Hz, 1H), 7.58 (d, J = 16.4 Hz, 1H), 7.40–7.24 (m,
2H), 7.17 (s, 1H), 6.97 (s, 1H), 4.73 (s, 2H), 3.85 (s, 3H),
3.80 (s, 3H), 2.61 (s, 6H); 13C NMR (126 MHz, DMSO-d6) δ
166.0, 163.2, 151.9, 151.8, 147.2, 146.4, 139.6, 131.1, 130.5,
128.0, 126.0, 118.2, 116.1, 115.5, 112.7, 55.9, 36.8, 27.7 (1
carbon is missing due to overlapping); ESI-MS m/z for
C22H22FN5NaO4S2 [M+Na]+ calcd 526.10, found 525.90.
2-((4-Amino-5-Mercapto-4H-1,2,4-Triazol-3-Yl)
Methyl)-N,N-Dimethyl-4,5-Dimethoxybenzene
Sulfonamide, 5
To a flask containing compound 4 (180 mg, 0.57 mmol), abs
EtOH (8.5 mL) and KOH (48 mg, 0.85 mmol) were succes-
sively added. Then, CS2 (50 μL, 0.85 mmol) was added
dropwise at 0°C and the reaction mixture was allowed to
warm to room temperature and stirred for 24 hours.
Subsequently, diethyl ether (8.5 mL) was added and the
solid product was filtered and washed with cold diethyl
ether. The product S1 was dried and used in the next reaction
without further purification. To a sealed vial the previous salt
S1 and aqueous hydrazide 80% (2.76 mL, 45.41 mmol) were
added and the mixture was refluxed for 2 hours. Acidification
with 10% aq. HCl was followed and the pH was adjusted to
7. The product was formed, filtered through a Gooch funnel,
washed with cold water, and dried. Compound 5 was
(E)-2-((6-(4-Chlorostyryl)-[1,2,4]Triazolo[3,4-b]
[1,3,4]Thiadiazol-3-Yl)Methyl)-4,5-Dimethoxy-N,
N-Dimethylbenzenesulfonamide, KA26
Yellow solid, 72% yield, mp = 229–231°C; 1H NMR (500
MHz, DMSO-d6) δ 7.84 (d, J = 8.6 Hz, 2H), 7.64 (s, 2H),
7.53 (d, J = 8.6 Hz, 2H), 7.30 (s, 1H), 7.15 (s, 1H), 4.72 (s,
2H), 3.84 (s, 3H), 3.79 (s, 3H), 2.61 (s, 6H); 13C NMR
(126 MHz, DMSO-d6) δ 165.7, 151.8, 147.2, 146.3, 139.4,
134.8, 133.3, 129.8, 129.0, 127.9, 126.1, 119.0, 115.5,
112.7, 55.9, 36.8, 27.6 (2 carbons are missing due to
overlapping); ESI-MS m/z for C22H22ClN5NaO4S2 [M
obtained as a grey solid in 68% yield (over two steps). 5: +Na]+ calcd 542.07, found 541.85.
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concentration of 15 mM. The final volume of DMSO did not
2-((6-(2,5-Dinitrophenyl)-[1,2,4]Triazolo[3,4-b]
[1,3,4]Thiadiazol-3-Yl)Methyl)-4,5-Dimethoxy-N,
N-Dimethylbenzenesulfonamide, KA39
exceed 1% of the culture medium.
1
Brown solid, 29% yield, mp = 162–164°C; H NMR (500
Cell Lines and Culture Conditions
MHz, DMSO-d6) δ 8.75 (s, 1H), 8.70 (d, J = 8.9 Hz, 1H),
8.50 (d, J = 9.0 Hz, 1H), 7.29 (s, 1H), 7.13 (s, 1H), 4.74 (s,
2H), 3.84 (s, 3H), 3.79 (s, 3H), 2.58 (s, 6H); 13C NMR
(126 MHz, DMSO-d6) δ 161.2, 153.8, 151.9, 150.5, 149.3,
147.3, 146.3, 128.2, 127.6, 126.9, 126.1, 124.3, 115.4,
112.8, 55.9, 55.8, 36.7, 27.6 (1 carbon is missing due to
overlapping); ESI-MS m/z for C20H19N7NaO8S2 [M+Na]+
calcd 572.06, found 571.75.
1H-NMR and 13C-NMR spectra as well as LC/ESI-MS
analysis data for compounds S1(1), 2, 3, 4, 5, KA39, KA26,
and KA25, as indicated in Scheme 1, are presented as
Supplementary Material, in Figures S1–S15, respectively.
Our study was conducted in four human cell lines, of which
three were of colorectal cancer origin (DLD-1, HT-29, and
LoVo) and one from normal lung (MRC5). All cell lines were
obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA) and cultured in different cul-
ture mediums according to the supplier instructions. All
growth mediums were supplemented with 10% fetal bovine
serum and 1% penicillin/streptomycin. All cancer cell lines
were cultured as monolayers and maintained at 37°C in
a humidified 5% CO2 atmosphere.
In vitro Anticancer Activity
The in vitro anticancer activity of the three disubstituted
triazolo[3,4-b]thiadiazole derivatives (KA25, KA26, and
KA39) against DLD-1, HT-29, and LoVo cancer cells was
evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide] MTT assay, a well-known quantita-
tive colorimetric method, which associates cell viability with
Drug Preparation
Three novel triazolo[3,4-b]thiadiazole derivatives, KA25,
KA26, and KA39 (Figure 1), were synthesized according to
a previously described experimental procedure.17,26–28 Stock
solutions were prepared by dissolving the required amount of
the substances in DMSO solvent in order to reach the desired color formation (absorbance). Moreover, in order to gain
Figure 1 Chemical structures of KA25 (2-((6-(4-fluorostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-4,5-dimethoxy-N,N-dimethylbenzenesulfonamide), KA26
(2-((6-(4-chlorostyryl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-4,5-dimethoxy-N,N-dimethylbenzene sulfonamide), and KA39 (2-((6-(2,5-dinitrophenyl)-[1,2,4]tria-
zolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)-4,5-dimethoxy-N,N-dimethylbenzenesulfonamide),triazolo[3,4-b]thiadiazole derivatives.
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a better insight into the tumor specificity of KA25, KA26, and and cells were exposed to KA39 (IC50=9 μM) and etoposide
KA39, we explored their cytostatic and cytotoxic properties on (IC50=27 μΜ) as reference drug compound, for 48 and 72
healthy lung (MRC5) cells as well. Etoposide (a known topIIα
poison) was included in our experiments in order to evaluate
the in vitro anti-tumor activity of the newly synthesized tria-
zolo[3,4-b]thiadiazole derivatives by comparing them to eto-
poside’s therapeutic effectiveness. Briefly, cells were seeded
into a 96-well plate at a density of 8×103 cells per well and
maintained for 72 hours at 37°C in humidified 5% CO2. After
24 hours of cell growth, cells were treated with the tested
compounds in concentrations of 1–100 μΜ. Following 48
hours of drug exposure, culture medium was discarded and
replaced with 100 μL of fresh medium. To determine cell
viability, 50 μL of MTT (5 mg/mL) were added in each well
and cells were incubated for 3 hours. Incubation time was
followed by discard of supernatant and addition of 100 μL of
DMSO so the formazan crystals could be solubilized. The
absorbance of the converted dye was recorded at
a wavelength of 540 nm on an ELISA reader (Versamax,
Orleans, USA).29, 30 The MTT assay provides the required
absorption values for determining three dose-response para-
meters, GI50, TGI, and IC50, using the linear progression
method. According to the National Cancer Institute (NCI),
GI50 and TGI are the drug concentrations which signify the
cytostatic effect of a tested compound and induce 50% and
100% of cell growth inhibition, respectively; IC50 is the drug
concentration which implies the cytotoxic effect of a tested
compound and leads to a 50% decrease of cell viability.31,32
The three parameters are determined using the mean of cell
survival in all nine absorbance measurements including con-
trol 24h (Ct24), control 72h (Ct72) and the seven drug con-
centrations (Tt72). The percentage of growth inhibition
was calculated as NCI indicates: [(Tt72x)-(Ct24)/(Ct72)-
(Ct24)]×100 for concentrations for which Tt72x>Ct24
and [(Tt72x)-(Ct24)/Ct24]×100 for concentrations for
which Tt72x<Ct24; GI50 was calculated from [(Tt72x)-
(Ct24)/(Ct72)-(Ct24)]×100=50, TGI from [(Tt72x)-(Ct24)/
(Ct72)-(Ct24)]×100=0, and IC50 from [(Tt72x)-(Ct24)/
Ct24]×100=50. All the experiments were carried out in
triplicate.
hours, while unexposed cells served as controls. For apop-
tosis assay, cells were washed with ice-cold PBS (pH 7.4)
and detached enzymatically with standard trypsinization.
All required centrifugations were carried out in 1500 rpm
for 5 minutes, including culture medium discard and two
washing steps with 2 mL of cold cell staining buffer
(BioLegend, San Diego, CA, USA). Subsequently, pellets
were re-suspended with 150 μL of Annexin V Binding
Buffer and 100 μL of cell suspension were transferred.
Afterwards, cells were stained with 5 μL of FITC Annexin
V and 5 μL of 7-AAD Viability Staining Solution and then
cells were incubated for 15 minutes at room temperature in
the dark. Cell staining was followed by the addition of 400
μL of Annexin V Binding Buffer and 400 μL of cell staining
buffer before analyzing on the flow cytometer (CyFlow®,
SL, Partec, GmbH, Germany). For each sample, 1×104
events were acquired and analysis was carried out in tripli-
cate. Flow cytometric analysis was performed using the
Partec FloMax software.
Flow Cytometric Analysis of Cell Cycle
Growth Arrest
Cell cycle progression was analyzed by the CyStain PI
Absolute
T reagent kit (Sysmex, Partec, GmbH,
Germany). HT-29 cancer cells were seeded in a 6-well
plate and maintained for 24 hours at 37°C in humidified
5% CO2. After 24 hours of cell growth, culture medium
was replaced with fresh medium; untreated cells repre-
sented controls whilst cells were treated with KA39 at
TGI and IC50 concentrations of 15.9 and 19.5 μΜ, respec-
tively. Treated cells were allowed to grow for 48 and 24
hours, respectively. In order to compare cell cycle progres-
sion under treatment with KA39, HT-29 cancer cells were
treated with etoposide at a TGI concentration of 69 μΜ for
48 hours and IC50 concentration for 24 hours. An MTT
assay was performed in order to determine the required
IC50 concentration of etoposide in HT-29 cancer cells. To
analyze cell cycle, cells were washed with ice-cold PBS
(pH 7.4) and detached enzymatically with standard trypsi-
nization. As previously described, all centrifugations were
carried out at 1500 rpm for 5 minutes and cells were
washed each time with 2 mL of cold cell staining buffer
(BioLegend, San Diego, CA, USA). Subsequently, pellets
Flow Cytometric Analysis of Apoptosis
Apoptosis was conducted by the FITC Annexin
V Apoptosis Detection Kit with 7-AAD (Biolegend, San
Diego, California, USA). DLD-1 cancer cells were seeded
(1.0×106 cells/well) in a 6-well plate and maintained for 24
hours at 37°C in humidified 5% CO2. After 24 hours of cell were resuspended with 150 μL of nuclei extraction buffer
growth, culture medium was replaced with fresh medium and cells were incubated for 15 minutes in the dark with
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gentle shaking. Following DNA extraction, DNA contents LifeSpan Biosciences, Seattle, USA). The inhibitory
were stained with 2 mL of cell staining solution (contain-
ing staining buffer, PI and RNAse) and incubated at least
for 30–60 minutes while they were not exposed to light
before analyzing on the flow cytometer (CyFlow®, SL,
Partec, GmbH, Germany). For each sample, 1×104 events
were acquired and analysis was carried out in triplicate.
Flow cytometric analysis was performed using Partec
FloMax software.
impact of the three disubstituted triazolo[3,4-b]thiadiazole
derivatives (KA25, KA26 and KA39) in the phosphoryla-
tion of topIIα was tested in two colorectal cancer cell lines,
LoVo and HT-29. In order to determine the required TGI
concentrations of KA25 and KA26 derivatives in both
cancer cell lines, further MTT assays were performed, as
previously described. According to manufacturer’s instruc-
tions, cells were seeded onto a 96 well plate at a density of
15–20×103 cells per well and maintained for 24 hours at
37°C in humidified 5% CO2. After 24 hours of cell culture,
cells were treated with KA39, KA25, and KA26 at TGI
and IC50 concentrations (μΜ) for 6 and 24 hours. Cells
were washed with 1×TBS and fixed with 4% fixing solu-
tion for 20 minutes. Afterwards, cells were washed three
times with 1× Wash Buffer and incubated with 1×
Quenching Buffer for 1 hour. Then, cells were washed
three times with 1× Wash Buffer and incubated with 1×
Blocking Buffer for 1 hour. Once cells were washed three
times with 1× Wash Buffer, 50 μL of 1× primary antibo-
dies (Anti-TOP2A (Phospho-Ser1106), Anti-TOP2A and
GAPDH antibody) were added and cells were incubated
for 16 hours at 4°C. Incubation with primary antibodies
was followed by three washing steps with 1× Wash Buffer.
Then, 50 μL of 1× secondary antibodies were added
(HRP-Conjugated with Anti-Rabbit IgG antibody or HRP-
Conjugated with Anti-Mouse IgG antibody) to the corre-
sponding wells and cells were incubated for 1.5 hours.
After washing the cells three times with 1× Wash Buffer,
50 μL of 1× Ready to Use Substrate were added and cells
were incubated in the dark for 30 minutes with gentle
shaking. The reaction was terminated by the addition of
50 μL of 1× Stop Solution and the absorption was imme-
diately measured at 450 nm on an ELISA reader
(Versamax, Orleans, USA). After reading the absorbance
at 450 nm, the plate was washed twice with 200 μL of 1×
Wash Buffer and twice with 200 μL of 1× TBS. After
letting the plate dry for 5 minutes, cells were stained with
50 μL of crystal violet and incubated for 30 minutes at
room temperature. Subsequently, the plate was well-
washed with continually running and distilled water as
well, and wells were allowed to dry for 30 minutes.
Then, 100 μL of SDS solution were added to each well
and the plate was incubated at room temperature for
1 hour. Finally, the absorbance was measured at 595 nm
on an ELISA reader (Versamax, Orleans, USA). The
DNA Topoisomerase II Assay
The inhibition of the catalytic activity of topoisomerase II
was estimated by the plasmid-based topoisomerase II
drug screening kit (TopoGen, Buena Vista, CO, USA).
The inhibitory effect of KA39 against topoisomerase II
was tested in a supercoiled DNA relaxation assay in
a range of concentrations (0.1, 1.0, 7.5, and 20 μΜ).
According to manufacturer’s recommendations, each
reaction included 150 ng of pHOT1 supercoiled DNA, 4
μL complete buffer [0.5 M Tris-HCl, pH (8.0), 150
M NaCl, 100 mM Mg2Cl, 5 mM Dithiothreitol, 300 μg
BSA/mL and 20 mM ATP], nuclease free water whose
volume was variable depending on the case and 6 units of
topoisomerase II. Concerning the tested compounds, 2 μL
were added to the mixture before reaction was started by
the addition of topoisomerase II. All reactions were incu-
bated at 37°C for 30 minutes and terminated by adding
0.1 volume of 10% SDS. To digest DNA/topoisomerase
II complexes, 50 μg/mL of proteinase K were added and
samples were further incubated at 37°C for 15 minutes.
Afterwards, 0.1 volume of loading buffer was added and
samples were cleaned up by adding chloroform/isoamyl
alcohol (24:1, v/v). After a brief vortex mixing, samples
were spun in a microcentrifuge for 5 seconds. The upper
aqueous phase of DNA samples as well as 2 μL of each
marker (supercoiled and linear DNA) were loaded onto
0.8% agarose gel in TBE buffer and run at 50 V for 120
minutes (electrophoresis system, BioRad, USA). As
a non-ethidium bromide gel, the agarose gel was stained
with 0.5 μg/mL of ethidium bromide and destained with
water prior to the photo documentation of DNA electro-
phoresis. The non-ethidium bromide gel was photodocu-
mented in Geldoc It2 imager UVP (Cambridge, UK).
DNA Topoisomerase IIα Phosphorylation
TopIIα phosphorylation (Ser1106) was determined by the
LSBioTM Human Phospho-TOP2A/Topoisomerase II
Alpha Cell-Based Phosphorylation ELISA Kit (LSBio, experiment was carried out in triplicate.
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and induces cytotoxicity to a greater extent than KA25,
KA26, and etoposide (Tables 1 and 2). In addition, the
selectivity of all three derivatives was assessed in a lung
normal cell line as well. As Tables 1 and 2 demonstrate, the
three triazolo[3,4-b]thiadiazole derivatives induced signifi-
cant cytostatic and cytotoxic activities against all cancer cell
lines while they exhibited rather low activity in non-
tumorigenic (MRC5) cells. Based on our results, GI50,
TGI, and IC50 values of KA25, KA26, and KA39 com-
pounds were 3–5 times higher than those in MRC5 cells,
indicating significant selectivity of the tested triazolo[3,4-b]
thiadiazole derivatives.
Computational Studies for Determining
Direct Effects on Topoisomerase IIα
Molecular docking was performed into the ATP-binding
pocket of the human topIIα ATPase in an attempt to
explore the possible binding modes of the synthesized
compounds KA25, KA26, and KA39 with the ATPase
domain while etoposide was also tested. More specifically,
the alpha isoform of human topoisomerase II structure,
containing ANP as the co-crystallized ligand (PDB ID:
1ZXM) was selected and retrieved from the RCSB
Protein Data Bank (www.rcsb.org).33 The co-crystallized
ligand and non-bonded heteroatoms were removed, the
polar hydrogens were added and the non-polar were
merged through AutoDockTools (1.5.6 package). In addi-
tion, Gasteiger’s charges were added and the structure was
saved in the pdbqt format. The structures of the ligands
were generated and energy minimized. AutoDock vina
program was used for the docking study following the
standard procedure.34 The three-dimensional grid with
20×20×20 points and a spacing of 1 Å was created and
centered at the area surrounding the co-crystallized ligand
AMP-PNP (X=36.071; Y=−0.286; Z=38.215). The
exhaustiveness value was set to 100. Figures for best
scoring docking complexes were generated by PyMOL
viewer and Discovery Studio visualizer.
Apoptotic Analysis
DLD-1 cancer cells were treated with KA39 at IC50 concen-
tration (μM), for 48 and 72 hours, while its apoptotic
potency was compared to that of etoposide, also utilized at
IC50 concentration (μM) for 48 and 72 hours. Untreated
DLD-1 cells (control) were also cultured for 48 and 72
hours as well. As flow cytometric analysis indicated, treat-
ment with ΚΑ39 at IC50 concentration (μM) for 48 hours
displayed high rates of early (17.35%) and late apoptosis
(13.12%) compared to the corresponding untreated cells
(P<0.001) (Figure 2). On the other hand, exposure to
KA39 (at the same concentration for 72 hours) led to
a significant decrease of the percentage of viable cells and
consequently to an increase of early (20.68%) and late
(20.96%) apoptosis (Figure 2). As expected, treatment with
KA39, for 72 hours, contributed to an enhanced late apop-
tosis (20.96%) in contrast to treatment for 48 hours (13.12%;
P<0.01) (Figure 2). On the other hand, cell exposure to
etoposide at IC50 concentration (μM), for 48 hours, remark-
ably decreased cell viability, thereby yielding high rates of
early (51.12%) and late (18.84%) apoptosis (P<0.001).
Treatment with etoposide at the same concentration, for 72
hours, significantly increased the rates of late apoptosis
Statistical Analysis
Student’s t-test was used to compare the level of sign-
ificance between the experimental groups. Differences
with a P-value less than 0.05 were considered significant.
Microsoft Excel (Microsoft Hellas, Athens, Greece).
Results
In vitro Anticancer Activity
In order to determine the in vitro anti-proliferative activity
of the three novel triazolo[3,4-b]thiadiazole derivatives
(KA25, KA26, and KA39), the MTT assay was conducted
in three human colorectal cancer cell lines (DLD-1, HT-29,
and LoVo) while etoposide was utilized as a reference drug
compound. Briefly, KA39 showed the most potent antic-
ancer activity against all cancer cell lines, demonstrating
significant cytostatic and cytotoxic effects (P<0.001).
Moreover, KA25 displayed higher cytostatic activity than
KA26 derivative (P<0.01), while etoposide induced cyto-
static activity against all three colorectal cancer cell lines
and cytotoxic activity against DLD-1 cancer cells only.
Overall, it is suggested that KA39 inhibits cell proliferation
Table 1 In vitro Antiproliferative Activity of KA25 and KA26
Against Four Human Cell Lines (Three Colorectal Cancer Cell
Lines and One Normal)
Cell Lines
KA25
GI50
KA26
GI50
TGI
IC50
TGI
IC50
DLD-1
HT-29
LoVo
11.4 0.5
1 0.3
65 0.5
>100
>100
>100
>100
>100
1.2 0.81
2 0.76
15 0.8
30 0.9
>100
>100
>100
>100
>100
>100
>100
>100
8 0.76
15 0.9
13.8 0.52
>100
MRC5
Abbreviations: IC50, Half maximal cytotoxic concentration; GI50, 50% growth
inhibition; TGI, Total 100% growth inhibition.
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Table 2 In vitro Antiproliferative Activity of KA39 and Etoposide Against Four Human Cell Lines (Three Colorectal
Cancer Cell Lines and One Normal)
Cell Lines
KA39
GI50
Etoposide
GI50
TGI
IC50
TGI
IC50
DLD-1
HT-29
LoVo
3 0.52
5 0.76
9 0.76
1.8 0.39
9.5 0.5
14 0.5
25 0.3
8 0.5
69 0.9
>100
27 0.28
>100
11.5 0.8
2.2 0.2
15 0.36
15.9 0.55
5.5 0.1
27 0.76
19.5 0.9
10.5 0.15
40.5 0.76
>100
MRC5
>100
>100
Abbreviations: IC50, Half maximal cytotoxic concentration; GI50, 50% growth inhibition; TGI, Total 100% growth inhibition.
(30.48%) with a concomitant decrease of those of early arrest, in S phase, while exposure for a greater time interval
apoptosis (42.01%; P<0.001). As Table 2 shows, the IC50 enhanced the percentage of growth arrested cells in the G2/
value of etoposide in DLD-1 cancer cells is 3-fold higher M phase (P<0.001).
compared to that of KA39 (Figure 2).
DNA Topoisomerase II Assay
To investigate whether KA39 derivative inhibits the cata-
Cell Cycle Analysis
lytic activity of topoisomerase II, the DNA decatenation
HT-29 cancer cells were treated with KA39 at TGI and IC50
assay was performed by using plasmid DNA as a substrate
concentrations (μΜ), for 48 and 24 hours, respectively. In
while VP-16 was used as a reference compound.
order to determine the effect of KA39 on cell cycle distribu-
According to the data in Figure 4, in the absence of any
tion, untreated HT-29 cells were also cultured for 48 and 24
drug, topoisomerase II catalyzes the reaction cycle through
hours, thereby acting as controls. In addition, etoposide was
which normal circular relaxed DNA products are formed,
also utilized under the same experimental conditions. Based
known as topoisomers (lane 3). During DNA electrophor-
on flow cytometric analysis, the highest percentage of
esis, KA39 induced the formation of supercoiled DNA in
untreated HT-29 cells, at 24 hours, were distributed in the
all concentrations used (eg, 0.1, 1.0, 7.5, and 20 μΜ) in
G1 phase (65.32%) whereas treatment with KA39, at IC50
contrast to VP16-induced linear DNA (lanes 4–8). The
concentration (μΜ) for 24 hours, induced significant cell
KA39-induced supercoiled DNA reaction product indi-
cycle growth arrest indicated by cellular distribution at the
cates that KA39 acts as a topoisomerase II inhibitor, either
S phase (21.52%; P<0.001). At the same time, treatment
by blocking the DNA binding activity of the enzyme or by
with etoposide, at IC50 concentration for 24 hours, led to cell
affecting the ATPase action and consequently preventing
cycle growth arrest at the G2/M phase, a 2-fold increase
any necessary conformational transitions of topoisome-
when compared to untreated cells (at G2/M; P<0.001)
rase II.
(Table 3, Figure 3). On the other hand, untreated HT-29
cells, at 48 hours, were shown to be distributed at the G1
Phosphorylation of DNA Topoisomerase
phase (64.68%) while exposure to KA39, at TGI concentra-
tion (μΜ) for 48 hours, displayed a relative increase in the IIα
G2/M phase (9.48%) (P<0.001). Upon treatment with etopo- The inhibitory impact of KA25, KA26, and KA39 in phos-
side, at TGI concentration (μΜ) for 48 hours, the highest phorylating topIIα was conducted in two human colorectal
percentage of HT-29 cells was recorded in SubG1 phase cancer cell lines, LoVo and HT-29. According to our data, the
(55.80%) with a concomitant decrease in G1 phase phosphorylation of topIIα was significantly inhibited by
(P<0.001) (Table 3, Figure 3). Nevertheless, the percentages KA39, at TGI and IC50 concentrations of 5.5 μΜ and 10.5
of cell cycle distribution in the S phase were quite similar in μΜ respectively, in LoVo cells when treated for 24 hours
HT-29 cancer cells, treated and untreated for 48 hours (P<0.001). Anti-topIIα normalization indicated that topIIα
(Table 3). All treatment conditions induced a decrease in phosphorylation was decreased upon treatment with KA39, at
G1 phase with a significant increase (P<0.001) in SubG1 IC50 concentration, after exposure for 24 hours (Figure 5A).
probably due to apoptotic induction by KA39 and etoposide Regarding GAPDH normalization, results were in line with
in HT-29 cancer cells. Overall, treatment with KA39 led to those of anti-topIIα normalization, that is greater inhibition of
inhibition of cell proliferation as well as cell cycle growth topIIα phosphorylation by KA39 at IC50 concentration, for 24
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Figure 2 Apoptotic induction in DLD-1 cancer cells, upon treatment with KA39 and etoposide, at IC50 concentration (μΜ), for 48 and 72 hours, compared with those of
untreated cells (controls). The percentages of viable, early-apoptotic, late-apoptotic, and necrotic cells are demonstrated.
hours (Figure 5B). By contrast, treatment with the same concentration, whilst no significant alteration was observed, at
compound, at TGI and IC50 concentrations for 6 hours, IC50 concentration, when compared to the untreated cells
enhanced the phosphorylation status of topIIα. More specifi- (Figure 5A). Furthermore, GAPDH normalization indicated
cally, according to anti-topIIα normalization, a considerable a remarkable increase in topIIα phosphorylation for both
increase of topIIα phosphorylation was demonstrated, at TGI treatment concentrations, at 6 hours (Figure 5B).
Table 3 Cell Cycle Distribution in Untreated (Control) and Treated HT-29 Cancer Cells with KA39 and Etoposide, at IC50 and TGI
Concentrations (μΜ), for 24 and 48 hours, Respectively
% of Cells in Each Phase of the Cell Cycle at:
Phases
Control
KA39
Etoposide IC50 24 hours
Control 48 hours
KA39
Etoposide
24 hours
IC50 24 hours
TGI 48 hours
TGI 48 hours
SG
G1
S
18.21%
65.32%
12.00%
3.56%
27.06%
46.64%
21.52%
3.74%
20.23%
57.28%
16.50%
5.99%
15.41%
64.68%
15.59%
4.67%
33.51%
40.95%
14.66%
9.48%
55.80%
26.06%
13.10%
4.37%
G2/M
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Figure 3 Cell cycle growth arrest in HT-29 cancer cells induced by KA39 and etoposide. HT-29 cells were arrested in S- and G2/M phase after being treated at IC50
concentration (μΜ), for 24 hours, respectively. Treatment with both compounds at TGI concentration (μΜ), for 48 hours, significantly increased SubG1 phase.
Figure 4 Supercoiled and linear DNA are reactions products, induced by KA39 and VP-16, respectively. The samples were loaded onto the agarose gel as follows: lane 1
represents pHOT1 supercoiled DNA (marker), lane 2 linear pHOT1 DNA (marker), lane 3 relaxed circular DNA (topoisomers), lane 4 VP16-induced linear DNA, and lanes
5–8 KA39-induced supercoiled DNA. All topoisomerase II reaction products are resolved on a non EB gel.
As Figure 5A shows, ΚΑ25 inhibited the phosphorylation after cells being exposed to KA25, for 24 hours (Figure 5A
of topIIα in LoVo cancer cells treated at TGI=13.8 μΜ, for 24
and B). However, treatment with KA25 at the same
hours (P<0.001). According to GAPDH and anti topIIα nor- drug concentration, for 6 hours induced less significant inhibi-
malization, topIIα phosphorylation was sufficiently reduced tion of topIIα phosphorylation (P<0.01). Particularly, the
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Figure 5 Inhibition of topIIα phosphorylation in LoVo and HT-29 cancer cell lines, treated with KA25, KA26, and KA39, at TGI and IC50 concentrations, for 6 and 24 hours.
(A) The diagram shows inhibition of topIIα phosphorylation, according to anti-topIIα normalization, in LoVo cancer cells. (Β) The diagram illustrates inhibition of topIIα
phosphorylation, according to GAPDH normalization, in LoVo cancer cells. (C) The diagram demonstrates inhibition of topIIα phosphorylation, according to anti-topIIα
normalization, in HT-29 cancer cells. (D) The diagram displays inhibition of topIIα phosphorylation, according to GAPDH normalization, in HT-29 cancer cells. Anti-topIIα
normalization is expressed by the ratio phosphotopIIα/topIIα whereas GAPDH normalization by the rate of phosphotopIIα/GAPDH. * P<0.05, ** P<0.01, *** P<0.001.
proportion of phosphorylated and total topIIα is decreased TGI concentration over 6 and 24 hour treatment periods
under treatment with KA25, for 6 hours, while greater inhibi- (Figure 5D). Nevertheless, the most outstanding inhibitory
tion was shown through GAPDH normalization (Figure 5A effect of KA39 was displayed at IC50 concentration when
and B). Moreover, KA26-induced inhibition of topIIα phos- cells were treated for 24 hours (P<0.001). Based on both
phorylation was not statistically significant since treatment at ways of normalization, the phosphorylated form of topIIα
TGI=100 μΜ, for 6 and 24 hours, did not affect the phosphor- was highly reduced compared to the untreated cells.
ylation status of topIIα. More specifically, the ratio of phos- However, inhibition of topIIα phosphorylation was
phorylated and total topIIα remained similar with that of observed upon treatment with KA39, at IC50 concentration
untreated cells although GAPDH normalization demonstrated for 6 hours, which was not shown to be statistically sig-
quantitative alterations in the phosphorylated form of topIIα nificant (Figure 5C and D). In general, the greatest inhibi-
(Figure 5B).
tion of topIIα phosphorylation was induced by KA39 at
Regarding HT-29 cells, the inhibition in the phosphor- IC50 concentration under all treatment times (P<0.001).
ylation of topIIα was significantly induced by KA39 in all On the other hand, KA25 and KA26 did not show statis-
treatment conditions. More precisely, the inhibition of tically significant inhibition in topIIα phosphorylation
topIIα phosphorylation was statistically significant upon though anti-topIIα and GAPDH normalizations indicated
treatment with KA39 at TGI and IC50 concentrations of that both compounds contributed to a reduction of the
15.9 and 19.5 μΜ respectively, for 24 hours (P<0.001). phosphorylated form of topIIα at TGI concentrations of
Concerning TGI, the ratio of phosphorylated with total 120 μΜ (for KA25) and 200 μΜ (for KA26) over 6 and
topIIα was considerably decreased in all treated cells at 6 24 hour treatment periods (Figure 5C and D). According to
and 24 hours (Figure 5C). In addition, GAPDH normal- our data, the maximum inhibitory effects of KA39, KA25,
ization also confirmed the inhibitory impact of KA39 at and KA26 on topIIα phosphorylation, at either TGI or IC50
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concentrations over 6 or 24 hour treatment periods, in showed comparable binding scores and were found to
LoVo cells were at 60%, 47%, and 18%, respectively, occupy the active site in a manner similar to that of etoposide,
whereas they were at 76%, 33%, and 50% in HT-29 cells as illustrated in Figures 6 and 7. The binding affinity scores
for the same compounds, respectively.
and the interacting residues are shown in Table 4.
Computational Studies of Direct Effects
on Topoisomerase IIα
Discussion
TopIIα is a versatile molecule whose functionality may
As previously described, KA25, KA26, KA39, and etoposide be affected by its cellular abundance, binding interac-
were all docked, using the AutoDock vina software, with tions, changes in DNA topology, and post-translational
only the lowest energy conformations being considered for modifications.35 With respect to posttranslational modifi-
analysis. According to the results, all three compounds cations, phosphorylation seems to play a leading role in
Figure 6 Predicted docking modes and 2D interactions of KA39 (magenta) and etoposide (blue) in the ATP-binding domain of human topIIα (PDB: 1ZXM).
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Figure 7 3D interactions between KA25 (pink sticks) and KA26 (yellow sticks) with the ATP-binding site of human topIIα.
the catalytic activity of the enzyme with numerous pro- like Ser-29 and Ser-1106 are located in the ATP-binding
teins being involved including casein kinase IIβ, protein domain within the N-terminal region and catalytic core of
kinase C (PKC), extracellular signal-related kinase 2 topIIα, respectively.43 Prior studies showed that the phos-
(ERK2), p38γ, and members of the polo-like kinase phorylation at Ser-1106 activates the decatenation function
(Plk) family. Casein kinase IIβ, PKC, and ERK2 augment of topIIα, suggesting that the phosphorylation site Ser-1106
the catalytic activity of topIIα via its phosphorylation, plays a major role in the catalytic activity of topIIα while
while p38γ ensures the activity as well as the stability mutation of Ser-1106 to alanine leads to decreased activity of
of the enzyme by phosphorylating Ser-1542.36 In addi- topIIα.9 Later studies revealed two kinases, casein kinase
tion, members of the Plk family like Plk1, Plk2, and Plk3 I (CKI) and II (CKII), which catalyze the phosphorylation
activate topIIα through its phosphorylation in specific at Ser-1106. However, further investigation demonstrated
serine and threonine residues, for example Plk1 phos- that the isoenzymes of CKI superfamily, CKIδ/ε, play
phorylates Ser-1337 and Ser-1524, whereas Plk3 phos- a more essential role in the phosphorylation at this site than
phorylates Thr-1342.37,38
CKII. Grovaz et al43 showed that CKIδ/ε kinases are speci-
fically involved in the phosphorylation at Ser-1106, enhan-
cing the in vivo activity of topIIα and as a result leading to
increased topIIα-DNA cleavage. On the other hand, inhibi-
tion or downregulation of CKIδ/ε decreases the phosphoryla-
tion at Ser-1106, implying that a lack of CKIδ/ε kinase
activity or absence of phosphorylation at Ser-1106
Due to the fact that most of the phosphorylation sites of
topIIα are located into the C-terminal domain without influ-
encing its enzymatic activity, but only being responsible for
its nuclear localization, deletion of this region or mutations in
such phosphorylation sites like Ser-1376 and Ser-1524 do not
affect the activation of topIIα.10,39–42 Phosphorylation sites
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Table 4 Docking Interactions Between Human topIIα ATPase
and KA39, KA25, KA26, and Etoposide
proliferation, chromosomal rearrangements, abortive cell-
cycle arrest and higher sensitivity to topIIα inhibitors.44,45
In our study, the inhibition of topIIα phosphorylation was
conducted in LoVo and HT-29 cancer cells which were
treated with KA25, KA26, and KA39 at TGI and IC50 con-
centrations, for 6 and 24 hours. Of utmost significance was
the KA39-induced inhibition of topIIα phosphorylation in
both cancer cell lines treated, at TGI and IC50 concentrations,
for 24 hours. Nonetheless, the greatest inhibition was
observed in HT-29 cells whose topIIα phosphorylation was
significantly reduced. However, the two colorectal cancer
cell lines do not express the same p53 status as LoVo cancer
cells express the wild-type p53 whilst HT-29 cancer cells
express the mutated p53 (p.R273H) (Table 5). In addition,
LoVo cells express mutated Plk2 (p.S636N) leading to
decreased topIIα phosphorylation.48 Consequently, the com-
binatory effect of wild type p53 and mutated Plk2 can reduce
the expression levels of total and phosphorylated topIIα, in
LoVo cancer cells. On the other hand, in HT-29 cancer cells,
the loss of functional p53 results in overexpression of topIIα
which, in turn, may enhance the KA39-induced inhibition of
topIIα phosphorylation. In reference to the crystal violet
stained cells, no alterations in topIIα expression were
observed either in transcriptional or translational level (data
not shown).
Compound Binding
Score
Hydrogen Interacting Residues
Bonds
KA39
−6.8
−7.6
−7.6
−6.9
Ser149
Ile141, Thr147, Lys168,
Ser148, Asn91, Tyr34,
Ser149, Asn150, Ile33,
Glu155, Arg98, Thr159,
Lys157, Asp94, Ile125,
Asn95, Asn120, Thr215,
Phe142
KA25
Asn120
Thr215
Asn150
Ile118, Asn120, Asn95,
Thr215, Ile125, Asp94,
Arg98, Lys157, Thr159,
Gln97, Asn150, Ser149,
Gly161, Ile141, Asn91,
Phe142, Phe177, Ala167,
Ile88, Ala92, Ile217
KA26
Asn120
Thr215
Asn150
Ile118, Asn120, Asn95,
Thr215, Ile125, Asp94,
Arg98, Thr159, Lys157,
Gln97, Asn150, Ser149,
Ile141, Asn91, Phe142,
Ala167, Phe177, Ile88,
Ala92, Ile217
Etoposide
Asn91
Asp94
Ser149
Tyr34
Asn95, Asn120, Ala92,
Thr215, Ile125, Phe142,
Asn91, Gly161, Val90,
Asp94, Glu87, Arg162,
Lys378, Gln376, Gly166,
Tyr165, Asn163, Ala167,
Gly164, Thr147, Ile141,
Ser148, Asn150, Ser149,
Lys168, Arg98, Thr159,
Tyr34, Lys157, Ile33
The catalytic activity of topIIα can be inhibited either
by targeting the strand DNA passage reaction or prevent-
ing the required ATP hydrolysis. The double-strand DNA
passage reaction can be blocked in several steps including
DNA binding to topIIα, formation of a topIIα-DNA clea-
vage complex and DNA religation. TopIIα blockers, like
etoposide, trap topIIα cleavage complexes, constituting
DNA religation as impossible, whereas DNA intercalators
prevent topIIα from binding to DNA through alterations in
DNA structure.49–51 With reference to ATP cofactor, clos-
ing and opening of the topIIα clamp are coupled with ATP
binding and hydrolysis. Inhibition of ATP hydrolysis does
not allow the ATPase domain to be reopened, thus trapping
topological DNA complexes inside the enzyme.52 As
Figure 4 shows, KA39-induced supercoiled DNA and
VP16-induced linear DNA underline the different pharma-
cological mechanisms through which KA39 and VP16
inhibit topIIα activity. According to the data from the
DNA decatenation assay, the formation of KA39-induced
supercoiled DNA indicates that this compound interferes
suppresses the function of topIIα.43 Experimental studies
using cell lines strongly support a linkage of p53 and levels
of topIIα expression. Given that the wild-type p53, as a tumor
suppressor protein, regulates the transcriptional activity of
several genes including topIIα, a mutated p53 may cause
unregulated expression of the genes involved in the regula-
tion of topIIα. Wang et al44 demonstrated that the reduction
of both mRNA and protein levels arise through p53-induced
inhibition of topIIα gene transcription. More precisely, wild-
type p53 suppresses the activity of human topIIα gene
promoter while loss of the p53 transcriptional regulator sti-
mulates the expression of topIIα 5–10 times higher in tumor with the catalytic activity of topIIα either by blocking the
cells. Therefore, mutated p53 leads to overexpression of access of topIIα to its plasmid substrate or by inhibiting
topIIα which in turn contributes to accelerated cell the ATPase action of the enzyme.
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So far, we demonstrated that all three derivatives inhibit completed, topIIα is rapidly decreased.53,54 The phosphor-
topIIα phosphorylation which is critical for topIIα activity.
ylation of topIIα is increased in a cell-cycle dependent
Furthermore, we showed that KA39 specifically inhibits manner as well, with most of topIIα being phosphorylated
in the G2 phase.53 Our data from cell-cycle analysis suggest
topIIα catalytic activity, thus inducing the formation of
that the KA39 derivative constitutes a potent topIIα inhibi-
tor since 21.52% of HT-29 cancer cells were arrested in the
S phase upon treatment at IC50 concentration for 24 hours.
Increased percentages in the SubG1 phase were also
recorded in all treatment conditions, signifying the apopto-
tic activity of our compound (Table 3). However, the apop-
totic potency of KA39 was confirmed by flow cytometry
analysis in DLD-1 cells where apoptosis was at its highest
levels at 72 hours (Figure 2).
supercoiled DNA which may be due to the inhibition of
critical steps of the catalytic cycle. Nonetheless, it is unclear
whether the triazolo[3,4-b]thiadiazole derivatives affect
topIIα in a direct or indirect manner. In silico studies
revealed that all three compounds may act directly through
binding to the ATPase domain of topIIα (Figures 6 and 7).
According to the results, the binding scores of KA25,
KA26, KA39, and etoposide (used as a positive control)
were quite similar (Table 4). For both KA25 and KA26
compounds, they share the same hydrogen bonds, whereas
the interacting residues are pretty similar, indicating that the
two derivatives interact with the ATPase domain in the
same way. In contrast, KA39 interacts with the ATPase
domain in a very different way since the hydrogen bonds
and interacting residues are unlike those of KA25 and
KA26. It is noteworthy that KA39 develops hydrogen
bonds with the ATPase domain only through one residue,
Ser149, indicating a highly specific way of interaction
(Table 4). Comparing with the three triazolo[3,4-b]thiadia-
zole derivatives, etoposide interacts with the ATPase
domain through different hydrogen bonds and interacting
residues, sharing only one common residue with KA39 at
Ser149. With respect to the most potent anticancer agent,
KA39 derivative, it is possible that it alters the ATPase
action of topIIα, leading to the formation of supercoiled
DNA as DNA decatenation assay demonstrated.
Furthermore, Tables 1 and 2 indicate that KA39 exhi-
bits the greatest cytostatic and cytotoxic effect in compar-
ison with those of KA25 and KA26. According to the IC50
values, of great interest is the sensitivity of the MMR-
deficient cells, DLD-1 and LoVo, after exposure to KA39
when compared to the HT29-MMR proficient cell line
(Table 5). It is very likely that a correlation between
mismatch repair system (MMR) and drug sensitivity to
topIIα inhibitors may exist. In fact, later studies have
shown enhanced drug sensitivity in MSI-H colorectal can-
cer cell lines that have been treated with etoposide,
a known topIIα inhibitor that interrupts the catalytic
cycle by targeting to the topIIα cleavage complex.25
Conclusion
DNA topoisomerases, known for resolving topological
issues and relaxing supercoiled DNA, are introduced as
potent chemotherapeutic drug targets attracting more and
more attention in the area of drug discovery.6,55 In our
study, we successfully demonstrated that all three 3.6-dis-
Being a cell-cycle dependent enzyme, the expression of
topIIα varies during the different phases of cell-cycle. More
specifically, the expression of topIIα is at its lowest levels
during the G1 phase while it starts to increase before the
S phase, remains stable during most of the S and raises
again in the late S phase. The expression of topIIα peaks
in the late G2 phase due to its necessity in cellular events
ubstituted-1,2,4-triazolo[3,4-b]thiadiazole
derivatives
exhibited in vitro anticancer activity and induced the inhi-
bition of topIIα phosphorylation. Nevertheless, among the
three triazolo[3,4-b]thiadiazole derivatives, the most
like chromatin condensation, chromosome assembly, and potent one was shown to be KA39 in terms of an
segregation of sister chromatids. Once mitosis has been in vitro anti-proliferative and inhibitory activity in topIIα
Table 5 Description of Histotypes and Special Characteristics of the Three Human Colorectal Cancer Cell Lines Involved in the
Investigation of the Biological Activity of Triazolo[3,4-b]thiadiazole Derivatives KA25, KA26, and KA39
Cancer Type
Human Cell Line Designation
p53 Status
MSI Status
References
46,47
46,47
46,47
Colorectal adenocarcinoma, Dukes’ type C
Colorectal adenocarcinoma
DLD-1
HT-29
LoVo
p. S241F
p. R273H
Wild type
MSI-H (MSH6 deficiency)
MSS
Colorectal adenocarcinoma, Duke’s type C, grade IV
MSI-H (MSH2 deficiency)
Abbreviations: MSI-H, Microsatellite instability; MSS, Microsatellite stable.
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Sagredou et al
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phosphorylation. The inhibition of topIIα phosphorylation
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arabjc.2016.09.015
This work was supported by the GALENICA S.A. and
ENERGON BIOTECHNOLOGIES S.A. and granted by
the Hellenic Society of Medical Oncology (HESMO) and
Athena Institute of Biomedical Sciences, Greece.
Disclosure
18. Chowrasia D, Karthikeyan C, Choure L, Sahabjada GM, Arshad M,
Trivedi P. Synthesis, characterization and anticancer activity of some
fluorinated 3,6-diaryl-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazoles. Arab
J Chem. 2017;10:S2424–S2428. doi:10.1016/j.arabjc.2013.08.026
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(10):8244–8256. doi:10.3390/molecules16108244
The tested investigational compounds were covered in the
international patent application WO2018/011414 (PCT/
EP2017/067908). The authors declare no conflicts of inter-
est in this work.
20. Gomha S, Abdel-aziz H. Synthesis and antitumor activity of
1,3,4-thiadiazole derivatives bearing coumarine ring. Heterocycles.
2015;91(3):583–592. doi:10.3987/COM-14-13146
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