Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro
Aim: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. Materials and Methods: The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MD...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2017
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| Zitieren: | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro / I.V. Zalutski, N.Y. Lukianova, D.M. Storchai, A.P. Burlaka, Y.V. Shvets, T.V. Borikun, I.M. Todor, V.S. Lukashevich, Y.A. Rudnichenko, V.F. Chekhun // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 106–111. — Бібліогр.: 21 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859950365346103296 |
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| author | Zalutski, I.V. Lukianova, N.Y. Storchai, D.M. Burlaka, A.P. Shvets, Y.V. Borikun, T.V. Todor, I.M. Lukashevich, V.S. Rudnichenko, Y.A. Chekhun, V.F. |
| author_facet | Zalutski, I.V. Lukianova, N.Y. Storchai, D.M. Burlaka, A.P. Shvets, Y.V. Borikun, T.V. Todor, I.M. Lukashevich, V.S. Rudnichenko, Y.A. Chekhun, V.F. |
| citation_txt | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro / I.V. Zalutski, N.Y. Lukianova, D.M. Storchai, A.P. Burlaka, Y.V. Shvets, T.V. Borikun, I.M. Todor, V.S. Lukashevich, Y.A. Rudnichenko, V.F. Chekhun // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 106–111. — Бібліогр.: 21 назв. — англ. |
| collection | DSpace DC |
| container_title | Experimental Oncology |
| description | Aim: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. Materials and Methods: The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Results: Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of “free” iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231 and MDA-MB-468 cells. These changes resulted in increased expression of proapoptotic Bax protein (p < 0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4–1.7-fold, p < 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14–24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G₂/M phase, especially in receptor-negative cell lines. Conclusion: The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, “free” iron and NO generation rate, resalting in the blocking of cell cycle at G₂/M-phase and apoptosis of malignant cells.
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106 Experimental Oncology 39, 106–111, 2017 (June)
INFLUENCE OF EXOGENOUS LACTOFERRIN ON THE OXIDANT/
ANTIOXIDANT BALANCE AND MOLECULAR PROFILE
OF HORMONE RECEPTOR-POSITIvE AND -NEGATIvE HUMAN
BREAST CANCER CELLS IN VITRO
I.V. Zalutski1, N.Y. Lukianova2, D.M. Storchai2, A.P. Burlaka2, Y.V. Shvets2, T.V. Borikun2, I.M. Todor2,
V.S. Lukashevich2, Y.A. Rudnichenko2, V.F. Chekhun2, *
1State Scientific Institution “Institute of Physiology”, NAS of Belarus, Minsk 220072, Republic of Belarus
2R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
Aim: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive
and receptor-negative breast cancer cells in vitro. Materials and Methods: The study was performed on receptor-positive (MCF-7,
T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining,
flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Results: Upon treatment
with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase
(p < 0.05) and the level of “free” iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced
in receptor-negative MDA-MB-231 and MDA-MB-468 cells. These changes resulted in increased expression of proapoptotic
Bax protein (p < 0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial
cardiolipin (1.4–1.7-fold, p < 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14–24%, p < 0.05).
Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle
at G2/M phase, especially in receptor-negative cell lines. Conclusion: The study showed that exogenous lactoferrin causes a viola-
tion of an antioxidant balance by increasing the level of ROS, “free” iron and NO generation rate, resalting in the blocking of cell
cycle at G2/M-phase and apoptosis of malignant cells.
Key Words: lactoferrin, breast cancer, oxidative status, antioxidant status, apoptosis.
Breast cancer (BC) is a heterogeneous disease that
varies by the morphological and molecular structure
as well as by the clinical course, and therefore requires
different approaches to diagnosis and treatment.
To date, the expansion of knowledge about the role
of metalloproteins in malignant transformation and
tumor progression as important components of me-
tabolism is a great interest. In this respect explora-
tion of iron metabolism disorders in cancer patients
become increasingly relevant in recent years [1–3].
The role of such violations in the tumorigenesis
is already proven by clinical and experimental studies.
There is evidence that the concentration of iron and
iron-regu lating proteins correlates with tumor ag-
gressiveness of BC [4]. Among the least studied iron-
containing proteins is lactoferrin (LF) — a protein with
a unique set of biological properties of different nature.
An important feature of LF is its ability to bind to nucleic
acids and to regulate cell cycle, cell proliferative acti-
vity, and affect the oxidant/antioxidant balance in the
cells. The synthesis and secretion of LF may be con-
stitutional (e.g., secretory glands) or controlled by sex
hormones [5].
Nowadays the search and development of means
of drug therapy with antioxidant properties is per-
formed worldwide. Experimental studies have shown
that natural antioxidant proteins (ceruloplasmin, su-
peroxide dismutase (SOD), and alkaline phosphatase)
have the highest activity compared with synthetic
analogs. However, in modern oncology practice, only
ceruloplasmin-based drugs are used.
The lack of studies aimed at assessing the impact
of LF on the growth of malignant tumors, because of its
ability under certain conditions to exercise prooxidative
properties is one of the reasons why LF is not applied
in practice. The existence of functionally different
phenotypic and molecular genetic characteristics
of BC cells with different biological aggressiveness
is the basis for the development of methods for selec-
tive therapeutic intervention, which should be based
on the results of experimental LF studies. Thus, our
aim was to investigate the mechanisms of cytotoxic
activity and pro/antioxidant effect of LF on hormone
receptor-positive and -negative BC cells.
MATERIALS AND METHODS
Cell culture. Hormone-positive MCF-7 and T47D and
hormone-negative MDA-MB-231 and MDA-MB-468 cell
lines were used in our study. Cells were provided by the
Bank of Cell Lines of Human and Animal Tissues
at R.E. Kavetsky Institute of Experimental Patho logy,
Oncology, and Radiobiology of NAS of Ukraine. All cells
were cultured in DMEM (Sigma, USA), supplemented
with recombinant human insulin (Life Technologies,
USA; 0.01 mg/ml) and 10% fetal bovine serum (Sigma
Submitted: May 15, 2017.
*Correspondence: E-mail: chekhun@onconet.kiev.ua
Abbreviations used: BC — breast cancer; EPR — electron para-
magnetic resonance; LF — lactoferrin; ROS — reactive oxygen
species; SOD — superoxide dismutase.
Exp Oncol 2017
39, 2, 106–111
ORIGINAL CONTRIBUTIONS
Experimental Oncology 39, 106–111, 2017 (June) 107
Aldrich, USA). All cultures were incubated in humidified
atmosphere with 5% CO2 at 37 °C.
Measurement of cell viability, using the MTT as-
say was performed as described previously [6]. The
cytotoxic effect of exogenous LF, wich was synthesized
at the State Scientific Institution “Institute of Physio-
logy” (Minsk, Republic of Belarus) was measured in the
dose range of 2–300 μg/ml.
Immunocytochemical study of the expression
of apoptosis regulators in BC cells was performed
using monoclonal mouse anti-human Bcl-2 (clone
124) and polyclonal rabbit anti-human Bax antibo dies
(DakoCytomation, Denmark). For the detection reac-
tion visualization, UltraVision Quanto Detection System
HRP DAB (Thermo Fisher Scientific, USA) was used.
Cells for immunocytochemical studies were
grown on glass cover slips, fixed in the cooled mix-
ture of methanol:acetone (1:1) at –20 °C for 120 min,
washed in PBS and incubated with 1% BSA for 20 min.
Primary monoclonal antibodies were diluted in the
blocking buffer and kept at a room temperature for one
hour, followed by incubation with UltraVision Quanto De-
tection System for 10 and 15 min; after the washing, the
immune reaction was visualized by using DAB Quanto.
When immunocytochemical reactions were completed,
the cells were stained by Mayer’s hematoxylin for
10–15 s and placed in Faramount Aqueous Mount-
ing Medium (DakoCytomation, Denmark). Evaluation
of the results was carried out in 3 visual fields by light
microscopy (x 1000, oil immersion) using the classical
H-Score method:
S = 1×N1
+ + 2×N2
+ + 3×N3
+,
where S is “H-Score” index, N1
+, N2,+ and N3
+ are
numbers of cells with low, medium or high levels
of marker expression.
Low -temperature electron paramagne-
tic resonance. The level of “free” iron and rate
of NO gene ration by inducible NO-synthase were
estimated by low-temperature electron paramagnetic
resonance (EPR). To determine the level of “free” iron,
samples were kept during spectra recording at liquid
nitrogen temperature (77 K). For low-temperature EPR
the following parameters were used: stripe width was
1525 G, frequency — 9.15 GHz, microwave power —
40 mW, amplitude modulation — 10.0 G, frequency
modulation — 100 kHz. g-Factor was calculated using
a standard formula:
g = hν/βH,
where h is Plank’s constant; ν — frequency,
β — Bohr’s magneton; H — external magnetic field
in resonance.
The evaluation of speed of NO generating by induc-
ible NO-synthase in BC cells was conducted at room
temperature using technology SpinTrap in the tem-
perature of liquid nitrogen (77 K). The level of NO was
determined using a diethyldithiocarbomate spin trap
(Sigma, USA) and EPR technology. The generation
of NO was registered during 5 min followed by ces-
sation of NO generation by liquid nitrogen. The levels
of NO were calculated in nmol/2.5•105 cells.
Genotoxic activity of LF assessment was per-
formed using a standard classical method of alkaline
gel electrophoresis of isolated cells (Comet assay)
as described in [7]. After drying the samples were
stained with acridine orange (Sigma Aldrich, USA;
2 mg/ml). Microscopic analysis was performed using
a fluorescent microscope MC 300x FS (Micros, Aus-
tria). For each analyzed specimens at least 100 “DNA-
comets”. Digital images were analyzed by the com-
puter program “CometScore” (TriTek Corp., USA).
Measurement of SOD. SOD activity was detected
as described earlier [8]. Briefly, cells were homo genized
in the glass homogenizer with 2 ml of 0.1 М PBS. After
centrifugation at 3000 rpm for 20 min supernatant was
analyzed for SOD activity using radiospectrometer
ЕPR-1307 (ZEPS AN, Russia) at room temperature.
Flow cytometry. Determination of the reactive
oxygen species (ROS), cardiolipin and glutathione
content, the study of apoptosis and DNA status of cells
were conducted using specific dyes for flow cyto metry
on Beckman Coulter EPICS® XL Flow Cytometer (Beck-
man Coulter, USA). To study of ROS generation by flow
cytometry CM-H2DCFDA dye (Thermo Fisher Scientif-
ic, USA) was used following the manufacturer’s recom-
mendations. Assessment of the content of glutathione
in BC cells was performed using dye CellTracker™
GreenCMFDA dye (Thermo Fisher Scientific, USA),
following the manufacturer’s recommendations. To vi-
sualize the distribution of cardiolipin in mitochondria
acridine orange 10-nonyl bromide (NAO; Invitrogen,
USA) dye was used as described in [9]. To determine
the number of apoptotic cells the staining with An-
nexin A5-FITC Kit (Beckman Coulter, France) was used
as described in [10]. Charts of density distribution
of the studied parameters were analyzed using FCS
Express V3 (De Novo Software, USA). DNA status was
assessed using ModFit v3.2 software (Verity Software
House, USA).
Statistical analysis. Experimental data were
analyzed using the Student’s t-test. p-values less than
0.05 were considered statistically significant. Statisti-
cal analysis of the obtained data was performed using
the STATISTICA 6.0 software (Dell, USA).
RESULTS AND DISCUSSION
BC is a heterogeneous disease that varies
by the morphological and molecular features as well
as by the clinical course, and therefore requires dif-
ferent approaches to diagnosis and treatment. Know-
ledge about the role of metalloproteins in malignant
transformation and tumor progression as important
components of metabolism attaches great importance
to date. In this respect in recent years become increas-
ingly relevant studying of disorders of iron metabolism
and possibilities of its modification by exogenous
agents [11]. LF is very perspective agent for this pur-
pose, because it is normal iron-regulating protein,
possessing immune and antitumor activities [12].
We established more pronounced cytotoxic ac-
tivity of LF on receptor-negative MDA-MB-231 and
108 Experimental Oncology 39, 106–111, 2017 (June)
MDA-MB-468 cells compared to receptor-positive
MCF-7 and T47D cells. As seen from the data present-
ed in Fig. 1, IC30 dose for MCF-7 and T47D cells was
10 μg/ml, while for MDA-MB-231 and MDA-MB-468 cells
the similar dose of exogenous LF caused the death
of 45% and 40% of cells, respectively. The dose of LF,
that causes death in 30% of the cells with negative
receptor status, was 5 μg/ml. To study the molecular
effects of LF we used IC30 doses; cells were treated
with LF for 24 h.
300
MCF-7
T47D
MDA-MB-231
MDA-MB-468
0 30 60 90 120 150 180 210 240 270
LF dose, µg/ml
Am
ou
nt
o
f d
ea
d
ce
lls
, %
100
80
60
40
20
0
Fig. 1. Cytotoxic activity of exogenous LF on human BC cells
with different receptor status
It is known that one of the main functions
of LF is the regulation of the concentration of iron
ions in the cells. Iron is a key element for the normal
processes of proliferation and cell growth. Intra-
cellular iron is carefully maintained and controlled
at a certain level by many mechanisms. It is known
that cancer cells have a significant increase in iron
content due to their enhanced metabolic and pro-
liferative needs [13]. On the other hand, the content
of “free” iron in cancer cells may decrease because
of its exhaustion, which also indicates an increased
need for this element in cells that rapidly grow and
divide. However, the excess iron can lead to the for-
mation of dangerous ROS, which creates conditions
for the initiation and progression of tumors. Increased
number of ROS also can be caused by any other cell
response to stress. This is the adaptation to extreme
conditions, in which ROS act as second messengers
participating in signal transduction and activation
of transcription factors of certain genes, including
those encoding enzymes and antioxidants.
The physiological level and rate of generation
of ROS in the cells are maintained at a constant level
due to the existence of multi-antioxidant protection
system. Higher concentrations of ROS due to shifting
the balance between the processes of their formation
and detoxification are a precondition for infringement
of functional activity of cells and pathological pro-
cesses [14]. Therefore, the next series of experiments
were aimed at studying changes on the level of “free”
iron in LF-treated BC cells with different hormone
receptor status.
We found a significant increase of “free” iron content
after treatment of BC cells with LF (Fig. 2). Thus, in the
treated receptor-positive MCF-7 and T47D cells, the
levels of “free” iron increased to 1.7–1.9-fold and were
0.98 ± 0.11•1016 spin/ml and 0.88 ± 0.1•1016 spin/ml.
Treatment of MDA-MB-231 and MDA-MB-468 cells
under similar conditions led to increased levels of
“free” iron in 2.3–2.5-fold (see Fig. 2).
"F
re
e"
ir
on
le
ve
l,
·1
016
s
pi
n/
m
l
0
1
2
3
4
5
6
7
8
9
MCF-7 T47D MDA-MB-231 MDA-MB-468
Control
After LF treatment
*
*
**
Fig. 2. The levels of “free” iron in BC cells with different receptor
status under the influence of exogenous LF. *р < 0.05 compared
to untreated cells
Culvation of BC cells with exogenous LF also
led to an increase in ROS generation (Fig. 3). Thus,
in treated MCF-7 and T47D cells intracellular le-
vels of ROS increased by 2 times and amounted
up to 3.32 ± 0.24 r. u. and 5.21 ± 0.67 r. u., respectively.
Treatment of MDA-MB-231 and MDA-MB-468 cells
with LF also led to an increase in intracellular levels
of ROS at 1.76- and 1.63-fold, respectively.
MCF-7 T47D MDA-MB-231 MDA-MB-468
Control
After LF treatment
*
*
*
*
0
5
10
15
20
25
Le
ve
l o
f R
O
S,
r.
u
.
Fig. 3. The levels of ROS in BC cells with different receptor
status under influence of exogenous LF. *р < 0.05 compared
to untreated cells
During the study of NO generation speed by in-
ducible NO-synthase in BC cells after cultivation with
LF, we established an increase of this indicator
in all studied cell lines. As can be seen from the data
presented in Fig. 4, in the hormone-responsible
MCF-7 and T47D cells the rate of NO-generation
by exogenous LF increased in 1.5–1.6-fold and
amounted up to 271.75 ± 10.45 nmol/2.5•105 and
256.70 ± 11.81 nmol/2,5•105, respectively. In recep-
tor-negative MDA-MB-231 and MDA-MB-468 cells
NO generation rate raised of more than 90%.
Thus, LF treatment of receptor-positive and recep-
tor-negative BC cells resulted in oxidative stress. This
is evidenced by the increase of “free” iron, intracellular
levels of ROS, and NO generation speed by inducible
Experimental Oncology 39, 106–111, 2017 (June) 109
NO-synthase after LF treatment. The most serious
violations of oxidative balance rates after LF effect
we observed in receptor-negative BC cells.
There is evidence about LF ability to disrupt
the mitochondrial membrane, which is associated
with increased levels of ROS and release of cyto-
chrome C [15]. ROS oxidize polyunsaturated acyl
chains of cardiolipin, which is a signal for triggering
apoptosis [16]. As mentioned above, the system
of glutathione is one of the most powerful intracellular
antioxidative mechanisms. The thiol groups of gluta-
thione in the reduced state are in a high concentration
in cells, which allows recovering disulfide bonds. The
balance between restored and oxidized forms of glu-
tathione is an important parameter that determines
the level of oxidative stress in cells [17].
Another powerful antioxidant enzyme is oxide-
reducing SOD that catalyzes the dismutation of O2–
radicals and prevents the transformation of super-
oxide anion radicals into the hydroxyl radical. Also,
SOD is an acceptor of free oxygen radicals, thereby
preventing lipid and protein peroxidation. Normally
the SOD supports basic concentration of superoxide
radicals at a certain level, thereby protecting cells from
damaging effect of O2– and hydroxyl radicals that can
be generated from O2– and HO [18].
In our study, we tested the reduced form of gluta-
thione using dye CellTrackerGreen (CMFDA), which
interacts with SH-groups of glutathione, by flow
cyto metry. On Fig. 5 are represented typical histo-
grams of reduced glutathione in the BC cells. As can
be seen from the data presented in Fig. 6, LF treat-
ment in a dose corresponding to IC30, for 24 h re-
sulted in a decrease in intracellular glutathione content
in studied BC cell lines, indicating a transition of glu-
tathione in the oxidized state. The most significant
decrease (in 4.3- and 2.3-fold) glutathione content
was observed in MCF-7 and MDA-MB-468 cells.
In the T47D and MDA-MB-231 cells the glutathione
level decreased by 1.9- and 1.4-fold, respectively,
and amounted to 3.57 ± 0.82 r. u. and 3.32 ± 0.45 r. u.,
respectively. Established differences in levels of glu-
tathione in MCF-7 and MDA-MB-468 cells are likely
caused by the high proliferative activity of these cells.
MCF-7 T47D MDA-MB-231 MDA-MB-468
Control
After LF treatment
*
*
*
*
0
2
4
6
8
10
12
14
16
18
20
Re
du
ce
d
gl
ut
at
io
ne
le
ve
l,
r.u
.
Fig. 6. The reduced glutathione levels in BC cells with different
receptor status under influence of exogenous LF. *р < 0.05 com-
pared to untreated cells
LF treatment also led to reduced activity of SOD
in 1.2–1.7-fold compared to baseline in all studied
cell lines (Fig. 7).
MCF-7 T47D MDA-MB-231 MDA-MB-468
Control
After LF treatment
* *
*
*
0
2
4
6
8
10
12
SO
D
ac
tiv
ity
, r
.u
.
Fig. 7. SOD activity in BC cells with different receptor status
under influence of exogenous LF. *р < 0.05 compared to un-
treated cells
According to the literature, one of the mechanisms
of exogenous LF action is initiating apoptosis in malig-
nant cells [19]. Thus, Fujita et al. found that treatment
of colorectal cancer cells with LF increases the expres-
sion of death receptor CD95 and proapoptotic Bax
and Bid proteins [19]. We established that exposure
to exogenous LF of studied BC cells caused cell death
mainly by apoptosis. As it is shown in Table 1, under
the influence of LF 25.5% and 22.6% of the MCF-7 and
MCF-7 T47D MDA-MB-231 MDA-MB-468
Control
After LF treatment
*
*
**
0
100
200
300
400
500
600
700
Ra
te
o
f
N
O
g
en
er
at
io
n,
n
m
ol
/2
,5
·1
05 c
el
ls
Fig. 4. The rate of NO generation by inducible NO-synthase
in human BC cells with different receptor status under influence
of exogenous LF. *р < 0.05 compared to untreated cells
Fig. 5. Typical flow cytometry histograms of glutathione content
in BC cells: a — untreated cells; b — LF-treated cells
Control
MCF-7
Control
MCF-7 + LF
Co
un
t
Co
un
t
FL1 LOG FL1 LOG
50
38
25
13
0
50
38
25
13
0
100 101 102 103 10 100 101 102 103 104
a b
110 Experimental Oncology 39, 106–111, 2017 (June)
T47D cells, respectively, were in a state of apoptosis.
The highest percentage of apoptotic cells under exoge-
nous LF treatment was observed in receptor-negative
MDA-MB-468 and MDA-MB-231 cells (43.2 and 41.3%,
respectively). Therefore, LF treatment resulted in an in-
crease of apoptotic cells in receptor-positive BC cells
(by 14% in MCF-7 and 17% in T47D cells) as well as in re-
ceptor-negative BC cells (by 33% in MDA-MB-231 cells
and 29% in MDA-MB-468 cells).
Table 1. Percentage of apoptotic cells in BC cell lines before and after
LF treatment
Cell line Apoptotic cells, %
Control After LF treatment
MCF-7 8.1 ± 3.1 25.5 ± 2.8*
T47D 8.7 ± 2.9 22.6 ± 3.2*
MDA-MB-231 10.6 ± 3.7 43.2 ± 2.7*
MDA-MB-468 12.2 ± 4.0 41.3 ± 3.8*
Note: *р < 0.05 compared to untreated cells.
Since it is known that a key role in the initiation
of apoptosis belongs to cardiolipin — an essential
phospholipid component of the mitochondria mem-
branes — on the next step we studied the changes
in its content in the BC cells under the influence
of exogenous LF. As can be seen from the data pre-
sented in Fig. 8, 24 h LF expose resulted in a decrease
in NAO fluorescence intensity in all investigated cells
lines. So, upon LF treatment the levels of non-oxidized
cardiolipin in MCF-7 and T47D cells decreased by 1.6-
and 1.7-fold, respectively. In MDA-MB-231 cells
cardiolipin levels decreased 2.4-fold and amounted
to 57.29 ± 7.65 r. u., whereas in MDA-MB-468 cell line
cardiolipin levels decreased by 2.1-fold and amounted
to 136.07 ± 74.20 r. u., indicating the development
of oxidative stress. Unidirectional reducing of NAO
fluorescence in the cells of all studied lines under the
LF impact demonstrates the ability of LF to oxidize mi-
tochondrial cardiolipin. The most significant changes
in cardiolipin content we observed in receptor-nega-
tive MDA-MB-231 and MDA-MB-468 cells.
MCF-7 T47D MDA-MB-231 MDA-MB-468
Control
After LF treatment
*
*
*
0
50
100
150
200
250
300
350
No
t-
ox
id
ize
d
m
ito
ch
on
dr
ial
c
ar
di
ol
ip
in
le
ve
l,
r.u
.
Fig. 8. The levels of not-oxidized mitochondrial cardiolipin
in BC cells with different receptor status under the influence
of exogenous LF. *р < 0.05 compared to untreated cells
We determined that the LF is able to initiate the
apoptotic program in a more severe form in receptor-
negative cells not only due to the oxidation of cardioli pin,
but also by influencing the level of expression of pro-
and antiapoptotic proteins. Thus, in the LF-treated
MCF-7 and T47D cells expression level of proapoptotic
Bax protein increased by 10–13% to 223.4 ± 2.6 and
187.3 ± 4.8 H-score points, respectively, while in the
MDA-MB-231 and MDA-MB-468 cells expression level
of Bax after LF treatment increased by 16–17% and
amounted to 180.7 ± 3.8 and 167.4 ± 4.7 H-score
points, respectively (Table 2). The study of the expres-
sion of antiapoptotic Bcl-2 protein after exogenous LF in-
fluence showed a decrease of Bcl-2 in cells of all studied
lines (see Table 2). Thus, Bcl-2 expression in receptor-
positive BC cells after LF treatment decreased by 16–
18%, while the level of Bcl-2 in the MDA-MB-231 and
MDA-MB-468 cells decreased at 21–23%.
Table 2. The levels of pro- and anti-apoptotic proteins in BC cells
with different receptor status
Cell line
Protein level, H-score
Proapoptotic Bax protein Antiapoptotic Bcl-2 protein
Control After
LF treatment Control After LF
treatment
MCF-7 195.1 ± 4.5 223.0 ± 2.6* 298.1 ± 2.1 252.0 ± 0.8*
T47D 165.0 ± 3.7 187.3 ± 4.8* 286.1 ± 5.8 240.2 ± 6.1*
MDA-MB-231 156.2 ± 2.8 180.0 ± 3.8* 258.0 ± 2.7 208.1 ± 3.6*
MDA-MB-468 142.1 ± 4.6 167.2 ± 4.7* 247.2 ± 3.1 190.4 ± 4.8*
Note: *р < 0.05 compared to untreated cells.
It is known that an important LF feature is its
ability to bind to nucleic acids and thus affect the
proliferative activity of cells. Also, LF is able to stop
cell growth by modulating the expression and activity
of G1 cyclin-dependent kinases and activation of trans-
cription [20, 21]. There are also reports of a possible
genotoxic effect of LF. Given the above, the next step
we investigated the changes of cell cycle in LF-treated
BC cells. As seen from the data presented in Table 3,
cytotoxic effects of LF followed by blocking cell cycle
at G2/M phase, particularly in receptor-negative cells
and 1.4–2.5-fold decrease in cell number in the syn-
thetic phase of the cell cycle of all studied BC cell lines.
At the same time, we have confirmed the LF genotoxic
action using the Comet assay. We identified a signifi-
cant increase (p < 0.05) the percentage of DNA in the
comet tail in 1.9- and 2.3-fold, respectively, under the
influence of LF in MDA-MB-468 and MDA-MB-231 cells
with negative receptor status (Table 4).
Table 3. Cell cycle distribution of receptor-positive and receptor-negative
BC cells
Cell line Control, % After LF treatment, %
G1 G2 + M S G1 G2 + M S
MCF-7 51.4 ±
2.8
11.8 ±
1.3
36.8 ±
3.6
72.6 ±
3.1*
9.1 ±
2.0
18.3 ±
1.6*
T47D 55.3 ±
4.1
19.8 ±
3.4
24.9 ±
2.6
71.2 ±
2.9*
11.4 ±
1.9*
17.4 ±
2.1*
MDA-MB-231 51.2 ±
4.8
27.5 ±
4.2
21.3 ±
1.8
76.6 ±
4.2
13.9 ±
0.4*
9.5 ±
2.7*
MDA-MB-468 56.7 ±
3.9
22.6 ±
1.9
20.7 ±
2.1
74.8 ±
4.0*
17.3 ±
0.7*
8.07 ±
1.9*
Note: *р < 0.05 compared to untreated cells.
Table 4. Percentage of DNA in the comet tail of BC cells with different
receptor status
Cell line Tail DNA, %
Control After LF treatment
MCF-7 5.33 ± 1.41 9.80 ± 2.50
T47D 5.20 ± 1.10 9.70 ± 1.90
MDA-MB-231 6.10 ± 1.52 14.03 ± 2.11*
MDA-MB-468 5.80 ± 0.90 11.02 ± 1.90*
Note: *р < 0.05 compared to untreated cells.
Experimental Oncology 39, 106–111, 2017 (June) 111
CONCLUSION
Exogenous LF causes the increase of ROS levels,
speed of NO generation by inducible NO-synthase
and the levels of “free” iron in BC cells. Moreover
LF effects are more pronounced in receptor-negative
MDA-MB-231 and MDA-MB-468 cells. Also, LF treat-
ment causes the decrease in the content of reduced
glutathione. We also established the ability of LF to in-
crease the percentage of apoptotic cells and to re-
duce cardiolipin levels in mitochondria. This, in turn,
leads to increased expression of proapoptotic Bax
protein and reduction of the expression antiapop-
totic Bcl-2 protein. Cytotoxic effects of LF followed
by blocking cell cycle at G2/M phase, particularly
in receptor-negative cells this process was accompa-
nied by a decrease the number of cells in the synthetic
phase of the cell cycle.
So, we can conclude that LF can be used as an exo-
genous modifier of such cancer features as oxidant/
antioxidant balance, apoptotic rates, and cell cycle
progression. Our results demonstrated that LF could
be considered as a promising agent, which can
be used in low doses and the obtained data create the
basis for further in vivo investigations.
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Copyright © Experimental Oncology, 2017
|
| id | nasplib_isofts_kiev_ua-123456789-137977 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T16:16:54Z |
| publishDate | 2017 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Zalutski, I.V. Lukianova, N.Y. Storchai, D.M. Burlaka, A.P. Shvets, Y.V. Borikun, T.V. Todor, I.M. Lukashevich, V.S. Rudnichenko, Y.A. Chekhun, V.F. 2018-06-17T20:07:53Z 2018-06-17T20:07:53Z 2017 Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro / I.V. Zalutski, N.Y. Lukianova, D.M. Storchai, A.P. Burlaka, Y.V. Shvets, T.V. Borikun, I.M. Todor, V.S. Lukashevich, Y.A. Rudnichenko, V.F. Chekhun // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 106–111. — Бібліогр.: 21 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/137977 Aim: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. Materials and Methods: The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Results: Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of “free” iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231 and MDA-MB-468 cells. These changes resulted in increased expression of proapoptotic Bax protein (p < 0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4–1.7-fold, p < 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14–24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G₂/M phase, especially in receptor-negative cell lines. Conclusion: The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, “free” iron and NO generation rate, resalting in the blocking of cell cycle at G₂/M-phase and apoptosis of malignant cells. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro Article published earlier |
| spellingShingle | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro Zalutski, I.V. Lukianova, N.Y. Storchai, D.M. Burlaka, A.P. Shvets, Y.V. Borikun, T.V. Todor, I.M. Lukashevich, V.S. Rudnichenko, Y.A. Chekhun, V.F. Original contributions |
| title | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro |
| title_full | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro |
| title_fullStr | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro |
| title_full_unstemmed | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro |
| title_short | Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro |
| title_sort | influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/137977 |
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