In-vitro antitumor activity of new quaternary phosphonium salts, derivatives of 3-hydroxypyridine

Article abstract of DOI:10.1097/CAD.0000000000000642

This work presents the results of in-vitro biological activity studies of three novel anticancer agents, phosphonium salts based on the 3-hydroxypyridine scaffold, including one derivative of 4-deoxypyridoxine. Proliferation and viability of cells treated with these compounds was assessed by the colony formation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Effects of the compounds on apoptosis and cell cycle were studied by flow cytometry using annexin V-FITC/propidium iodide and propidium iodide staining, respectively. The influence of the compounds on mitochondrial membrane potential and intracellular reactive oxygen species was evaluated using tetramethyl rhodamine ethyl and DCFHA staining. Western blot analysis was used to study the changes in the expression of Bcl-xL, Bax, and caspase-3 apoptotic proteins. The treatment of ovarian adenocarcinoma cells OVCAR-4 with the tested compounds inhibited the growth and induced cell cycle arrest in the G1 phase. 3-Hydroxypyridine derivatives induced apoptosis by hyperexpression of Bax and caspase-3, whereas 4-deoxypyridoxine derivative induced cell death partly by reactive oxygen species generation and caspase-3 hyperexpression. These results indicate that the quaternary phosphonium salts studied represent potential therapeutic agents for the treatment of ovarian cancer.

Full text of DOI:10.1097/CAD.0000000000000642

Preclinical report
1
In-vitro antitumor activity of new quaternary phosphonium
salts, derivatives of 3-hydroxypyridine
Alfiya G. Iksanovaa, Raylya R. Gabbasovaa, Tatyana V. Kupriyanovaa,
Almaz A. Akhunzyanova, Michail V. Pugacheva, Ruzalia M. Vafivaa,
Nikita V. Shtyrlina, Konstantin V. Balakina,b and Yurii G. Shtyrlina
This work presents the results of in-vitro biological activity
studies of three novel anticancer agents, phosphonium
salts based on the 3-hydroxypyridine scaffold, including one
derivative of 4-deoxypyridoxine. Proliferation and viability of
cells treated with these compounds was assessed by the
colony formation and 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide assays. Effects of the
compounds on apoptosis and cell cycle were studied by
flow cytometry using annexin V-FITC/propidium iodide and
propidium iodide staining, respectively. The influence of the
compounds on mitochondrial membrane potential and
intracellular reactive oxygen species was evaluated using
tetramethyl rhodamine ethyl and DCFHA staining. Western
blot analysis was used to study the changes in the
expression of Bcl-xL, Bax, and caspase-3 apoptotic
proteins. The treatment of ovarian adenocarcinoma cells
OVCAR-4 with the tested compounds inhibited the growth
and induced cell cycle arrest in the G1 phase.
hyperexpression of Bax and caspase-3, whereas
4-deoxypyridoxine derivative induced cell death partly by
reactive oxygen species generation and caspase-3
hyperexpression. These results indicate that the quaternary
phosphonium salts studied represent potential therapeutic
agents for the treatment of ovarian cancer. Anti-Cancer
Drugs 00:000–000 Copyright © 2018 Wolters Kluwer
Health, Inc. All rights reserved.
Anti-Cancer Drugs 2018, 00:000–000
Keywords: apoptosis, cell cycle, cytotoxicity, 4-deoxypyridoxine,
3-hydroxypiridine, OVCAR-4
aScientific and Educational Center of Pharmaceutics, Kazan (Volga region)
Federal University, Kazan and bDepartment of Analysis and Scientific Forecasting,
I.M. Sechenov First Moscow State Medical University, Moscow, Russia
Correspondence to Yurii G. Shtyrlin, PhD, DSc, Scientific and Educational Center
of Pharmaceutics, Kazan (Volga region) Federal University, PO Box 420008,
Kazan, Russia
Tel/fax: + 7 843 233 7363; e-mail: yurii.shtyrlin@gmail.com
Received 25 October 2017 Revised form accepted 14 April 2018
3-Hydroxypyridine derivatives induced apoptosis by
Introduction
a mitochondria-targeted Hsp90 inhibitor that increases
cell death in HeLa and MCF-7 cells [5].
Cancer is one of the leading causes of morbidity and
mortality worldwide, with ∼ 5.4 million deaths in 2017
(http://www.worldometers.info/). Ovarian cancer is the
eighth most common cancer among women, and it
accounts for about 4% of all cancers in women [1].
According to global estimates, 225 000 new cases are
detected each year, and 140 000 individuals die annually
from the disease [1]. Subsequently, novel approaches are
required for improvement of ovarian cancer therapies.
Some pyridine derivatives also showed anticancer activ-
ity. For example, 2,6-diaryl substituted pyridine showed
moderate cytotoxic against different human cancer cell
lines [6]. 3-Cyanopyridine compounds with different
alkyl or (hetero)aryl groups were found to possess
anticancer activity [7]. 2,6-Dibenzylamino-3,5-dicyano-
pyridines showed potential and promising anticancer
activity toward cell lines of nine different types of human
cancer [8]. Pyridine derivative LTU-27 (8-methyl-2-
(morpholine-4yl)-7-(pyridine-3-methoxy)-4H-1,3-benzox-
acine-4-one) was shown to sensitize cancerous cells,
specifically lung cancer and colon cancer, to the effects of
radiation [9].
Phosphonium salts are one of the promising classes of
compounds with antitumor activity. Thus, a combination
of ampicillin with phosphonium cations forms active ionic
liquids that show potent antiproliferative activities
against five different human cancer cell lines and low
cytotoxicity against two primary cell lines − skin and
gingival fibroblasts [2]. A series of triphenylphosphonium
salts with remarkable activity against a panel of cancer
cell lines as well as in a mouse model of human breast
cancer has been reported [3]. Betulinic acid derivatives
with one or two triphenylphosphonium moieties
show considerable antitumor activities against the
tested cancer murine and human tumor cell lines [4].
Gamitrinib modified by triphenylphosphonium cation is
In our previous work, different phosphonium salts based
on the 3-hydroxypyridine scaffold were synthesized.
Some of them possessed pronounced antitumor activity
against MCF-7 breast cancer cells [10]. This work pre-
sents the results of systematic in-vitro biological activity
studies of three leading compounds from this novel
interesting chemotype of anticancer agents.
0959-4973 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.
DOI: 10.1097/CAD.0000000000000642
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2
Anti-Cancer Drugs 2018, Vol 00 No 00
Methods
Colony formation assay
Compounds
Cells (1000 cells/well) were cultured in 980 μl of the
culture medium according to the culture conditions in a
six-well plate for 24 h. The medium was then changed to
980 μl of a new culture medium, and 20 μl of compounds
were added and incubated under standard conditions for
7 days. The concentration of stock solutions was 300
μmol/l. After incubation, the colonies were washed three
times with a cold solution of PBS and fixed in a fixing
buffer (glacial acetic acid, methanol, distilled water in a
10 : 10 : 80 ratio). The colonies were then stained with a
0.4% alcoholic solution of crystal violet. The results were
presented as the percentage of control values (untreated
cells).
Phosphonium salts 1–3 studied in this work (Fig. 1) were
synthesized according to our recently reported approa-
ches [10]. The reaction of 3-hydroxypyridine with
paraformaldehyde in alkaline medium led to 2-mono-
hydroxymethyl and 2,6-bis-hydroxymethyl derivatives,
which were converted into the corresponding chlorides
by reaction with thionyl chloride. At the last step, the
chlorides were treated with an excess of tris(p-tolyl)
phosphine to yield the target phosphonium salts 1 and 2.
Compound 3 was synthesized from 4-deoxypyridoxine,
which was treated with thionyl chloride to yield the
corresponding 5-chloromethyl derivative. Acylation of
the aromatic hydroxy group with acetyl chloride, followed
by reaction of the resulting intermediate with tris(p-tolyl)
phosphine yielded phosphonium salt 3. The developed
syntheses are straightforward and well reproducible.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide-assay
The cytotoxicity of compounds on OVCAR-4 culture was
assessed using a proliferative 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide test (MTT)
(Promega, Madison, Wisconsin, USA). Cells (1000 cells/
well) were cultured in 90 μl of the culture medium
according to the culture under standard conditions in a
96-well plate for 24 h. Then, 10 μl of the test preparations
were added and incubated under standard conditions for
6, 24, 48, and 72 h. The concentrations of stock solutions
were 300 μmol/l for 1 and 2; 1800 μmol/l for 3; and 100
μmol/l for doxorubicin. The medium with the tested
samples was replaced with a nutrient medium (80 μl), and
then 20 μl of MTT reagent (5 mg/l) was added, the
mixture was incubated for 3.5 h, the medium was
removed, and 100 μl of DMSO was added. After 10 min,
the optical density of the cell solutions was measured at
555 nm (reference wavelength 650 nm) on a TECAN
plate reader (Switzerland). Data were presented as per-
centage of MTT reduction compared with the control
cells. For the tested drugs, a dose–response curve was
obtained, and IC50 values (P < 0.05) were calculated.
Cell culture
Human ovarian adenocarcinoma cells OVCAR-4
(GSM136312) and primary human foreskin fibroblast cells
(HSF) isolated from the skin explant according to conven-
tional protocol [11] were cultured in α-MEM (PanEko,
Moscow, Russia) supplemented with 10% fetal bovine
serum (PAA; PAA Laboratories, Morningside QLD,
Australia), L-glutamine, and 1% penicillin–streptomycin at
37°C in a 5% humidified CO2 atmosphere in air. Cells were
removed from the culture substrate by treatment with
trypsin-EDTA with subsequent inactivation of trypsin by
adding α-MEM-containing serum. Cell suspensions were
precipitated by centrifugation at 500g and pellet was
resuspended in PBS. Cell viability and density were mea-
sured in a Neubauer chamber using a 0.4% solution of
Trypan blue. Suspensions with the amount of viable cells
no less than 90% were used in the experiments.
Fig. 1
Structures of phosphonium salts based on 3-hydroxypyridine and 4-deoxypyridoxine: 3-hydroxy-2-[tris(p-tolyl)phosphoniomethyl] pyridinium dichloride
(1), 3-hydroxy-2,6-bis-[tris(p-tolyl)phosphoniomethyl] pyridinium dichloride (2), 3-acetoxy-2,4-dimethyl-5-[tris(p-tolyl)phosphoniomethyl] pyridinium
chloride (3).
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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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4
Anti-Cancer Drugs 2018, Vol 00 No 00
Cytotoxicity in 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide-assay
cell cycle progression in OVCAR-4 cells, the cells were
treated for 48 h with concentrations equal to IC50 and
then DNA was stained with PI, followed by FACS ana-
lysis (Fig. 2).
The cytotoxic activity of compounds against OVCAR-4
and HSF cell lines was examined using the MTT assay.
All compounds induced time-dependent and dose-
dependent growth inhibition (Table 2). After 72-h
exposure, the calculated IC50 values ranged from 0.91 to
16 µmol/l for OVCAR-4 cells and from 0.76 to 65 µmol/l
for HSF. Compounds 1 and 3 have better selectivity
indexes (SIs, 5.1 and 4.1, respectively) compared with
doxorubicin and 2. The IC50 values obtained after 72-h
exposure were used for subsequent experiments.
The treatment of cells with 1, 2, and 3 for 48 h resulted in
increased percentage of cells in the G1 phase, with a
concomitant reduction in cell numbers in the S phase.
After the treatment of OVCAR-4 cells with compounds 1,
2, and 3, the cell population in the G0/G1 phase was 67,
69, and 70%, respectively, compared with 49% cell
population in the G0/G1 phase in the control group. At
the same time, the cell population in the S phase was 25,
23, and 21% after the treatment with compounds 1, 2,
and 3, respectively, compared with 42% cell population
in the S phase in the control group. However, the dif-
ferences between the experimental groups in the G2/M
phase were not statistically significant.
Apoptosis detection by flow cytometry
The effect of compounds on apoptosis was evaluated using
the PI and annexin V-FITC biparametric cytofluorimetric
analysis. After treatment for 48 h with the tested com-
pounds in concentrations equal to IC50, OVCAR-4 cells
were stained with PI and annexin V-FITC, and then
analyzed using flow cytometry. The results are presented
in Table 3. No early or delayed manifestations of apoptosis
were found in cells treated with compound 3, but the
treatment of cells with compounds 1 and 2 for 48 h resulted
in increased percentage of the late-stage apoptotic cells by
∼1.9 and 3 times, respectively.
Mitochondrial membrane potential (ΔΨ)
The effect of compounds on the MMP was studied using
TMRE that was accumulated within the mitochondria in
inverse proportion to MMP according to the Nernst
equation. The polarized mitochondria are capable of
accumulating more cationic dye than depolarized ones.
Figure 3 shows that the mitochondrial membrane
potential of OVCAR-4 cells was decreased after treat-
ment with compound 2. The intracellular fluorescent
intensity of cells was 8100 relative fluorescence units
(RFU), lower than that of the control group
(10 000 RFU). The results indicated that compound 2
induced mitochondrial dysfunction and apoptosis as
shown in Fig. 4 and Table 3. At the same time, com-
pounds 1 and 3 did not alter the mitochondrial mem-
brane potential of OVCAR-4 cells.
Inhibition of cell growth induced by the studied qua-
ternary phosphonium salts has been associated with a
significant level of cellular death, but this assay does not
distinguish between cells that have undergone apoptotic
death versus those that have died as a result of a necrotic
pathway as in both cases, the dead cells are stained with
both FITC annexin V and PI.
Cell cycle analysis by flow cytometry
It was shown that all compounds reduce the number
of viable OVCAR-4 cells in a dose-dependent manner.
To determine whether the studied compounds regulate
ROS generation in OVCAR-4 cells
To evaluate whether the tested compounds could increase
ROS levels, OVCAR-4 cells were treated with 1, 2, and 3
in concentrations equal to IC50 for 48 h. As shown in Fig. 5,
1 and 3 boosted ROS generation. After the treatment of
OVCAR-4 cells with compounds 1 and 3, the intracellular
dichlorofluorescein fluorescence intensity was 9200 and
11 000 RFU, respectively, compared with 8000 RFU in the
Table 2 Cytotoxicity of compounds in the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide-assay
Cytotoxicity, IC50 (μmol/l)
Compounds
6 h
24 h
48 h
72 h
OVCAR
HSF
1
2
3
DOX
1
2
3
DOX
1
> 30
> 30
> 180
> 10
> 30
> 30
> 180
> 10
–
4.4 1.0 1.1 0.04
1.9 0.02
8.4 0.6
16 6
0.91 0.1
9.6 0.5
> 30
> 30
Table 3 Effect of compounds on apoptosis of OVCAR-4 cells in
> 180
49 15
concentrations equal to IC50 (1–1.9 μmol/l, 2–8.5 μmol/l, 3–9 μmol/l)
3.9 0.5 3.1 0.5
> 30
> 30
> 180
> 10
–
> 30
> 30
> 180
> 10
–
%Gated
19
65
8
8
1
2
3
Control
0.76 0.05
5.1
Selectivity index
IC50 HSF/IC50
Live (LL)
Apoptotic (LR)
77.6 1.3
0.02 0.02
81.8 1.0
0.01 0.0
84.5 0.5
0.03 0.02 0.02 0.02
92.1 0.7
Apoptotic/dead (UR) 0.28 0.08* 0.44 0.06* 0.13 0.05 0.15 0.02
Dead (UL) 22.1 1.4* 17.9 1.1* 15.3 0.5* 7.8 0.7
OVCAR-4
2
3
DOX
–
–
–
–
–
–
–
–
–
2.3
4.1
0.9
Apoptosis was measured using flow cytometric analysis of annexin V binding 48 h
after treatment. The presented values are calculated from the flow cytometry plots
(Fig. 3). LL, lower left; LR, lower right.; UL, upper left; UR, upper right.
*Values indicate P < 0.05.
The data represent the mean SEM of three independent experiments.
HSF, human skin fibroblast.
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In-vitro antitumor activity of new quaternary phosphonium salts Iksanova et al.
5
Fig. 2
Effect of compounds 1–3 on OVCAR-4 cell cycle arrest. Cells were treated with compounds and harvested after 48 h. Then, they were fixed with
ethanol, stained with propidium iodide, and analyzed by flow cytometry. (a) Cell distribution histograms. (b) Percentage of cells in the G0/G1, S, and
G2/M phases (data are presented as mean SD of three independent experiments). *Values indicate P < 0.05.
control group. These results suggested that 1 and 3
induced cellular toxicity in OVCAR-4 cells, at least partly
by ROS generation. Interestingly, under the same experi-
mental conditions, compound 2 did not increase the
intracellular ROS concentration.
the compounds in concentrations equal to IC50 for 48 h; as
a control, untreated cells were used. Lysates of the treated
and untreated cells were prepared and processed for wes-
tern blot analysis to study the changes in the levels of
apoptosis-related proteins. The level of Bcl-xL protein did
not change, whereas the level of Bax protein increased
after the treatment with compounds 1 and 2. The relative
ratio of Bax to Bcl-2 was increased. Furthermore, the
expression of caspase-3 protein was significantly increased
with all compounds (Fig. 6).
Expression of apoptosis-related proteins
In the next experiment, we attempted to elucidate the role
of Bcl-xL, Bax, and caspase-3 in the antitumor activity of
the tested compounds. OVCAR-4 cells were treated with
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6
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 3
In comparison with the clonogenic test, the MTT assay
enables the assessment of the metabolic cellular activity.
It is known that the duration of the OVCAR-4 cell divi-
sion cycle is 43 h on average. For this reason, in the MTT
cytotoxicity assay, incubation with the tested compounds
has been performed for 72 h, during which time the dose
and time kinetics of the cell survival-modulating action of
the tested compounds were measured (Table 2). After
72-h exposure, doxorubicin showed the highest anti-
tumor activity, but the lowest SI, which may explain its
expressed side effects. At the same time, all the studied
phosphonium salts had a relatively good SI (from 2.3 to
5.1). Interestingly, there were no cytotoxic actions of the
studied compounds 2 and 3 before 72 h, which indicates
their indirect action by triggering the apoptotic pathways.
It is accepted widely that the charged molecules are unable
to move freely across the cellular membranes without the
aid of active transport systems. The distribution of positive
charge across the large lipophilic surface of the phospho-
nium cation significantly lowers the activation energy, thus
facilitating passage through the membranes [3]. The posi-
tive charge also leads to the accumulation of phosphonium
salts in mitochondria because of their negative membrane
potential. Given these important features of the triphe-
nylphosphonium cation, it has been used for the targeted
mitochondrial delivery of spin traps, fluorescent dyes, and
antioxidants. It is important to note that many solid tumors
have a more negative mitochondrial membrane potential
compared with normal cells [13], and this feature can be
used for selective delivery of the phosphonium-modified
agents to tumor cells.
Effect of compounds 1–3 on the mitochondrial membrane potential
(Dψm). The OVCAR-4 cells treated were stained with TMRE and the
intensity of tetramethyl rhodamine ethyl fluorescence in each sample
was analyzed using a TECAN plate reader. Data are presented as
mean SD (relative fluorescence units, RFU) of three independent
experiments. *Values indicate P < 0.05.
The obtained results suggest that compounds 1 and 2
induce apoptosis by hyperexpression of Bax and caspase-
3, but compound 3 probably acts by alternative
mechanisms associated with triggering of cell death by
ROS generation and caspase-3 hyperexpression.
Discussion
Phosphonium salts show great promise for the diagnosis
and treatment of neoplasms [3]. Several phosphonium
salts, because of their ability to disrupt mitochondrial
structure and alter cellular lipid content, have shown
antiproliferative activity in a panel of cancer cell lines and
in an in-vivo model of ovarian cancer [3]. The diagnostic
imaging studies [12] have elucidated two key features of
phosphonium salts: (a) they are capable of preferentially
accumulating within tumor cells and (b) the phospho-
nium cation itself is not cytotoxic.
At the same time, one should not exclude the possibility of
direct effects of the phosphonium-containing compounds
on the activity of both mitochondrial reductases, which
catalyze the reduction of tetrazolium salts to colored for-
mazan, and on direct transport of the MTT reagent.
Therefore, it may be suggested that the affinity of phos-
phonium compounds to mitochondria positively correlates
with the observed IC50 values. Thus, compound 3, which
shows the highest activity in the clonogenic test, shows the
minimal cytotoxicity in the MTT test; the opposite
dependences are observed for compound 1.
In this study, we assessed the effects of three quaternary
phosphonium salts on the basis of 3-hydroxypyridine and
4-deoxypyridoxine (Fig. 1) on OVCAR-4 ovarian cancer
cells. Treatment of these cells with tris(p-tolyl)phosphine
1 and bis-tris(p-tolyl)phosphine 2 derivatives of 3-hydro-
xypyridine, and acetoxy-dimethyl-tris(p-tolyl)phospho-
niomethyl derivative 3 of 4-hydroxypyridine was found
to inhibit the growth and to induce cell cycle arrest in the
G1 phase. According to the results of the clonogenic
assay, compound 3 had the highest antiproliferative
activity, which was comparable to that of the reference
drug doxorubicine. Introduction of an additional phos-
phonium fragment resulted in increased cytotoxicity.
Thus, bis-phosphonium compound 2 (IC50 0.36 0.08
μmol/l) was 5.7-fold more cytotoxic than monopho-
sphonium compound 1 (IC50 2.06 0.13 μmol/l).
The subsequent evaluation of biological activity was based
on IC50 values obtained in the MTT experiment (Table 2).
Analysis of action of compounds 1–3 on the cell cycle
showed that all the tested compounds induced a decreased
percentage of cells in the S phase and a proportionally
increased percentage of cells in the G1 phase, which indi-
cates the cell cycle arrest in the G1 phase.
One of the key strategies for anticancer drug develop-
ment is to induce apoptotic events in cancer cells while
minimizing the negative effects in normal cells [14,15].
In this work, we have shown that a proportion of cells are
killed after incubation with the tested compounds for
48 h, as evidenced by increased percentage of cells
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In-vitro antitumor activity of new quaternary phosphonium salts Iksanova et al.
7
Fig. 4
Flow cytometry analysis of the apoptotic effects of compounds 1–3 on OVCAR-4 cells. The scatter dot plots indicating the percentage of live [annexin
V negative, propidium iodide (PI) negative], early (annexin V positive, PI negative), and late (annexin V positive, PI positive) apoptotic cells after
treatment with medium (control) (a), compound 1 (b), compound 2 (c), and compound 3 (d) for 48 h using annexin V and PI assay.
stained positively with FITC annexin V and PI. It should
be noted that the cell death caused by compound 3 is
because of an increase in the intracellular level of ROS
and also because of overexpression of caspase-3.
Activation of caspase-3 enzymes is a marker of apoptosis
that can be used in cellular assays to quantify the efficacy
of activators and inhibitors of this ‘death cascade’.
Caspase-3 enzymes are activated in apoptotic cells both
by extrinsic (death ligand) and by intrinsic (mitochon-
drial) pathways. In view of the absence of the influence
of compound 3 on the mitochondrial potential and the
expression of Bax and Bcl-xL proteins, it can be assumed
that this agent induces the externally mediated caspase
pathway of apoptosis because of an elevated ROS level.
apoptosis-induced cascade of intracellular signals invol-
ving the Bcl-2 protein family. The latter includes both
antiapoptotic (e.g. Bcl-xL, Bcl-2) and proapoptotic (e.g.
Bax, Bid, tBid, Bad, Bcl-xs) proteins [16]. We have found
that compounds 1 and 2 increase the expression of the
proapoptotic Bax protein and the main marker of apop-
tosis, caspase-3. These compounds, in addition to cell
death, cause an increase in the percentage of cells in the
late stages of apoptosis. Nevertheless, there are differ-
ences in the hypothetical mechanism of action of
compounds 1 and 2. Thus, compound 2 has a bis-phos-
phonium fragment in its structure, which reduces the
negative membrane mitochondrial potential. It has been
shown that the decrease of MMP leads to structural
changes in the mitochondria, such as matrix condensation
and cristal unfolding [16], which result in the redis-
tribution of cytochrome c from the cristae to the
It was reported that the extrinsic pathway can activate
the intrinsic pathway by triggering a mitochondrial
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8
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 5
structural similarity, there are some differences in the
mechanisms of anticancer action of compounds 1–3.
Specifically, the monophosphonium fragment in compound
1 does not lead to a decrease in the membrane mitochondrial
potential, and this agent exerts its action mainly by increasing
the level of ROS. Our experimental results also show that
compound 1 induces apoptosis by hyperexpression of Bax
and caspase-3. Compound 2 has a bis-phosphonium fragment
in its structure, which reduces the negative membrane
mitochondrial potential, thus leading to the release of
cytochrome C to the intermembrane space and initiation of
apoptosis by hyperexpression of Bax and caspase-3.
Compound 3 has the highest antiproliferative activity, at least
partly by ROS generation. It also induces apoptosis by
hyperexpression of caspase-3, rather than proapoptotic pro-
teins. It should be stressed that the mechanisms described
are just hypothetical, and further studies are required to
validate these. In general, these results suggest that new
quaternary phosphonium salts based on 3-hydroxypyridine
derivatives represent potential therapeutic agents for the
treatment of ovarian cancer.
Effect of compounds 1–3 on ROS concentration. The treated OVCAR-
4 cells were stained with DCFH-DA, and the intensity of DCF
fluorescence in each sample was analyzed using a TECAN plate reader.
Data are presented as mean SD (relative fluorescence units, RFU) of
three independent experiments. *Values indicate P < 0.05.
Acknowledgements
Fig. 6
This work was funded by Programs of Competitive
Growth of Kazan Federal University and I.M. Sechenov
First Moscow State Medical University.
Conflicts of interest
There are no conflicts of interest.
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