Inorganic Chemistry
Article
low field with respect to the starting metal chlorido species, was
293 and HEK-293T cells were cultured in DMEM GlutaMAX. Both
media were supplemented with 10% FBS and 1% penicillin/
5
MeMe
2+
attributed to the solvato species [Ir(η -C Me )(L
)(solv)]
5
5
S
2+
6
MeMe
2+
S 2+
(
)(solv)] ([7 ] ) (solv =
streptomycin. The cells were cultured at 37 °C with CO (5%).
2
D O, DMSO-d ; Scheme S1). No solvolysis of metal−chloride
The resistance in A2780cisR cells was maintained by routine additions
2
6
bonds occurred before Ag(CF SO ) addition, as indicated by the
of cisplatin (2 μM) to the medium. The cytotoxicity was determined
3
3
absence of a 35Cl NMR signal and comparison with H NMR data for
1
35
using the MTT assay. Cells were seeded in flat-bottomed 96-well
S
2+
S 2+
the solvato species [6 ] and [7 ] .
plates as suspensions in a prepared medium at a density of 4300 cells/
well in 100 μL. Stock solutions of metal complexes were prepared in
DMSO and sequentially diluted in the medium up to the desired
concentration (0−200 μM range) and 0.5% DMSO. The cytotoxic
effects of RAPTA-C and cisplatin were also assessed as negative and
positive control experiments. The compounds were added to the cells
following a 24 h preincubation in 100 μL aliquots, and the plates were
incubated for an additional 72 h. MTT (20 μL, 5 mg/mL in
Dulbecco’s phosphate buffered saline) was then added to the cells,
and the plates were incubated for another 4 h. The medium was
removed, and the purple formazan products, formed by the
mitochondrial dehydrogenase activity of vital cells, were dissolved in
DMSO (100 μL/well). The absorbance directly proportional to the
number of surviving cells was read at 590 nm using a SpectroMax
M5e multimode microplate reader (using SoftMax Pro software,
version 6.2.2). The percentage of surviving cells in treated wells vs
untreated control wells was calculated; the resulting IC50 values are
reported as means ± standard deviation from two independent
experiments, each comprising four tests per concentration level.
Stability in Methanol. A freshly prepared solution of the selected
−
3
1
compound in CD OD (0.6 mL; 5 × 10 M) was analyzed by H and
3
19
1
F{ H} NMR. Next the orange (Ru) or yellow/orange (Ir) solution
was maintained at room temperature (∼25 °C) and periodically
analyzed by H and F{ H} NMR. Once equilibrium was reached
no further change observed in NMR spectra), 1D and 2D NMR
1
19
1
(
1
13
19
14
13
1
1
experiments ( H, C, F, N, C-DEPT 135, H− H COSY,
1
13
1
13
H− C gs-HSQC, H− C gs-HMBC) were performed for a
pure compounds and by ESI-MS(+) spectrometry on CH OH
3
solutions in the case of [3]+ and [4]+. The relative amount of
1
compounds in solution (%) was calculated by H NMR integration.
Experimental details and MS and NMR data (CH OH and CD OD,
3
Stability in Cell Culture Medium. A stock solution of the selected
Ru/Ir compound (ca. 3 × 10−3 mmol/g) was prepared in DMSO and
6
Cellular Uptake. A2780 cells were seeded at a density of 2 × 10
stored at room temperature. An RPMI 1640 cell culture medium
cells/Petri dish and incubated overnight. The cells were treated with
the tested compounds at the concentrations and times given in the
respective table. Following the treatment, the cells were harvested
(
Sigma-Aldrich) was treated with NaH PO /Na HPO (c = 190
2 4 2 4 PO
4
mM, pH 7.35) and then stored at 4 °C under N . In three 2 mL vials,
2
the stock solution (20 μL) was added to the cell culture medium (2.0
mL) and the final mixtures (Ru/Ir ca. 45 μmol/g, 1% DMSO) were
heated at 37 °C for different times (0/24/48 h) with stirring. A fourth
reference solution was prepared by adding the stock solution (20 μL)
to HPLC water (2.0 mL). Next, the solutions were filtered on PTFE
syringe filters (0.45 μm) and analyzed by HPLC/ESI(+)-MS. LC
separation was conducted on an analytical reversed-phase Poroshell
(
trypsin), washed, and pelleted. The pellets were digested with HCl
using a microwave digestion system (CEM Mars). The Ru/Ir amount
was determined with ICP-MS.
DNA Metalation. After the treatment (see above), the cells were
lyzed using DNAzol (DNAzol, MRC) reagent, following the
manufacturer’s protocol. The DNA content was determined
spectrophotometrically; the samples were lyzed in 30% HCl
1
20 EC-C18 column (3.0 × 75 mm, particle size 2.7 μm; Agilent
Technologies) with a Zorbax precolumn (4.6 × 12.5 mm, particle size
μm; Agilent Technologies) at 30 °C. The separation was achieved
using a gradient of formic acid (FA) 0.1% v/v in H O (eluent A) and
(Suprapur, Merck Millipore), and the Ru/Ir content was quantified
by ICP-MS.
5
Enzyme Inhibition Assays. Preparation of Cell Lysates for
2
Analysis of Enzyme Activities. HeLa cells were seeded at a density
5
FA 0.1% v/v in CH CN (eluent B), both LC-MS grade; the flow rate
3
of 3 × 10 cells/dish and incubated at 37 °C. The next day, the cells
was 0.4 mL/min, and the injection volume was 4 μL. The elution
program started from 95% A, hold for 2.6 min, then a linear gradient
to 50% B in 13 min, then to 70% B in 5 min, and then to 100% B in 6
min, held for 5 min. Re-equilibration took 5 min. ESI operating
were treated with the investigated compounds or mixtures of their
respective components at concentrations corresponding to the IC50
values previously determined with MTT assays after 72 h. Following a
further 18 h treatment, the cells were scraped, washed with PBS, and
counted. Identical cell counts were pelleted and lyzed in PBS-based
lysis buffer (1% NP40, 1 mM PMSF, cocktail of protease inhibitors)
on ice for 15 min. The supernatant was cleared and immediately
subjected to an enzyme activity analysis.
conditions: drying gas (N , purity >98%) 350 °C and 10 L/min;
2
capillary voltage 4.5 kV; nozzle voltage 1 kV; nebulizer gas 35 psig;
sheath gas (N , purity >98%) 375 °C and 11 L/min. The fragmentor
2
was at 175 V, the skimmer at 65 V, and the OCT 1 RF at 750 V.
High-resolution MS spectra were achieved from 5 to 30 min in
positive mode in the range 100−1700 m/z; the mass axis was
calibrated daily using the Agilent tuning mix HP0321 (Agilent
Technologies) prepared in acetonitrile and water. Calibration curves
for E-CO H, B-CO H, and F-CO H were derived in the 0.4−10 μM
Analysis of Glutathione S-Transferase activity. The enzyme
activity was evaluated with a Glutathione S-Transferase Fluorescent
Activity Kit (Invitrogen) following the manufacturer’s protocol.
Briefly, the cell lysates were plated in several dilutions in the Assay
Buffer. Master mix containing Detection Reagent and Glutathione was
added to the wells. The fluorescence was read in a kinetic mode at
460 nm with excitation at 390 nm. After blank subtraction, the data
were plotted as ΔRFU in the initial 5 min of the reaction. Data from
three to five measurements were normalized to the data of nontreated
control cells.
Analysis of Cyclooxygenase (COX) Activity. The enzyme activity
was evaluated with a Cyclooxygenase Activity Assay Kit (Abcam)
following the manufacturer’s protocol. Briefly, the cell lysates were
plated in several dilutions in Assay Buffer. Then the Reaction Mix
containing COX Probe and COX Cofactor was added to each well.
The reaction was initiated with the addition of an arachidonic acid/
NaOH solution. Fluorescence data were read in a kinetic mode at 587
nm with excitation at 535 nm. After blank subtraction, the data were
plotted as ΔRFU in the initial 5 min of the reaction. Data from three
to five measurements were normalized to the data of nontreated
control cells.
2
2
2
range from stock solutions containing the three analytes ca. 80 μmol/
g in water, appropriately diluted in HPLC-grade water.
Biological Studies. Cytotoxicity. Human cell lines A2780 and
A2780cisR (ovarian carcinoma) were obtained from the European
Collection of Cell Cultures, HEK-293 (embryonic kidney) was
obtained from the ATCC (Merck, Buchs, Switzerland), and MCF-7
(
breast carcinoma) and HeLa (cervical carcinoma) were obtained
from the ECACC (European Collection of Authenticated Cell
Cultures, Salisbury). HEK-293T cells were provided by the biological
screening facility (EPFL, Switzerland). CHO-K1 and MMC-2
(
derived from CHO-K1) Chinese hamster ovary cell lines were
provided by Miroslav Pirsel (Institute of Experimental Oncology SAS,
Bratislava). Cell culture media RPMI 1640 GlutaMAX and DMEM
GlutaMAX and penicillin/streptomycin were purchased from Life
Technologies, and fetal bovine serum (FBS) was obtained from
Merck. Cancer cells were cultured in RPMI 1640 GlutaMAX;HEK-
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Inorg. Chem. 2021, 60, 9529−9541