In-vitro antitumor activity of new quaternary phosphonium salts Iksanova et al.
3
Apoptotic assay
precipitated by centrifugation at 500g and the pellet was
resuspended in PBS to a final density of 2 × 106 cells/ml.
To 100 μl of cell suspensions, 1 μl ТМRЕ solution (17
μmol/l) or 1 μl DCFH-DA solution (200 μg/ml) was added
to each well, followed by 20 min of incubation at 20°C.
Subsequently, the medium was removed and washed
twice with PBS. Then, the fluorescence of TMRE (ex.
488 nm, em. 575 nm) or dichlorofluorescein, which is the
oxidized product of DCFH-DA (ex. 485 nm, em.
530 nm), was measured using the TECAN plate reader.
Data were expressed as the percentage of the ROS level
in the control group.
OVCAR-4 cells were seeded in 25 cm2 flasks till nearly
80% confluence. Media were changed to media containing
compound 1, 2, or 3 in a concentration equal to IC50. After
a 48-h incubation, the cells were harvested by trypsiniza-
tion to detach the cells without affecting the integrity of
the cell membrane. The cells were washed in PBS and
1 × 105 cells were collected by centrifugation. Then, cells
were resuspended in 300 µl of 1 × binding buffer, and 1 µl
of annexin V-FITC and 1 µl of propidium iodide (PI)
(50 µg/ml) were added. The cells were incubated at room
temperature for 30 min in the dark. The fluorescence
intensity of annexin V-FITC was determined by the
FL1-H channel and PI by the FL3-H channel on the
Guava easyCyte 8HT Flow Cytometer (Millipore,
Millipore Corporation, Burlington, Massachusetts, USA).
Immunoblot analysis and source of antibodies
The cells were harvested, washed in PBS, and lysed in
ice-cold RIPA buffer (50 mmol/l Tris pH 7.4, 150 mmol/l
NaCI, 1 mmol/l EDTA, 0.25% sodium deoxycholate,
1.0% NP-40, and protease inhibitors) at 4°C for 20 min.
Cell lysates were normalized (Bradford assay) and equal
protein loading was confirmed with Ponceau S staining.
Total protein (20–30 μg) was resolved by denaturing
12% SDS-PAGE gel electrophoresis before transfer to
Cell cycle
Assessment of cell cycle distribution and cell proliferation
is important to study cell growth differentiation and
apoptosis. OVCAR-4 cells were seeded in 25 cm2 flasks
till nearly 80% confluence. Media were changed to media
containing compound 1, 2, or 3 in IC50. After a 48-h
incubation, the cells were harvested by trypsinization to
detach the cells without affecting the integrity of the cell
membrane. The cells were washed in PBS, pelleted by
centrifugation at 1500 rpm, and fixed in ice-cold 70%
ethanol while vortexing. Cells were fixed for 4 h at 4°C
and washed in PBS twice by centrifugation at 1500 rpm.
Then, 50 µl of a 10 mg/ml stock of RNase, 500 µg/ml PI,
and 10% Triton-X10 were added and incubated at room
temperature for 30 min in the dark. The PI fluorescence
intensity in the cells was measured using a Guava
easyCyte 8HT Flow Cytometer at excitation and emis-
sion wavelengths of 536 and 617 nm, respectively, using
an Fl2-H filter.
a
nitrocellulose membrane (Inverclyde Biologicals,
Bellshill, Lanarkshire, UK) and probed with antibody
overnight at 4°C. Membranes were incubated with the
corresponding secondary HRP antibodies for 1 h at room
temperature before
a signal was detected using
Amersham ECL Prime Western Blotting Detection
Reagent (GE Healthcare, Chicago, Illinois, USA)
and the Bio-Rad ChemiDOC system. The following
antibodies were used in this study: monoclonal anti-
bodies against Bax (Abcam, Cambridge, UK), Bcl-2
(Abcam), caspase-3 (Santa Cruz Biotechnology Inc.,
Santa Cruz, California, USA), and anti-β-actin (Abcam).
The secondary antibodies included HRP-conjugated
anti-rabbit IgG (Abcam).
Determination of mitochondrial membrane potential and
reactive oxygen species
Results
Colony formation assay
A fluorometric assay using intracellular dye tetramethyl
rhodamine ethyl (TMRE) for the measurement of
mitochondrial membrane potential (MMP) and intracel-
lular oxidation of 2,7-dichlorohydrofluorescein diacetate
(DCFH-DA) for the measurement of reactive oxygen
species (ROS) in cells was performed. OVCAR-4 cells
were seeded in T-25 flasks at a density of 5 × 106 cells/
flask and were incubated at 37°C under a humidified
atmosphere containing 5% CO2 for 24 h before treat-
ment. The cells were incubated in full α-MEM media
containing compounds in a concentration equal to IC50.
As a negative control sample, we used Milli-Q water,
which was obtained using a water filtration station
(Millipore Corporation, Burlington, Massachusetts,
USA). After 48 h, the medium was removed and washed
with PBS. Then, cells were harvested with trypsin-
EDTA with subsequent inactivation of trypsin by add-
ing α-MEM-containing serum. Cell suspensions were
The ability of the OVCAR-4 cells to maintain their clo-
nogenic capacity and form colonies after treatment was
evaluated using a clonogenic assay. Compounds 1, 2, 3
and DOX induced a dose-dependent growth inhibition
after 7 days of exposure. The calculated IC50 values
ranged from 0.09 to 2.1 μmol/l (Table 1). Doxorubicin
and compound 3 had slightly higher antiproliferative
activity compared with 1 and 2.
Table 1 Effect of compounds on colony-forming capacity in
OVCAR-4 cells by a clonogenic assay
Compounds
IC50 (μmol/l)
1
2
3
DOX
2.06 0.13
0.36 0.08
0.10 0.00
0.09 0.00
The data represent the mean SEM of three independent experiments.
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