Antiproliferative and proapoptotic activity of molecular copper(II) complex of N-1-tosylcytosine

Article abstract of DOI:10.1016/j.jtemb.2017.10.009

In an attempt to enhance the previously observed antiproliferative capacity of 1-(p-toluenesulfonyl)cytosine (N-1-tosylcytosine, ligand 1), its copper(II) complex (Cu(1-TsC-N3)₂Cl₂, complex 2) was prepared and tested in vitro on vari

Full text of DOI:10.1016/j.jtemb.2017.10.009

JournalofTraceElementsinMedicineandBiologyxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology
journal homepage: www.elsevier.com/locate/jtemb
Biochemistry
Antiproliferative and proapoptotic activity of molecular copper(II) complex
of N-1-tosylcytosine
Ljubica Glavaš-Obrovaca,⁎, Marijana Jukića, Katarina Miškovića, Ivana Markovićb, Dijana Saftićc,
Željka Banc, Josipa Matićc, Biserka Žinićc,⁎
a
Department of Chemistry, Biochemistry and Clinical Chemistry, Faculty of Medicine, Josip Juraj Strossmayer University of Osijek, Huttlerova 4, HR-31000 Osijek,
Croatia
b
Department of Clinical Laboratory Diagnostics, Clinical Hospital Centre Osijek, Huttlerova 4, HR-31000 Osijek, Croatia
Laboratory for Biomolecular Interactions and Spectroscopy, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10 000 Zagreb,
c
Croatia
A R T I C L E I N F O
A B S T R A C T
Keywords:
In an attempt to enhance the previously observed antiproliferative capacity of 1-(p-toluenesulfonyl)cytosine (N-
1-tosylcytosine, ligand 1), its copper(II) complex (Cu(1-TsC-N3)2Cl2, complex 2) was prepared and tested in vitro
on various carcinoma and leukemia cells. The comparative in vitro studies using the ligand 1, the complex 2,
CuCl2 x 2H2O salt (salt 3) and the 1:2 mixture of the salt 3 and ligand 1 (mixture 4) were performed on normal
(WI38), human carcinoma (HeLa, CaCo2, MiaPaCa2, SW620), lymphoma (Raji) and leukemia (K562) cell lines.
Significantly elevated concentration of the intracellular copper after treatment of K562 cells and HeLa cells
during 2 h with complex 2 (7.83 vs. 5.4 times) was detected by atomic absorption spectroscopy. Cytotoxicity was
analyzed by MTT assay. We found that antiproliferative capacity of the tested compounds varies (IC50 after 72 h
of exposure: 0.6 × 10−6 M to > 100 × 10−6 M). Leukemia and lymphoma cells were found the most sensitive
to complex 2 which showed more than 100 times higher in vitro activity against K562 cells than ligand 1.
Apoptotic morphological changes, an externalization of phosphatydilserine, and changes in the mitochondrial
membrane potential of treated cells were found. The caspase-3 activity in HeLa and K562 cells was measured by
caspase-3 colorimetric assay kit. Caspase-3 was not activated in the treated K562 cells while salt 3 and the
mixture 4 in the HeLa cells significantly increased tested enzyme activity. These findings suggest that copper(II)
in the molecular complex 2 by improving entry of the N-1-tosylcytosine 1 into cells increases its antiproliferative
capacity.
N-1-tosylcytosine
Copper(II) complex
In vitro antitumor activity
Apoptosis
In summary, the present study demonstrated that complex 2 possesses an antileukemic effect on K562 cells,
and its anticancer activity was attributed with induction of apoptosis. The exact mechanism of apoptosis in-
duction by complex 2 must be further investigated.
1. Introduction
than the ligands themselves [12]. Generally the use of metal complexes
as new chemotherapeutics or diagnostic agents has rapidly increased in
Sulfonamides are very useful pharmaceutical compounds exhibiting
a wide range of biological activities such as antibacterial [1,2], antic-
ancer and antiviral [3,4], carbonic anhydrase inhibitory [5,6], and anti-
inflammatory activity [7]. Also, a large number of structurally diverse
sulfonamide derivatives have been studied as candidates for develop-
ment of anticancer drugs [8,9]. It has been reported that the biological
activity of some sulfur containing compounds could be enhanced by
coordination with metals [10,11]. Metal-chelates of some sulfonamide
derivatives have been found to exhibit stronger bacteriostatic activity
recent times [13–15]. In the anticancer research, high attention is paid
to non-platinum metal complexes such as those of ruthenium, gold,
copper and silver, which showed strong in vitro anticancer activity and
also overcome negative side-effects encountered with cisplatinum and
other platinum-based anticancer drugs [16,17]. Highly important role
of copper in many biological processes including metalloenzymes,
redox related transformations [18] and anti-inflammatory effects [19]
inspired many investigations on Cu(II) complexes as potential antic-
ancer agents [20,21].
⁎
Corresponding authors.
E-mail addresses: lgobrovac@mefos.hr (L. Glavaš-Obrovac), marjukic@mefos.hr (M. Jukić), kmiskovic@mefos.hr (K. Mišković), markovic.ivanaa@kbo.hr (I. Marković),
dsaftic@irb.hr (D. Saftić), Zeljka.Ban@irb.hr (Ž. Ban), Josipa.Matic@irb.hr (J. Matić), bzinic@irb.hr (B. Žinić).
http://dx.doi.org/10.1016/j.jtemb.2017.10.009
Received 13 February 2016; Received in revised form 25 March 2017; Accepted 17 October 2017
0946-672X/©2017ElsevierGmbH.Allrightsreserved.
Pleasecitethisarticleas:Glavaš-Obrovac,L.,JournalofTraceElementsinMedicineandBiology(2017),
http://dx.doi.org/10.1016/j.jtemb.2017.10.009

L. Glavaš-Obrovac et al.
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We have reported on the synthesis of novel pyrimidine nucleobase
derivatives containing a sulfonamide pharmacophore [22–24]. The
compounds showed strong antitumor activity in vitro [25–27] and in
vivo [28–30] conditions. This type of nucleic base derivatives was found
to inhibit DNA, RNA, and protein syntheses and induce apoptosis in
human tumor cells [30,31]. Encouraged by the numerous reports on
anticancer activity of complexes, we examined the possibility to pre-
pare 1-(p-toluenesulfonyl)cytosine (ligand 1) metal complexes and as-
sess their biological activity. We have shown that ligand 1 is capable to
form palladium (II) [32] and other transition metal complexes in-
cluding Cu(II) and provided the X-ray crystallographic evidence of their
structure [33].
the RPMI 1640 medium (Gibco, EU) supplemented with 10% heat-in-
activated fetal bovine serum (FBS, Gibco, EU), 2 mM glutamine, 1 mM
sodium pyruvate, 10 mM HEPES and 100U/0.1 mg penicillin/strepto-
mycin was used. Cells were cultured in a humidified atmosphere at
37 °C, 5% of CO2 in the CO2 incubator (IGO 150 CELLlife™, JOUAN,
Thermo Fisher Scientific, Waltham, MA, USA). The trypan blue dye
exclusion method was used to assess cell viability.
2.2.2. Cytotoxicity evaluation by MTT assay [34]
Cytotoxic effects of ligand 1, complex 2, salt 3 and the mixture 4
were assessed by MTT assay, carried out in 96-well microtiter plates
(Greiner, Frickenhausen, Austria). The adherent cells (WI38, CaCo2,
HeLa, MiaPaCa2, and SW620) were seeded at a concentration of
2 × 104 cells/mL and left overnight in the CO2 incubator allowing
them to attach to the plate surface, while Raji and K562 where seeded
into 96-well plates immediately before compounds addition. Stock so-
lutions of ligand 1, complex 2, the mixture 4 were made in DMSO and
salt 3 in 10% DMSO, and used for preparation of working dilutions
prior to addition to cells. Final concentration range was from 10−7 M
up to 10−3 M. After expired time of incubation (72 h), growing medium
was discarded and 5 mg/mL of MTT solution was added to each well,
and incubated at 37 °C for 4 h. The reaction was determined by dis-
solving MTT-formazan crystals in DMSO. Absorbance was measured at
570 nm on Elisa micro plate reader (iMark, BIO RAD, Hercules, CA,
USA). Control non-treated cells were grown under the same conditions.
All experiments were performed at least three times in triplicates. The
IC50 value, defined as compound concentration leading to cellular
viability reduction by 50%, was calculated.
In this study the results of in vitro cytotoxicity screening and proa-
poptotic potential of free ligand 1, complex 2, salt 3 and the mixture 4
against human tumor cells are reported.
2. Experimental
2.1. Synthesis
2.1.1. Preparation of the ligand 1 (1-TsC)
Synthesis of 1-(p-toluenesulfonyl)cytosine (ligand 1) was performed
according to the procedure reported by Kašnar-Šamprec et al. [28].
A mixture of cytosine (1 mmol) and N,O-bis(trimethylsilyl)acet-
amide (BSA) (3 mmol) was heated under reflux in dry acetonitrile
(3.3 mL) for 1 h. The solution was cooled to 0 °C and tosyl chloride
(1.2 mmol) was added. The reaction mixture was heated for 16 h at
80 °C, cooled and treated with a small amount of methanol. The re-
sulting solid was filtered off and recrystallized from methanol yielding
N-1-tosylcytosine 1 (80%) as a white crystals: 1H NMR (DMSO-d6) δ/
ppm: 8.14 (d, 1H, J6,5 = 7.8 Hz, H-6), 7.95 (brs, 2H, NH2), 7.87 (d, 2H,
J = 8.1 Hz, Ph), 7.46 (d, 2H, J = 8.1 Hz, Ph), 5.98 (d, 1H,
J5,6 = 7.8 Hz, H-5), 2.42 (s, 3H, CH3); 13C NMR (DMSO-d6) δ/ppm:
166.27 (s, C-4), 151.22 (s, C-2), 145.61 (s, Ph), 139.73 (d, C-6), 134.47
(s, Ph), 129.80 (d, Ph), 129.02 (d, Ph), 97.50 (d, C-5), 21.20 (q, CH3).
Percent of live cells was calculated as follows:
OD sample − OD backgroud
% =
× 100
OD control − OD background
2.2.3. Measurement of the intracellular copper concentration
The K562 and HeLa cells were plated into six well plates at a density
of 8 × 106 cells in the cell culture medium and incubated in presence or
absence of 1 × 10−3 M of ligand 1, complex 2, salt 3 and the mixture 4
respectively for 2 h in the CO2 incubator. Cells were then collected in a
tube and centrifuged for 5 min at 1100 rpm. Obtained supernatant was
carefully removed and stored until analysis. Obtained pellet was wa-
shed three times with 10 mL PBS, centrifuged, and supernatant was
carefully removed from the cells. Subsequently, on the cells were added
500 μL of sterile H2O and after vortexing cells were frozen in liquid
nitrogen and thawed in the warm water. The process was repeated 5
times. Cells were then centrifuged at 14,000 rpm and obtained samples
were stored at −20 °C until analysis. Determination of intracellular
copper in lysates of HeLa and K562 cells and in the growth medium
were done by atomic absorption spectroscopy in an analytical system
Agilent AA FS240. For calibration were used a commercial calibrator
RECIPE ClinChem serum calibrator for trace elements. Calibration is
performed in four points (r > 0.99). To check the operation of the
analytical system and accuracy of calibration were used commercial
control samples RECIPE ClinChek serum control for trace elements at
two concentration levels. The measured values of copper in the control
samples are very close to the target values set by the manufacturer of
the control material), thus confirming the precision and accuracy of
measurements. Experiments were done at least in triplicate and re-
present independent replicates experiments.
2.1.2. Preparation of the Cu(1-TsC-N3)2Cl2 complex 2
Synthesis of Cu(1-TsC-N3)2Cl2 (complex 2) was performed by
modifying our method [33] from more concentrated methanol solution.
To a warmed solution of N-1-tosylcytosine 1 (245 mg, 0.92 mmol)
in methanol (20 mL) a hot solution of CuCl2 x 2H2O salt (78 mg,
0.46 mmol) in methanol (9 mL) was added. By cooling the solution
precipitation of blue-green crystals occurred. The solid was filtered off
and washed with a small amount of cold MeOH yielding 230 mg (75%)
of complex 2: 1H NMR (DMSO-d6) δ/ppm: 8.25 (brs, 1H, H-6), 7.89
(brs, 2.5H, Ph + part of NH2), 7.42 (brs, 3.5H, Ph + part of NH2), 2.34
(brs, 3H, CH3); 13C NMR (DMSO-d6) δ/ppm: 143.22 (s, Ph), 132.13 (s,
Ph), 127.58 (d, Ph), 126.75 (d, Ph), 19.27 (q, CH3).
2.2. Biological studies
2.2.1. Cell lines and cell culture conditions
All cell lines used in this work were obtained from the American
Type Culture Collection (ATCC, Rockville, MD, USA) and preserved in a
liquid nitrogen prior use. Cells were tested and authenticated using the
short tandem repeat profiling (STR) analysis.
Human fibroblasts (WI38) and tumor cell lines: pancreatic carci-
noma (MiaPaCa2), colon adenocarcinoma (CaCo-2), cervix adeno-
carcinoma (HeLa), and metastatic colon carcinoma cells (SW620) were
grown in a monolayer and cultured in the Dulbecco’s modified Eagle
medium – DMEM (Gibco, EU) supplemented with 10% heat-inactivated
fetal bovine serum (FBS, Gibco, EU), 2 mM glutamine, and 100U/
0.1 mg penicillin/streptomycin. To detach from the flask surface, cells
were trypsinized using 0.25% trypsin/EDTA solution. Raji (lympho-
blastoid cells derived from Burkitt lymphoma) and K562 (ery-
thromyeloblastoid leukemia cells) cell lines cultivation were grown in
2.2.4. Determination of apoptosis induction by flow cytometry
Based on cytotoxic results obtained by MTT test (Table 1), proa-
poptotic potential of investigated compounds ligand 1, complex 2, salt
3, and the mixture 4 were tested on HeLa and K562 cells using Annexin
V-FITC and propidium iodide dye (Annexin V-FITC Apoptosis Detection
Kit; Abcam, UK, EU). Cells were plated in 6-well plates at
a
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Table 1
Sensitivity of human tumor and normal cells towards investigated compounds.
Compounds
IC50 (10−6 M)
Normal cells
WI38
Leukemia cells
K562
Carcinoma cells
HeLa
Raji
MIAPaCa2
CaCo2
SW620
ligand 1
complex 2
salt 3
> 100
90.1
93.0
> 100
80
3.1
0.1
0.2
> 100
7.0
8.4
> 100
90.1
24.0
19.0
> 100
11.1
0.2
0.14
95.0
20.0
10.5
> 100
8.3
6.0
0.1
> 100
54.8
4.0
7.7
0.2
0.6
0.7
56.6
0.3
1.5
0.8
10
9.4
0.7
0.5
0.9
0.3
1.6
mixture 4
6.1
73.0
> 100
87.1
IC50–Drug concentration that inhibited cell growth by 50%. Data represents mean IC50 (μM) values
cells were treated with compounds during 72 h. Cytotoxicity was analyzed using MTT survival assay.
standard deviation (SD) of three independent experiments. Exponentially growing
concentration of 3 × 105 cells/well for HeLa cells and 5 × 105 cells per
well for K562 cells. After 24 h cells were treated with tested compounds
1–4 at the concentration of 10−5 M. After treatment cells were cen-
trifuged at 3000 rpm for 5 min, stained according to the manufacturer’s
protocol, and analysed by flow cytometry (FacsCanto II, BD Bios-
ciences, USA) using Flowlogic software (Inivai Technologies, USA).
2.2.5. Caspase-3 activity assay
Caspase-3 activity was measured according to the manufacturer's
instructions (Caspase 3 Assay Kit, Colorimetric, Sigma-Aldrich, EU).
HeLa and K562 cells were seeded at concentration of 3 × 105 cells per
well in six well plates and treated for 24 h with tested 10−5 M com-
pounds 1–4. Cells were then lysed with 100 μL of chilled cell lysis buffer
on ice for 15–20 min. After centrifugation (16,000 × g, 10–15 min,
4 °C), the supernatant was transferred in a new tube and analyzed at
405 nm using the ELISA reader (iMark, BIO RAD, Hercules, USA).
Caspase 3 activity is expressed as μmol of released p-nitroaniline (pNA)
per min per mL of cells’ lysate.
Scheme 1. Synthesis of Cu(1-TsC-N3)2Cl2 (complex 2).
3.2. Cytotoxicity
2.2.6. Measurement of mitochondrial membrane potential (ΔYm) by flow
cytometry
Cytotoxic potential of the ligand 1, molecular complex 2, salt 3 and
the mixture 4 was tested on normal human fibroblasts (WI38), tumor
cell lines growing in a monolayer, such as pancreatic carcinoma
(MiaPaCa2), colon adenocarcinoma (CaCo-2), cervix adenocarcinoma
(HeLa), and metastatic colon carcinoma cells (SW620), as well as on
Burkitt lymphoma (Raji) and erythromyeloblastoid leukemia cells
(K562) after 72 h of testing.
As shown in Table 1, cytotoxic potential of tested compounds de-
pended on treated cells’ type. Complex 2 displayed 50% growth in-
hibition of carcinoma cells in the concentration range
4.0–20.0 × 10−6 M. Leukemia and lymphoma cells were found to be
the most sensitive and 50% of K562 cells growth inhibition was ob-
served when 0.6 × 10−6 M complex 2 and 0.7 × 10−6 M of salt 3,
respectively, were applied. Ligand 1 was less efficient and showed si-
milar inhibitory potential against normal and tumor cells.
Changes in the mitochondrial membrane potential (ΔΨm) of treated
cells were measured using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetra-
ethylbenzimidazolocarbocyanine iodide (JC-1) dye. Briefly, tested cells
were plated in 6-well plates at a concentration of 3 × 105 HeLa cells
per well and 1.5 × 105 K562 cells per well, and incubated for 24 h with
10−5 M ligand 1, complex 2, salt 3 and the mixture 4, respectively.
After treatment, cells were centrifuged at 600 × g for 4 min and stained
according to the manufacturer’s protocol (Mitochondrial Membrane
Potential Kit, Sigma-Aldrich, EU). Changes in the mitochondrial mem-
brane potential were measured by flow cytometry (FacsCanto II, BD
Biosciences, USA) and analyzed using Flowlogic software (Inivai
Technologies, USA).
3. Results
3.3. Intracellular copper concentration
3.1. Synthesis
Intracellular copper concentration was significantly increased in
treated K562 cells after 2 h of incubation with tested compounds
compared to control cells (Fig. 1).
1-(p-Toluenesulfonyl)cytosine (ligand 1) was synthesized by con-
densation of silylated cytosine with p-toluenesulfonyl chloride as de-
scribed previously [28]. The molecular complex Cu(1-TsC-N3)2Cl2
2
Compared with nontreated cells, substantially higher concentration
of intracellular copper (by 7.83 times) were detected in by complex 2
treated cells, while in the cells treated by salt 3 the copper concentra-
tion increased by 4.37 times. A less increase in the intracellular copper
concentration (by 1.85 times) was measured in the K562 cells treated
by the mixture 4. In the HeLa cells treated with complex 2 significantly
increased concentrations of copper (by 5.4 times) were observed in
comparison with control cells. In the HeLa cells treated by salt 3
was prepared in 75% yield by modifying our method [33] in the re-
action of Cu(II) chloride with the ligand 1 in a 1:2 molar ratio of re-
actants and from the concentrated methanol solution (Scheme 1).
The ligand 1 and complex 2 are stable, non-hygroscopic, insoluble
in most organic solvent, but soluble in DMSO and DMSO/H2O mixture.
1H NMR study [33] and mass spectrometry (see Fig. S1 in Supplemental
materials) support the stability and formation of the above compounds.
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Fig. 1. Intracellular copper concentration in treated
K562 and HeLa cells and nontreated (control) cells
after 2 h of incubation with 1 × 10−3 M compounds
detected by atomic absorption spectroscopy. Data
represents mean values
standard deviation (SD)
of three independent experiments. *Statistically sig-
nificant, p < 0.05.
intracellular copper concentrations increased by 4.08 times and in HeLa
cells treated by the mixture 4 for 2.6 times. A statistically significant
difference between copper concentration in the K562 and HeLa cells
(7.83 times vs. 5.4 times) treated by complex 2 was observed.
4. Discussion and conclusions
Development of metal complexes, especially non-platinum com-
plexes, as new chemotherapeutics or diagnostic agents has rapidly in-
creased in recent times [35]. Complexes containing biologically im-
portant copper ions are promising agents in the cure of tumor diseases
and copper ions are also known as very important for many processes in
the high proliferative cells such are tumor cells [36,37].
3.4. Proapoptotic potential of tested compounds
3.4.1. Phosphatidylserine externalization
In an attempt toward the development of metal-based anticancer
drug the copper(II) complex of novel pyrimidine nucleobase derivative
containing a sulfonamide pharmacophore (Cu(1-TsC-N3)2Cl2, complex
2) was tested on antiproliferative capacity on carcinoma and leukemia
cell lines and obtained results were compared with cytotoxic effects of
free ligand 1, CuCl2 x 2H2O salt (salt 3) and the mixture 4 (salt 3 and
ligand 1 in a ratio 1:2). We found that molecular complex 2 sig-
nificantly improved inhibition of tumor cells’ growth in comparison
with effect of free ligand 1. Further, leukemia K562 cells were more
sensitive to complex 2 compared to lymphoma and carcinoma cell lines
(Table 1) though almost the same cytotoxic effects displayed the salt 3.
To shed some light into mechanisms of observed cytotoxicity we mea-
sured intracellular copper concentration in cells treated with complex
2, salt 3 and the mixture 4. Based on the cytotoxicity results (Table 1)
the K562 and HeLa cells were chosen and treated by tested compounds.
We found that the intracellular copper concentration significantly ele-
vated in by complex 2 treated K562 cells after 2 h of incubation by 7.83
times compared to control cells, by 2 times compared to the cells
treated with salt 3, and by 4 times compared to the cells treated by the
mixture 4. It should be emphasized that statistically significant differ-
ence between copper concentration in K562 and HeLa cells (7.83 times
vs 5.4 times) treated by complex 2 was observed. The intracellular
copper concentration also raised in Hela cells treated by all tested
compunds 1–4. In HeLa cells treated with salt 3 and with the mixture 4
copper concentrations increased as those in treated K562 cells. The
obtained results could be explained by the fact that copper enters into
cells in a form of molecular complex 2 by independent system which
enables biologically active copper compounds to penetrate the cell
surface without binding to other agents opposite to coordination com-
pounds of other metals [36]. This mechanism is different from the
mechanism of uptake of extracellular copper(II) ions [38,39], where the
copper(II) is reduced to copper(I) by metalo-reductases located on the
cell’s surface before entering the cell and susequently transported into
the cell mainly by specific copper transporter. The significantly lower
growth inhibition of the normal cells caused by salt 3 compared to its
effect on the tumor cells found in this study is in accord with earlier
observations [40,41].
For assessment of apoptosis, we examined the exposure of phos-
phatidylserine on the cell surface using Annexin-V/PI double staining
by flow cytometry. A capability of ligand 1 to induce apoptotic cell
death was detected in our previous studies [25]. Our current results
show that exposure of HeLa cells to tested compounds 1–4
(c = 1 × 10−5 M) during 24 h treatment slightly increased a number of
necrotic cells (Fig. 2A, quadrant Q1) while number of apoptotic cells
remained almost the same (Fig. 2A and B) in comparison with control
non treated cells. All investigated compounds significantly induced
externalization of phosphatidylserine at the outer membrane surface as
a universal process occurring during early apoptosis in K562 cells
(Fig. 2A and B). Necrotic features had 2–3% of treated K562 cells only
(Fig. 2A, quadrant Q1).
3.4.2. Detection of the caspase-3 activity
Cells, HeLa and K562, were treated with tested compounds for 24 h.
Obtained results, shown in Fig. 2C, indicate different answer of treated
cells on applied compounds 1–4. While in the treated leukemia cells
caspase-3 activity, expressed as μmol of released p-nitroaniline (pNA)
per min per mL of cells’ lysate, was similar as in control cells, in the
HeLa cells salt 3 and the mixture 4 increased enzyme activity for 40%
compared to nontreated control cells.
3.4.3. Changes in the mitochondrial membrane potential (ΔΨm)
Changes in the mitochondrial membrane potential (ΔΨm) were
measured in the treated K562 and HeLa cells and nontreated (control)
cells. The results show that all investigated compounds 1–4 induced
significant disruption of mitochondria (JC-monomers) in both cell lines
(Fig. 3; Supplementary material, Fig. S2) compared to control cells.
Leukemia cells (K562) show greater changes in ΔΨm than HeLa
cells. In K562 cells observed ΔΨm changes were between 73% (in the
cells treated by ligand 1) to 81.4% (in the cells treated by salt 3).
Similar percentages of JC-1 monomers were present in HeLa cells ex-
posed to salt 3 (65.77%) and in the cells exposed to the mixture 4
(66.88%) and in K562 cells (81.44% vs. 80.87%). Ligand 1 caused least
ΔΨm changes, in HeLa cells for 54.43% cells and 73.2% in K562 cells,
compared to other compounds.
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Fig. 2. Induction of apoptosis in HeLa and K562 cells. Cells were cultured 24 h in the presence of 1 × 10−5 M complex 2, free ligand 1, salt 3 and the mixture 4. respectively. Control cells
were nontretaed cells. After dying by annexin V-FITC and propidium iodide cells were analyzed using flow cytometry. (A) Dot plot of HeLa and K562 cells flow cytometry analysis with
Annexin V-FITC versus propidium iodide. The divisions of the plots distinguish late apoptotic/necrotic cells (Annexin V-/PI+, Q2 quadrant), from early apoptotic cells (Annexin V+/PI,
Q3 quadrant) and late apoptotic cells (Annexin V+/PI+, Q2 quadrant). The plots in the figure are representative of three independent experiments. (B) Percentage of cells in early
apoptosis after treatment and in control nontreated cells. (C) Caspase-3 activity, expressed as μmol of released p-nitroaniline (pNA) per min per ml of cells’ lysate. Data represents mean
values
standard deviation (SD) of three independent experiments. *Statistically significant, p < 0.05.
Fig. 3. Flow cytometry detection of changes in the
mitochondrial membrane potential (ΔΨm) in K562
and HeLa cells. Cells were treated with free ligand 1,
complex 2, salt 3, and the mixture 4 for 24 h re-
spectively. Data represents mean values
standard
deviation (SD) of three independent experiments.
*Statistically significant, p < 0.05.
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Numbers of studies found that copper in the form of organic com-
plexes exhibits cytotoxic activity through cell apoptosis or enzyme in-
hibition [20,37,42–45,47]. In our previous study a capability of ligand
1 to induce apoptotic cell death was detected [25]. Our current results
show that exposure of leukemia cells to tested compounds, during 24 h
leads to induction of apoptosis manifested by phosphatidylserine ex-
posure at the outer membrane surface of a cell as a universal process
occurring during early apoptosis. Although treated HeLa cells displayed
some features of early apoptotic death, more propidium iodide entered
into cells (Fig. 2A and B) that could be a sign of a late apoptosis or
necrosis.
Sport of Croatia through Grant Nos. 098-0982914-2935, 219-0982914-
2176, J.J. Strossmayer University supporting grant VIF-2015-MEFOS-2.
Financial support from the Croatian Science Foundation (grant number
HRZZ-1477) is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.jtemb.2017.10.009.
References
Apoptosis can be activated by two main signaling caspase depen-
dent pathways: the extrinsic or cytoplasmic pathway, in which cell
death receptors are an initiation point of the apoptotic process, and the
intrinsic or mitochondrial pathway, where several stimuli act directly
on an intracellular target and the mitochondria has a major role [45].
Equally, apoptosis may also be triggered through caspase independent
mechanisms, upon different stimuli [48]. Results of this study, pre-
sented in Fig. 2C, show that caspase-3 was not activated in the treated
K562 cells suggesting that in these cells apoptosis is triggered through
caspase independent mechanisms.
Since apoptosis is frequently accompanied by complex mitochon-
drial changes, alterations in the mitochondrial membrane potential may
be an early event in the apoptotic process or, on the contrary, may be a
consequence of the apoptotic signaling pathway [46]. In response to
multiple distinct intracellular stress conditions, mitochondrial mem-
branes can become permeabilized because of the pore-forming activity
of proapoptotic members of the Bcl-2 protein family [47]. Alternatively,
mitochondria can lose their structural integrity after the mitochondrial
permeability transition, a phenomenon that is initiated at the mi-
tochondrial inner membrane. In both cases, permeabilized mitochon-
dria allow for the release of propapototic proteins into the cellular
cytoplasm.
To monitor possible changes in the mitochondrial transmembrane
potential (ΔΨm), mitochondria were stained with the J aggregate-
forming lipophilic cation JC-1, which as a monomer emits green
fluorescence and in a reaction driven by the ΔΨm turns into a red
fluorescence-emitting dimer, thereby allowing the simultaneous ana-
lysis of the total mitochondrial mass per cell (green fluorescence) and of
ΔΨm (the quotient of red/green fluorescence). In our study, treatment
of both, K562 and HeLa cells with 1 × 10−5 M compounds 1–4 during
24 h increased of JC-1 red/green fluorescence that documents the sig-
nificant breakdown of the mitochondrial membrane potential (ΔΨm).
Given results suggest that disruption of mitochondrial membrane po-
tential produced by complex 2 and other tested compounds can lead to
cytotoxicity and cell death by apoptosis and/or necrosis as we shown in
Fig. 2. These observations can be explained by the fact that exposure to
excessive copper ions leads to necrosis or apoptosis through increased
generation of reactive oxygen species (ROS) production, oxidation of
lipids, or proteins via Fenton or Haber-Weiss reactions [38].
Obtained results lead to the conclusion that copper ions have a
significant role in apoptosis which is reflected in the increased anti-
proliferative activity of the complex 2 in comparison to the free ligand
1. They also indicate that induced apoptosis could be promoted by
mitochondria mediated way, but this step should be investigated in
more detail. The results of this study contribute to better understanding
of interaction of metal ions and cytosine derivatives and could be of
importance in future metallodrug design.
[1] H.J. Warshakoon, M.R. Burns, S.A. David, Structure-activity relationships of anti-
microbial and lipoteichoic acid-sequestering properties in polyamine sulfonamides,
Antimicrob. Agents Chemother. 53 (2009) 57–62.
[2] F. Zani, M. Incerti, R. Ferretti, P. Vicini, Hybrid molecules between benzenesulfo-
namides and active antimicrobial benzo[d]isothiazol-3-ones, Eur. J. Med. Chem. 44
(2009) 2741–2747.
[3] A. Scozzafava, T. Owa, A. Mastrolorenzo, C.T. Supuran, Anticancer and antiviral
sulfonamides, Curr. Med. Chem. 10 (2003) 925–953.
[4] C.T. Supuran, A. Innocenti, A. Mastrolorenzo, A. Scozzafava, Antiviral sulfonamide
derivatives, Mini-Rev. Med. Chem. 4 (2004) 189–200.
[5] C.T. Supuran, A. Scozzafava, B.C. Jurca, M.A. Iiies, Carbonic anhydrase inhibitors.
Part 49: synthesis of substituted ureido and thioureido derivatives of ar-
omatic:heterocyclic sulfonamides with increased affinities of isozyme I, Eur. J. Med.
Chem. 33 (1998) 83–93.
[6] G. Renzi, A. Scozzafava, C.T. Supuran, Carbonic anhydrase inhibitors: topical sul-
fonamide antiglaucoma agents incorporating secondary amine moieties, Bioorg.
Med. Chem. Lett. 10 (2000) 673–676.
[7] M. Gangapuram, K.K. Redda, Synthesis of substituted N-[4(5-methyl/phenyl-1, 3, 4-
oxadiazol-2-yl)-3, 6-dihydropyridin-1(2H)-yl]benzamide/benzene sulfonamides as
anti-inflammatory and anti-cancer agents, J. Heterocycl. Chem. 46 (2009) 309–316.
[8] A. Yokoi, J. Kuromitsu, T. Kawai, T. Nagasu, N.H. Sugi, K. Yoshimatsu, H. Yoshino,
T. Owa, Profiling novel sulfonamide antitumor agents with cell-based phenotypic
screens and array-based gene expression analysis, Mol. Cancer Ther. 1 (2002)
275–286.
[9] Z. Huang, Z. Lin, J. Huang, A novel kind of antitumour drugs using sulfonamide as
parent compound, Eur. J. Med. Chem. 36 (2001) 863–872.
[10] M. Sharma, H.L. Singh, S. Varshney, P. Sharma, A.K. Varshney, Some new co-
ordination compounds of organosilicon(IV) with schiff bases of sulpha drugs,
Phosphorus Sulfur Silicon Relat. Elem. 178 (2003) 811–819.
[11] H.L. Singh, J. Singh, A. Mukherjee, Synthesis, spectral, and in vitro antibacterial
studies of organosilicon(IV) complexes with schiff bases derived from amino acids,
Bioinorg. Chem. Appl. 2013 (2013), http://dx.doi.org/10.1155/2013/425832.
[12] W.E. Rudzinski, T.M. Aminabhavi, N.S. Biradar, C.S. Patil, Biologically active sul-
fonamide schiff base complexes of selenium (IV) and tellurium (IV), Inorg. Chim.
Acta 67 (1982) 177–182.
[13] T.W. Hambley, Metal-based therapeutics, Science 318 (2007) 1392–1393.
[14] B.K. Kepler, Metal Complexes in Cancer Chemotherapy, Wiley-VCH Verlag GmbH,
1993 (p. 12).
[15] M. Gielen, E.R.T. Tiekink, Metallotherapeutic Drugs and Metal-Based Diagnostic
Agents, The Use of Metals in Medicine, John Wiley & Sons, Ltd., 2005, 2017 (p. 6).
[16] E. Gallori, C. Vettori, E. Alessio, F.G. Vilchez, R. Vilaplana, P. Orioli, A. Casin,
L. Messori, DNA as a possible target for antitumor ruthenium(III) complexes: a
spectroscopic and molecular biology study of the interactions of two representative
antineoplastic ruthenium(III) complexes with DNA, Arch. Biochem. Biophys. 376
(2000) 156–162.
[17] M.J. Clarke, Ruthenium metallopharmaceuticals, Coord. Chem. Rev. 232 (2002)
69–93.
[18] B.R. Stern, M. Solioz, D. Krewski, P. Aggett, T.C. Aw, S. Baker, K. Crump,
M. Dourson, L. Haber, R. Hertzberg, C. Keen, B. Meek, L. Rudenko, R. Schoney,
W. Slob, T. Starr, Copper and human health: biochemistry, genetics, and strategies
for modeling dose-response relationships, J. Toxicol. Environ. Health B: Crit. Rev.
10 (2007) 157–222.
[19] K.R. Rupesh, A.M. Priya, B. Sundarakrishnan, R. Venkatesan, B.B.S. Lakshmi,
S. Jayachandran, 2,2′-Bipyridyl based copper complexes down regulate expression
of pro-inflammatory cytokines and suppress MAPKs in mitogen induced peripheral
blood mononuclear cells, Eur. J. Med. Chem. 45 (2010) 2141–2146.
[20] C. Marzano, M. Pellei, F. Tisato, C. Santini, Copper complexes as anticancer agents,
Anti-Cancer Agents Med. Chem. 9 (2009) 185–211.
[21] M. Belicchi Ferrari, F. Bisceglie, G. Pelosi, P. Tarasconi, R. Albertini, P.P. Dall’Aglio,
S. Pinelli, A. Bergamo, G. Sava, Synthesis, characterization and biological activity of
copper complexes with pyridoxal thiosemicarbazone derivatives. X-ray crystal
structure of three dimeric complexes, J. Inorg. Biochem. 98 (2004) 301–312.
[22] B. Kašnar, I. Krizmanić, M. Žinić, Synthesis of the sulfonylpyrimidine derivatives as
a new type of sulfonylcycloureas, Nucleosides Nucleotides 16 (1997) 1067–1071.
[23] B. Žinić, I. Krizmanić, D. Vikić-Topić, M. Žinić, 5-Bromo- and 5-iodo-N-1-sulfony-
lated cytosine derivatives. Exclusive formation of keto-imino tautomers, Croat.
Chem. Acta 72 (1999) 957–966.
Conflict of interest
There is no conflict of interest.
Acknowledgments
[24] B. Žinić, I. Krizmanić, M. Žinić, Sulfonylpyrimidine Derivatives with Anticancer
Activity, EP, 0 877 022 B1, (2003).
[25] Lj. Glavaš-Obrovac, I. Karner, B. Žinić, K. Pavelić, Antineoplastic activity of novel
This work was supported by the Ministry of Science, Education and
6

L. Glavaš-Obrovac et al.
N-1-sulfonylpyrimidine derivatives, Anticancer Res. 21 (2001) 1979–1986.
JournalofTraceElementsinMedicineandBiologyxxx(xxxx)xxx–xxx
2011/594529.
[26] Lj. Glavaš-Obrovac, I. Karner, M. Pavlak, M. Radačić, J. Kašnar-Šamprec, B. Žinić,
Synthesis and antitumor activity of 5-bromo-1-mesyluracil, Nucleosides Nucleotides
Nucleic Acid 24 (2005) 557–569.
[38] T. Furukawa, M. Komatsu, R. Ikeda, K. Tsujikawa, S. Akiyama, Copper transport
systems are involved in multidrug resistance and drug transport, Curr. Med. Chem.
15 (2008) 3268–3278.
[27] F. Supek, M. Kralj, M. Marjanović, L. Šuman, T. Šmuc, I. Krizmanić, B. Žinić, A
typical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in
vitro screening and computational analysis, Invest. New Drugs 26 (2008) 97–110.
[28] J. Kašnar-Šamprec, Lj. Glavaš-Obrovac, M. Pavlak, N. Štambuk, P. Konjevoda,
B. Žinić, Spectroscopic characterization and biological activity of N-1-sulfonylcy-
tosine derivatives, Croat. Chem. Acta 78 (2005) 261–267.
[39] S.B. Howell, R. Safaei, C.A. Larson, M.J. Sailor, Copper transporters and the cellular
pharmacology of the platinum-containing cancer drugs, Mol. Pharmacol. 77 (2010)
887–894.
[40] M. Panjehpour, M.-A. Taher, M. Bayesteha, The growth inhibitory effects of cad-
mium and copper on the MDA-MB468 human breast cancer cells, J. Res. Med. Sci.
15 (2010) 279–286.
[29] M. Pavlak, R. Stojković, M. Radačić-Aumiler, J. Kašnar-Šamprec, J. Jerčić,
K. Vlahović, B. Žinić, M. Radačić, Antitumor activity of novel N-sulfonylpyrimidine
derivatives on the growth of anaplastic mammary carcinoma in vivo, J. Cancer Res.
Clin. Oncol. 131 (2005) 829–836.
[30] J. Kašnar-Šamprec, I. Ratkaj, K. Mišković, M. Pavlak, M. Baus-Lončar, S. Kraljević
Pavelić, L.j. Glavaš-Obrovac, B. Žinić, In vivo toxicity study of N-1-sulfonylcytosine
derivatives and their mechanisms of action in cervical carcinoma cell line, Invest.
New Drugs 30 (2012) 981–990.
[31] Lj. Glavaš-Obrovac, I. Karner, M. Štefanić, J. Kašnar-Šamprec, B. Žinić, Metabolic
effects of novel N-1-sulfonylpyrimidine derivatives on human colon carcinoma
cells, Il Farmaco 60 (2005) 479–483.
[32] A. Višnjevac, M. Luić, R. Kobetić, D. Gembarovski, B. Žinić, Stabilization of the N-1-
substituted cytosinate iminooxo form in dinuclear palladium complexes,
Polyhedron 28 (2009) 1057–1064.
[41] R.G. Contreras, H. Sakagami, H. Nakajama, J. Shimada, Type of cell death induced
by various metal cations in cultured human gingival fibroblasts, In Vivo 24 (2010)
513–517.
[42] K.G. Daniel, D. Chen, S. Orlu, Q.C. Cui, F.R. Miller, Q.P. Dou, Clioquinol and pyr-
rolidine dithiocarbamate complex with copper to form proteasome inhibitors and
apoptosis inducers in human breast cancer cells, Breast Cancer Res. 7 (2005)
R897–R908.
[43] L. Tripathi, P. Kumar, A.K. Singhai, Role of chelates in treatment of cancer, Indian J.
Cancer 44 (2007) 62–71.
[44] F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Copper in diseases and
treatments, and copper-based anticancer strategies, Med. Res. Rev. 30 (2010)
708–749.
[45] K.G. Daniel, P. Gupta, R.H. Harbach, W.C. Guida, Q.P. Dou, Organic copper com-
plexes as a new class of proteasome inhibitors and apoptosis inducers in human
cancer cells, Biochem. Pharmacol. 67 (2004) 1139–1151.
[46] D. Li, Y. Ye, S. Lin, L. Deng, X. Fan, Y. Zhang, X. Deng, Y. Li, H. Yan, Y. Ma,
Evaluation of deoxynivalenol-induced toxic effects on DF-1 cells in vitro: cell-cycle
arrest, oxidative stress, and apoptosis, Environ. Toxicol. Pharmacol. 37 (2014)
141–149.
[33] A. Višnjevac, N. Biliškov, B. Žinić, Transition metal complexes of N-1-tosylcytosine
and N-1-mesylcytosine, Polyhedron 28 (2009) 3101–3109.
[34] G. Mickisch, S. Fajta, H. Bier, R. Tschada, P. Alken, Cross-resistance patterns related
to glutathione metabolism in primary human renal cell carcinoma, Urol. Res. 19
(1991) 99–103.
[35] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Advances in
copper complexes as anticancer agents, Chem. Rev. 114 (2014) 815–862.
[36] M. Plotek, K. Dudek, A. Kyziol, Selected copper(I) compexes as potential anticancer
agents, Chemik 67 (2013) 1181–1190.
[37] I. Iakovidis, I. Delimaris, S.M. Piperakis, Copper and its complexes in medicine: a
biochemical approach, Mol. Biol. Int. 2011 (2011), http://dx.doi.org/10.4061/
[47] S. Santos, A.M. Silva, M. Matos, S.M. Monteiro, A.R. Alvaro, Copper induced
apoptosis in Caco-2 and Hep-G2 cells: expression of caspases 3, 8 and 9, AIF and
p53, Comp. Biochem. Physiol. Part C 185–186 (2016) 138–146.
[48] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (2007)
495–516.
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