Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells
Aim: The aim of our study is to investigate the disorders of ferritin functioning in breast cancer (BC) cells of different molecular subtype. Materials and Methods: The cell lines used in the analysis include T47D, MCF-7, MDA-MB-231, MDA-MB-468, MCF-10A, and 184A1. Ferritin heavy chains (FTH) expres...
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| Опубліковано в: : | Experimental Oncology |
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| Дата: | 2014 |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2014
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells / S.V. Chekhun, N.Y. Lukyanova, Y.V. Shvets, A.P. Burlaka, L.G. Buchynska // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 179-183. — Бібліогр.: 31 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860151376346087424 |
|---|---|
| author | Chekhun, S.V. Lukyanova, N.Y. Shvets, Y.V. Burlaka, A.P. Buchynska, L.G. |
| author_facet | Chekhun, S.V. Lukyanova, N.Y. Shvets, Y.V. Burlaka, A.P. Buchynska, L.G. |
| citation_txt | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells / S.V. Chekhun, N.Y. Lukyanova, Y.V. Shvets, A.P. Burlaka, L.G. Buchynska // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 179-183. — Бібліогр.: 31 назв. — англ. |
| collection | DSpace DC |
| container_title | Experimental Oncology |
| description | Aim: The aim of our study is to investigate the disorders of ferritin functioning in breast cancer (BC) cells of different molecular subtype. Materials and Methods: The cell lines used in the analysis include T47D, MCF-7, MDA-MB-231, MDA-MB-468, MCF-10A, and 184A1. Ferritin heavy chains (FTH) expression was studied by immunohistochemical method. “Free iron” content and superoxide dismutase (SOD) activity were determined by means of EPR spectroscopy. Reactive oxygen species (ROS) level and peculiarities of microRNA expression in studied cell lines were evaluated using flow cytometry and PCR analysis, respectively. Results: It has been demonstrated that FTH expression directly correlates with proliferative activity of cells of both luminal (r = 0.51) and basal subtypes (r = 0.25), inversely correlates with expression of steroid hormones in cells of basal subtype (ER: r = −0.46; PR: r = −0.44) and does not depend on tumorigenic activity of both subtypes of studied cells (r = 0.12 and r = 0.9). Obtained data are the evidence that cells of luminal subtype B (MCF-7 cell line) and basal subtype (MDA-MB-231 and MDA-MB-468 cell lines) with high proliferative activity contain the highest level of free iron (2.9 ± 0.19·1016and 3.0 ± 0.22·1016) that can be consequence of intensive use of this element by cells, which actively divide and grow. Along with it, in cell of lines of basal subtype MDA-MB-231 and MDA-MB-468, high level of FTH (254 ± 2.3 and 270 ± 1.9) is being detected in consequence of increase of level of free iron, ROS (11.3 ± 1.05 and 7.27 ± 0.26) and SOD (9.4 ± 0.24 and 8.5 ± 0.18) as well as decrease of expression of microRNA 200b. In contrast, cells of luminal subtype B of MCF-7 line were distinguished by high expression of microRNA 200b and low ferritin level (125 ± 2.7). Conclusion: Obtained data demonstrate that tumor cells, which are referred to different molecular subtypes, are characterized by changes in system of support of balance of intercellular iron and certain associations of studied factors. Key Words: breast cancer, cell lines, luminal subtype, basal subtype, ferritin, “free iron”, ROS, SOD, miRNA 200b.
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Experimental Oncology 36, 179–183, 2014 (September) 179
SIGNIFICANCE OF FERRITIN EXPRESSION IN FORMATION
OF MALIGNANT PHENOTYPE OF HUMAN BREAST CANCER CELLS
S.V. Chekhun*, N.Y. Lukyanova, Y.V. Shvets, A.P. Burlaka, L.G. Buchynska
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine,
Kyiv 03022, Ukraine
Aim: The aim of our study is to investigate the disorders of ferritin functioning in breast cancer (BC) cells of different molecular sub-
type. Materials and Methods: The cell lines used in the analysis include T47D, MCF-7, MDA-MB-231, MDA-MB-468, MCF-10A,
and 184A1. Ferritin heavy chains (FTH) expression was studied by immunohistochemical method. “Free iron” content and superox-
ide dismutase (SOD) activity were determined by means of EPR spectroscopy. Reactive oxygen species (ROS) level and peculiarities
of microRNA expression in studied cell lines were evaluated using flow cytometry and PCR analysis, respectively. Results: It has been
demonstrated that FTH expression directly correlates with proliferative activity of cells of both luminal (r = 0.51) and basal subtypes
(r = 0.25), inversely correlates with expression of steroid hormones in cells of basal subtype (ER: r = −0.46; PR: r = −0.44) and does
not depend on tumorigenic activity of both subtypes of studied cells (r = 0.12 and r = 0.9). Obtained data are the evidence that cells
of luminal subtype B (MCF-7 cell line) and basal subtype (MDA-MB-231 and MDA-MB-468 cell lines) with high proliferative
activity contain the highest level of free iron (2.9 ± 0.19·1016
and 3.0 ± 0.22·1016) that can be consequence of intensive use of this
element by cells, which actively divide and grow. Along with it, in cell of lines of basal subtype MDA-MB-231 and MDA-MB-468,
high level of FTH (254 ± 2.3 and 270 ± 1.9) is being detected in consequence of increase of level of free iron, ROS (11.3 ± 1.05 and
7.27 ± 0.26) and SOD (9.4 ± 0.24 and 8.5 ± 0.18) as well as decrease of expression of microRNA 200b. In contrast, cells of luminal
subtype B of MCF-7 line were distinguished by high expression of microRNA 200b and low ferritin level (125 ± 2.7). Conclusion:
Obtained data demonstrate that tumor cells, which are referred to different molecular subtypes, are characterized by changes in sys-
tem of support of balance of intercellular iron and certain associations of studied factors.
Key Words: breast cancer, cell lines, luminal subtype, basal subtype, ferritin, “free iron”, ROS, SOD, miRNA 200b.
Breast cancer (BC) occupies the first place
in cancer-related morbidity among female population
in Ukraine [1]. In recent years, prognosis of BC clini-
cal course is based on determination of its molecular
subtypes namely, luminal A, luminal B, basal or “triple
negative”, “HER2/neu-positive”. These subtypes are
characterized by different response to the therapy,
differential course and prognosis of the disease [2–4].
Despite wide application of mentioned classification
in clinical practice, it has series of essential disadvan-
tages, since BC within the limits of even one subtype
is quite heterogeneous disease both by molecular
phenotype and clinical course [5, 6]. Last statement
can be confirmed by results of our previous studies,
according to which biomolecular markers which charac-
terize metastatic potential and invasive activity of BC cells
of certain molecular subtype, have been studied [7].
It is known iron plays an important role in patho-
genesis of many diseases, including cancer [8]. Iron-
containing proteins including heme-containing proteins
of respiratory chain and ribonucleotide reductase are
involved in wide range of biological processes: support
of respiratory function, DNA synthesis, cellular death
and oxidative stress [8–10]. However, “free” iron pro-
motes the formation of dangerous reactive oxygen spe-
cies (ROS) in cells [11]. Thus, delicate balance between
useful and harmful action of iron determines survival
and functioning of cells. One of the proteins, which
control this balance, is iron-containing protein ferritin.
Ferritin is macromolecular complex, which consists
of 24 subunits of two types: heavy (H) and light (L) [12].
Each organ or tissue, depending on physiological func-
tions, contains certain quantity of H- and L-subunits
in ferritin [13], and ferritin heavy chain (FTH) content
is increased in blood serum of BC patients before
clinical signs of BC. Therefore, FTH determination can
be used for determination of risk among patients when
carrying out screening tests [14].
The main function of ferritin is intracellular deposi-
tion of iron (4500 atoms of iron per ferritin molecule)
in soluble, nontoxic and physiologically available for
organism form [15–17]. Moreover, H-subunit of ferritin
acts as peroxidase, i.e. oxidizes iron with formation
of such side products as ROS, for instance, Н2О2,
which, as it has been stated above, has extremely
dangerous consequences for cell [18].
According to the data of literature, cancer deve-
lopment is accompanied with reprogramming of iron
metabolism in different ways that finally causes ac-
cumulation of iron directly in tumor tissue and cells
of its microenvironment [14]. In particular, levels
of iron and ferritin in BC biopsy material are 5–6 times
higher than in benign tumors. It has been shown that
ferritin content in blood serum is directly proportional
to the content of iron and is increased in the cases
of ovarian, prostate, testicular, pancreatic and he-
patocellular cancer, lymphogranulomatosis, acute
leukemia, etc. [16]. Also, it has been determined
that high serum ferritin in BC patients correlates with
dise ase stage [19]. Our previous in vitro studies have
Submitted: May 12, 2014.
*Correspondence: E-mail: chekhun@yahoo.com
Abbreviations used: BC — breast cancer; ER — estrogen recep-
tor; FTH — ferritin heavy chain; PR — progesterone receptor;
ROS — reactive oxygen species; SOD — superoxide dismutase.
Exp Oncol 2014
36, 3, 179–183
180 Experimental Oncology 36, 179–183, 2014 (September)
shown that the highest ferritin level and significant
decrease of microRNA200b expression are observed
in human BC cells, which are characterized by aggres-
sive mesenchymal phenotype [20]. Thus, the data
evidence on significance of ferritin for occurrence and
progression of BC.
The aim of present research was to study pecu-
liarities of disorders of ferritin functioning in BC cells
of different molecular subtype.
MATERIALS AND METHODS
Cell lines, cell culture and reagents. In the study,
6 BC cell lines (T47D, MCF-7, MDA-MB-231, MDA-
MB-468, MCF-10A and 184A1) were used.
T47D cells were cultured in RPMI-1640 medium
(Sigma), supplemented with 0.2 U/ml of bovine insulin
and 10% fetal bovine serum (FBS). MCF-7 cells were
cultured in Eagle’s Minimum Essential Medium (Sig-
ma), supplemented with 0.01 mg/ml of human recom-
binant insulin and 10% FBS. MDA-MB-231 and MDA-
MB-468 cells were cultured in Leibovitz’s L-15 medium
(Sigma), supplemented with 10% FBS. MCF-10A cells
were cultured in MEBM medium (Lonza), supple-
mented with 100 ng/ml cholera toxin. 184A1 cells were
cultured in MEBM medium (Lonza), supplemented with
0.005 mg/ml transferrin and 1 ng/ml cholera toxin.
Сells were received from the Bank of the Cell Lines
from Human and Animal Tissues of R.E. Kavetsky
Institute of Experimental Pathology, Oncology and
Radiobiology NAS of Ukraine.
Immunocy tochemical assay . The cel ls
grown up of cover slips were fixed in ice-cold methanol:
acetone (1:1) at −20 °C for 120 min and incubated with
1% BSA solution for 20 min. For immunocytochemistry,
primary monoclonal antibodies аnti-FTH (1:150; anti-
FTH (GTX62020; GeneTex, Irvine, CA, USA)), UltraVi-
sion LP Detection System (Lab Vision, Thermo Scien-
tific) and DAB Quanto (Thermo Scientific) were used
according to the instructions of the manufacturers.
When immunocytochemical reaction was completed,
the cells were stained with hematoxylin by Mayer and
placed in Faramount Aqueous Mounting Medium (Da-
koCytomation, Denmark). The acquisition of results
was made by light microscopy (х1000, oil immersion)
with the use of classical H-Score method:
S = 1•N1+ + 2•N2+ + 3•N3+,
where S — “H-Score” index, N1+, N2+ and N3+ —
number of cells with low, medium or high expression
of the marker [21]. Low level of studied markers
expression — from 0 to 100 H-scores, medium le-
vel — from 100 to 200 H-scores and high level — from
200 to 300 H-scores.
Low-temperature Fe(III) EPR. After 24 h of cul-
turing, the cells were scrapped, washed in PBS, cen-
trifuged at 1000 g for 10 min at 4 ˚C and the pellet was
re-suspended in PBS. The suspension containing
2•106 cells was transferred into EPR tubes and im-
mediately frozen in liquid nitrogen. The level of free
iron was determined by a low-temperature EPR
method [22]. Briefly, samples were maintained at
−196 ˚C during recording of the spectra using a finger
Dewar filled with liquid nitrogen. The following param-
eters were used for the low-temperature EPR: sweep
width 1525 G; frequency 9.15 GHz; microwave power
40 mW; modulation amplitude 10.0 G and modulation
frequency 100 kHz. The g-value was calculated using
the standard formula:
g = hv/βH,
where h — Planck’s constant, v — frequency,
β — Bohr magneton and H — external magnetic field
at resonance.
Measurement of intracellular ROS. CM-H2D-
CFDA, a lipid soluble membrane permeable dye upon
entering cells undergoes deacetylation by intracel-
lular esterases and forms the more hydrophilic, non-
fluorescent dye dichlorodihydrofluorescein (DCFH2).
This is subsequently oxidized by ROS with formation
of a highly fluorescent oxidation product, Dichlorofluo-
rescein (DCF). The generated fluorescence is directly
proportional to the amount of ROS. Fluorescence
was analyzed by flow cytometry. After centrifugation
(1500 rpm for 5 min) cells were resuspended in PBS,
incubated for 30 min at 37 °C with CM-H2DCFDA
(10 mM) for measurement of ROS. Positive control
with 25 μM H2O2 was also made (data not presented).
Cells were masked then and analysed by FC. Fluores-
cence was acquired in the log mode and expressed
as geometrical mean fluorescence channel (GMean).
Acquisition was performed on 10,000 gated events.
Measurement of SOD. SOD activity was de-
tected as described earlier [23]. Briefly, cells
were homo genized in 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 at room tempera-
ture as described in [23].
MicroRNA expression analysis. Total RNA
extraction from the cells was performed using
a commercial kit “Rhibo-zol” (Amplisense, Russia)
as per the manufacturer’s protocol. Concentration
of isolated RNA was determined using spectro-
photometer “NanoDrop 2000c” (ThermoScientific,
USA). Purity of isolated RNA is controlled by E260/
E280 ratio. RNA then was dissolved in TE buffer and
stored at −20 °C. RNA was reverse transcribed to cDNA
with gene specific primers according to the TaqMan
MicroRNA Assay protocol (PE Applied Biosystem)
using the TaqMan MicroRNA Reverse Transcription
Kit. Real-time PCR was performed using an Applied
Biosystems 7500 real-time PCR instrument equipped
with a 96-well reaction block. The 20 μl PCR mix in-
cluded 5 μl RT product, 10 μl TaqMan Universal PCR
master mix, and 1 μl of primers from TaqMan MicroRNA
Assay and was performed for 15 s at 95 °C and 1 min
at 60 °C for 40 cycles followed by the thermal dena-
turation protocol. The threshold cycle (Ct) was deter-
mined using default threshold settings. The Ct value
is defined as the fractional cycle number at which
the fluorescence passesthe fixed threshold. All ex-
periments were done in triplicates and repeated three
Experimental Oncology 36, 179–183, 2014 (September) 181
times. RNU48 miRNA was used as an internal control
to normalize RNA input in thereal-time RT-PCR assay.
The expression of microRNA relative to RNU48 miRNA
was determined using the 2-ΔCT method.
Statistical analysis. Statistical processing of the
obtained results was carried out with the help of mathe-
matical program of medical and biological statistics
STATISTIСA 6.0. Calculation and comparison of the sig-
nificance of differences between the average values
was carried out with usage of Student’s t-criterion;
correlation analysis was carried out using the Pearson
correlation coefficient. Significant were considered
the differences with the probability not less than 95%
(р < 0.05).
RESULTS AND DISCUSSION
Immunohistochemical study of FTH expression
has shown its different expression levels in BC cell
lines (Table 1). The highest FTH expression (2–3 times
higher compared with cells of luminal type) has been
detected in basal subtype cell lines, especially in MDA-
MB 231 and MDA-MB 468 cells. Low and medium FTH
expression have been observed in cells of luminal
subtype. Correlation analysis allowed us to determine
inverse dependence of FTH expression on receptor
status in basal subtype cells (Table 2).
Table1. Features of expression of FTH, level of “free iron” content, ROS
and SOD activity in BC cells of different molecular subtype
Cellular lines Molecular
subtype
Le vel
of ex-
pression
of FTH,
H-score
Quantity
of paramag-
netic centers
of “free iron”,
spins/ml
Le vel
of ROS
generation,
G/Mean
SOD
activity,
U/ml
T 47 D Luminal A 94±1.6 0.47±0.1·1016 2.71±0.45 5.8±0.18
MCF-7 Luminal B 125±2.7 0.56±0.1·1016 3.1±0.29 6.9±0.13
MDA-MB 231 Basal subtype 254±2.3 2.9±0.19·1016 11.3±1.05 9.4±0.24
MDA-MB 468 Basal subtype 270±1.9 3.0±0.22·1016 7.27 ±0.26 8.5±0.18
MCF-10 A Basal subtype 223±1.8 1.0±0.3·1016 3.9±0.15 6.5±0.31
184 A 1 Basal subtype 206±1.9 1.4±0.35·1016 4.94±0.22 7.3±0.11
Table 2. Coefficients of correlation between expression of FTH and other
studied indexes of BC cells of different molecular subtypes
Indexes Value of correlation coefficient
Luminal type Basal type
FTH/ER −0.12 −0.46*
FTH/PR −0.13 −0.44*
FTH/Ki-67 0.51* 0.25*
FTH/colony formation 0.12 0.09
FTH/number of paramagnetic centers of “free iron” 0.44* 0.39*
FTH/level of ROS generation 0.32* 0.22*
FTH/activity of SOD 0.36 * 0.14*
FTH/colony formation 0.12 0.09
FTH/number of paramagnetic centers of “free iron” 0.44* 0.39*
FTH/activity of SOD 0.36* 0.14*
Notes: ER — estrogen receptor; PR — progesterone receptor; *р < 0.05.
Since we have demonstrated in previous studies that
cells of luminal and basal subtypes of BC are essentially
different by tumorigenic properties in vitro, and their
proliferative activity varies within wide limits, we have
compared FTH expression and these in dexes [7]. It has
been determined that level of FTH expression correlates
with proliferative activity of BC cells of both luminal (r =
0.51) and basal (r = 0.25) subtypes. It has been dem-
onstrated that level of FTH expression does not corre-
lated with tumorigenic activity of cells of both studied
subtypes (Table 2). Thus, we have determined that cells
of luminal and basal BC are essentially different by FTH
expression, which within the limits of particular subtypes
is associated with cell proliferative activity.
It is known that the main function of ferritin
is deposition of iron ions, i.e. its expression level cor-
relates with content of “free iron” in tumor cells [6,
7]. Indeed, the highest “free iron” content among
studied BC cell lines has been observed in basal
molecular subtype cell lines MDA-MB 231and MDA-
MB468 and constituted 2.9 ± 0.19•1016 spins/ml and
3.0±0.22•1016 spins/ml, respectively. We emphasize
that these cells are characterized with highest FTH
expression, lack of steroid hormone receptors, high
proliferative potential and tumorigenicity.
In cells of T47D and MCF-7 lines, luminal
A and B molecular subtypes, content of “free iron”
was reliably lo wer — 0.47 ± 0.1•1016 spins/ml and
0.56 ± 0.1•1016 spins/ml, respectively (p < 0.05). When
analyzing features of cells of basal molecular subtype
MCF-10 A and 180A1, it should be mentioned that for
these cells mode rate proliferative activity and, along
with it, medium FTH expression, insignificant content of
“free iron” and high colony forming are typical. Analysis
of dependencies of FTH expression on content of free
iron allowed us to determine correlation of these indexes
in cells of both studied subtypes (Table 2).
Next, we have compared ROS and SOD indexes
as integral representation of the “free iron” level
in BC cells of different molecular subtypes. As it is shown
in Table 1, in cells luminal A (T47D) and B (MCF-7) mo-
lecular subtypes, the lowest le vels of ROS generation
and SOD activity has been observed. ROS content and
SOD activity in cells of luminal subtype was associated
with low expression of FTH, content of “free iron” and
proliferative activity of cells (Table 2).
In BC cells of basal subtype, medium and high
levels of ROS and SOD activity have been detected
(Table 1). The highest indexes of ROS and SOD, which
were associated with high “free iron” content, FTH
expression, proliferative activity of cells have been
determined in MDA-MB 231 and MDA-MB 468 cells
of basal molecular subtype (Table 2).
Thus, we have determined that there is a relation
between FTH expression, content of free iron, ROS
and SOD in BC cells of different molecular subtype.
It is known that BC malignancy grade is accom-
panied with certain epigenоmic changes in tumor
cells [3]. For evaluation of role of epigenomic com-
ponent in determination of FTH expression in cells
of studied lines, we have analyzed features of mi-
croRNA expression, which participates in regulation
of expression of this protein. Since in previous studies
we have shown that miR-200b plays important role
in post-transcriptional regulation of ferritin expression
(FTH is a target of miR-200b) [20], we have focus our
attention on study of miR-200b expression. As one may
see in the Figure, the highest level of miR-200b ex-
pression has been observed in T47D and MCF-7 cells
of luminal subtype. In 184A1 and MCF10A cells
of basal subtype, microRNA 200b expression level
reliably decreased compared with control and cells
182 Experimental Oncology 36, 179–183, 2014 (September)
of luminal subtype. The lowest indexes of microRNA
200b expression have been determined in BC cells
of basal subtype (MDA-MB-231 and MDA-MB-468).
The obtained data demonstrate that there is a depen-
dence between FTH expression and microRNA, which
participates in regulation of this protein.
0
20
40
60
80
100
120
140
T47D MCF-7 MDA-MB-
231
MDA-MB-
468
MCF-10A 184A1
Ch
an
ge
s
in
m
iR
NA
e
xp
re
ss
io
n,
%
RNU48
miR-200b
Figure. MicroRNA 200b expression in BC cells of different
molecular subtype
So, our data show that there are certain correla-
tions between FTH expression and such indexes
of BC malignancy as proliferation rate, receptor status
and colony forming activity (Table 2). In particular,
FTH expression directly correlates with proliferative
activity of BC cells of both luminal and basal subtypes,
inversely correlates with expression of steroid hor-
mones in cells of basal subtype and does not depend
on tumorigenic activity of both subtypes. The cells
of luminal subtype B of MCF-7 line and basal subtype
of MDA-MB-231 and MDA-MB-468 lines with high
proliferative activity contain the highest level of free
iron that can be consequence of intensive use of this
element by cells, which actively divide and grow.
In basal subtype MDA-MB-231 and MDA-MB-468 cell
lines, high levels of FTH, ROS and SOD are detected,
as well as decreased expression of microRNA 200b.
In contrast, cells of luminal subtype B MCF-7 line were
distinguished by high expression of microRNA 200b
and low ferritin level, what evidences that certain
molecular subtypes of BC are characterized by dif-
ferences in regulatory mechanisms of storage and
accumulation of endogenous iron.
Moreover, among four studied lines of basal sub-
types, two subtypes can be distinguished by determined
indexes: 1) cells of MDA-MB 231 and MDA-MB 468 lines
are characterized by high proliferative activity and
tumorigenicity in vitro, high level of FTH expres-
sion, content of “free iron”, ROS and SOD; 2) cells
of MCF-10 A and 184A1 lines are characterized
by moderate proliferative activity and tumorigenicity,
medium content of FTH, high content of “free iron”,
moderate levels of ROS and SOD activity.
The role of iron metabolism in tumor develop-
ment is one of the fundamental problems of modern
experimental oncology [8, 10, 24]. Such studies are
of special interest in the context of BC, since hormonal
status of woman is tightly associated with significant
shifts of iron content in organism. Both deficiency and
proficiency of iron can have negative consequences
for health. It is known that in developed countries, 20%
of women have iron deficiency that is an additional
factor, which causes increase of VEGF concentration
and, as the result, activation of angiogenesis in women
of premenopausal period [25]. Also, there is a syner-
gism of iron and estrogen metabolism disorders
in BC occurrence [26–28]. In the process of malignant
transformation the excess of iron contributes to ROS
formation causing DNA damage. At the same time,
estrogen can act as additional substrate in these reac-
tions due to adjunction of hydroxyl group and forma-
tion of catechol estrogen, which is one of the factors
of hormonal carcinogenesis.
We have analyzed certain mechanisms involved
in iron metabolism in BC cells of different molecular
subtypes. Our data demonstrate that BC cells, which
are referred to different molecular subtypes, are cha-
racterized by shifts in intercellular iron balance asso-
ciated of altered indexes mentioned above. Obtained
data are in accordance with the results of our previ-
ous studies concerning significant increase of FTH
in BC cells of aggressive mesenchymal subtype and
in BC cells with phenotype of drug resistance to cispla-
tin and doxorubicin [20, 30–31]. So, disorders of FTH
expression may reflect biological properties of luminal
and basal BC subtypes and play a role in clinical course
of breast cancer BC.
ACKNOWLEDGEMENT
This work was supported by interdisciplinary re-
search program of the NAS of Ukraine “Fundamentals
of molecular and cellular biotechnology”.
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| id | nasplib_isofts_kiev_ua-123456789-145363 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T17:52:16Z |
| publishDate | 2014 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Chekhun, S.V. Lukyanova, N.Y. Shvets, Y.V. Burlaka, A.P. Buchynska, L.G. 2019-01-20T20:54:49Z 2019-01-20T20:54:49Z 2014 Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells / S.V. Chekhun, N.Y. Lukyanova, Y.V. Shvets, A.P. Burlaka, L.G. Buchynska // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 179-183. — Бібліогр.: 31 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145363 Aim: The aim of our study is to investigate the disorders of ferritin functioning in breast cancer (BC) cells of different molecular subtype. Materials and Methods: The cell lines used in the analysis include T47D, MCF-7, MDA-MB-231, MDA-MB-468, MCF-10A, and 184A1. Ferritin heavy chains (FTH) expression was studied by immunohistochemical method. “Free iron” content and superoxide dismutase (SOD) activity were determined by means of EPR spectroscopy. Reactive oxygen species (ROS) level and peculiarities of microRNA expression in studied cell lines were evaluated using flow cytometry and PCR analysis, respectively. Results: It has been demonstrated that FTH expression directly correlates with proliferative activity of cells of both luminal (r = 0.51) and basal subtypes (r = 0.25), inversely correlates with expression of steroid hormones in cells of basal subtype (ER: r = −0.46; PR: r = −0.44) and does not depend on tumorigenic activity of both subtypes of studied cells (r = 0.12 and r = 0.9). Obtained data are the evidence that cells of luminal subtype B (MCF-7 cell line) and basal subtype (MDA-MB-231 and MDA-MB-468 cell lines) with high proliferative activity contain the highest level of free iron (2.9 ± 0.19·1016and 3.0 ± 0.22·1016) that can be consequence of intensive use of this element by cells, which actively divide and grow. Along with it, in cell of lines of basal subtype MDA-MB-231 and MDA-MB-468, high level of FTH (254 ± 2.3 and 270 ± 1.9) is being detected in consequence of increase of level of free iron, ROS (11.3 ± 1.05 and 7.27 ± 0.26) and SOD (9.4 ± 0.24 and 8.5 ± 0.18) as well as decrease of expression of microRNA 200b. In contrast, cells of luminal subtype B of MCF-7 line were distinguished by high expression of microRNA 200b and low ferritin level (125 ± 2.7). Conclusion: Obtained data demonstrate that tumor cells, which are referred to different molecular subtypes, are characterized by changes in system of support of balance of intercellular iron and certain associations of studied factors. Key Words: breast cancer, cell lines, luminal subtype, basal subtype, ferritin, “free iron”, ROS, SOD, miRNA 200b. This work was supported by interdisciplinary research program of the NAS of Ukraine “Fundamentals of molecular and cellular biotechnology”. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells Article published earlier |
| spellingShingle | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells Chekhun, S.V. Lukyanova, N.Y. Shvets, Y.V. Burlaka, A.P. Buchynska, L.G. Original contributions |
| title | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells |
| title_full | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells |
| title_fullStr | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells |
| title_full_unstemmed | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells |
| title_short | Significance of ferritin expression in formation of malignant phenotype of human breast cancer cells |
| title_sort | significance of ferritin expression in formation of malignant phenotype of human breast cancer cells |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/145363 |
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