O. Dömötör, et al.
Journal of Inorganic Biochemistry 202 (2020) 110883
2
.6. In vitro cell studies
compounds in 1 μM concentration (where the compounds are not toxic
to the cells). The increase in ROS caused by compounds applied was
measured at different time points – prior to treatment and 120 min after
the treatment.
Cell lines and culture conditions: All cell culture reagents were
obtained from Sigma-Aldrich and plastic ware from Sarstedt
(
Germany). Human colonic adenocarcinoma cell lines Colo 205 dox-
Catalase activity and GSH level assays: For both assays cells were
prepared in a same manner. The SUM159 and MCF-7 were seeded in 6-
orubicin-sensitive and Colo 320/MDR-LRP multidrug resistant, ex-
pressing ABCB1 (MDR1)-LRP, ATCC-CCL-220.1 (Colo 320) and CCL-
5
well plates at density of 5 × 10 cell/well, and were allowed 24 h to
2
22 (Colo 205) were purchased from LGC Promochem, Teddington, UK.
attach to the well. Then, cells were treated with 1 μM compound and
left for 24 h after which they were harvested, and the dry pellet was
stored at −80 °C until analysis. For analyses, cells were lysed in phos-
phate buffered saline (PBS) by 4 freeze/thaw cycles, and total protein
content was measured by Bradford method [43]. Total GSH was mea-
sured spectrophotometrically at 450 nm (ref. 620 nm) by modified
Tietze method based on reduction of DTNB (Ellman's reagent) to 2-
nitro-5-thiobenzoate (TNB anion) by GSH [44]. Catalase activity was
MRC-5 human embryonal lung fibroblast cell line (ATCC CCL-171) was
purchased from LGC Promochem, Teddington, UK. In addition, hepa-
tocellular carcinoma cell line (HepG2) and three subtypes of breast
cancer cell lines: the hormone-responsive MCF7, the HER2-positive
SkBr3 and the triple-negative SUM159 cancer cell lines were also used.
The cells were cultured in Roswell Park Memorial Institute (RPMI)
1
640 medium for Colo and MRC-5 or in Dulbecco's modified Eagle's
medium (DMEM) for HepG2 and breast cancer cell lines supplemented
with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM
sodium pyruvate and 100 mM HEPES. The cells were incubated at
assayed by measuring H
2
O decomposition by catalase in the whole cell
2
lysate by Góth method [45].
3
7 °C, in a 5% CO
2
, 95% air atmosphere. All cell lines were detached
3. Results and discussion
with Trypsin-Versene (EDTA) solution for 5 min at 37 °C.
MTT and EZ4U assays: Me-pyrTSC, Ph-pyrTSC, Ph-pyrSC and their
3.1. Synthesis, solid and solution phase characterization of Me-pyrTSC, Ph-
pyrTSC and Ph-pyrSC
6
copper(II) and Ru(η -p-cymene) complexes were dissolved in a 90% (v/
v) DMSO/H O mixture first using 10 mM ligand and 0, 5 or 10 mM
2
metal ion concentrations. The metal salts without ligands were also
tested. Cisplatin (Teva) was used as a positive control. Then stock so-
lutions were diluted in complete culture medium, and two-fold serial
dilutions of compounds were prepared in 100 μL of RPMI 1640, hor-
izontally. The semi-adherent colonic adenocarcinoma cells were treated
with Trypsin-Versene (EDTA) solution. They were adjusted to a density
Compounds Me-pyrTSC, Ph-pyrTSC and Ph-pyrSC (Chart 1) were
prepared by the condensation reaction of the corresponding 4-formyl
pyrazoles (pyrazole-4-carbaldehyde) and thiosemicarbazide or semi-
carbazide hydrochloride as described in references [27, 46], although
some modifications were applied, namely microwave-assisted reactions
were performed (Scheme S1). Acid-catalyzed condensation reaction of
acetone (1a) or acetophenone (1b) with phenylhydrazine in refluxing
ethanol led to the corresponding hydrazones (2a and 2b) in excellent
yields. Subsequent cyclization and simultaneous formylation with the
4
of 1 × 10 cells in 100 μL of RPMI 1640 medium, and were added to
each well, with the exception of the medium control wells. The final
volume of the wells containing compounds and cells was 200 μL. The
culture plates (Colo205, Colo320, MRC-5) were incubated at 37 °C for
Vilsmeier-Haack reagent (POCl /DMF) afforded 4-formyl-pyrazoles (3a
3
7
2 h; at the end of the incubation period, 20 μL of MTT solution (from a
and 3b) [46], which were then converted to thiosemicarbazones (Me-
stock solution of 5 mg/mL) were added to each well. After incubation at
7 °C for 4 h, 100 μL of sodium dodecyl sulfate solution (10% in 0.01 M
HCI) were added to each well and the plates were further incubated at
7 °C overnight. The EZ4U assay kit (Biomedica, Wien, Austria) was
pyrTSC and Ph-pyrTSC) and a semicarbazone (Ph-pyrSC), respectively,
in the presence of acetic acid (Me-pyrTSC and Ph-pyrTSC) or sodium
3
1
13
acetate (Ph-pyrSC) [27] in ethanol under MW irradiation. H and
C
3
NMR spectra were in agreement with the expected structures of the
1 13
used according to manufacturer's instructions in the case of cell lines
HepG2, MCF7, SkBr3 and SUM159. Samples and cells were prepared in
the similar way as in case of the traditional MTT assay. After the 24 h
incubation the compounds were diluted in a volume of 100 μL medium,
then the culture plates were incubated at 37 °C for 24 h; at the end of
the incubation period, 20 μL of MTT solution was added to each well
and incubated for 2 h incubation. The cell growth was determined using
both methods by measuring the optical density (OD) at 450 nm (ref.
intermediates and the final products and the H and C resonances
published previously [27,46–50] (see Supplementary Information).
The structure of Me-pyrTSC∙H O (I) was established by single crystal
2
X-ray diffraction (Fig. 1.a). Me-pyrTSC crystallized in monohydrate
form in triclinic P-1 space group. The molecule has a twisted con-
formation as ring A is twisted from ring B by 27.99(13)° and plane C is
twisted from ring B by 29.5° (Fig. 1.b). An intramolecular hydrogen
bond between N5-H5A…N3 stabilizes the molecular conformation. In
the crystal, dimers are formed between two molecules via the N5-
H5B…S1 hydrogen bonds, and vice versa (Fig. S7). The neighbouring
molecules are further connected through hydrogen bonds with solvent
water molecules. The acidic hydrogen, H2N4 is coordinating to a water
oxygen, and water hydrogens are connected to N2 and S1 atoms. The
packing arrangement is shown in Fig. S7. Selected H-bond distances
and angles are collected in Table S3. Apart from hydrogen bonds a π…π
(off-centered parallel stacking) interaction could be observed between
the two five membered rings for which the distance of 3.8687(14) Å can
be measured.
6
20 nm) with a Multiscan EX ELISA reader. Inhibition of the cell growth
(
expressed as IC50: inhibitory concentration that reduces by 50% the
growth of the cells exposed to the tested compounds) was determined
from the sigmoid curve where 100 − ((ODsample − ODmedium control) /
(
ODcell control − ODmedium control)) × 100 values were plotted against the
logarithm of compound concentrations. Curves were fitted by GraphPad
Prism software [42] using the sigmoidal dose-response model (com-
paring variable and fixed slopes).
Reactive oxygen species production assay: The ROS measurement
was performed in MCF7 and SUM159 cell lines with 2′,7′-di-
chlorodihydrofluorescein diacetate (DCFH-DA). While penetrating the
cell DCFH-DA is deacetylated by esterases and the forming DCFH inside
the cell reacts with ROS and transforms to the fluorescent 2,7-di-
chlorofluorescein (DCF) in turn. The emission intensity of DCF was
measured at 529 nm using 500 nm excitation wavelength. The SkBr-3
The molecular structure of Me-pyrTSC∙H O (I) was compared with
2
Ph-pyrTSC (PIKRUX) [51] and two other pyrazolyl TSC derivatives
(Ref. codes: CEHHEE and XEBCIS) defined previously [52,53]. CEHHEE
also crystallized with one water molecule, while PIKRUX and XEBCIS
crystallized without solvent inclusion in their crystal lattices. When the
pyrazole rings are overplayed high flexibility of the hydrazine-car-
bothioamide side chain can be seen for the different crystals (Fig. S8).
Since the TSCs and their metal complexes showed much more sig-
nificant cytotoxicity than the semicarbazone Ph-pyrSC (vide infra), our
solution studies were focused on the behaviour of Me-pyrTSC and Ph-
4
and HepG2 cancer cells were seeded 1 × 10 in 100 μL DMEM/10%
fetal bovine serum (FBS) into in 96-well microtiter plates. After al-
lowing cells to adhere for 24 h, 1 mM NAC was added to some wells
while 100 μM DCFH-DA was added to all wells. After removing the
media with the excess of DCFH-DA, cells were treated with the
4
D?m?t?r, Orsolya
