Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells
Aim: To assess the role of endogenous lactoferrin (LF) in the formation of the molecular phenotype of human breast cancer (BC) cell lines with varying degrees of malignancy, including cisplatin/doxorubicin resistant cell lines, and identify possible impact of exogenous LF. Materials and Methods: 5 b...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2015
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| Cite this: | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells / V.F. Chekhun, I.V. Zalutskii, L.A. Naleskina, N.Yu. Lukianova, T.M. Yalovenko, T.V. Borikun, S.O. Sobchenko, I.V. Semak, V.S. Lukashevich // Experimental Oncology. — 2015. — Т. 37, № 3. — С. 181-186. — Бібліогр.: 33 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859975408423796736 |
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| author | Chekhun, V.F. Zalutskii, I.V. Naleskina, L.A. Lukianova, N.Yu. Yalovenko, T.M. Borikun, T.V. Sobchenko, S.O. Semak, I.V. Lukashevich, V.S. |
| author_facet | Chekhun, V.F. Zalutskii, I.V. Naleskina, L.A. Lukianova, N.Yu. Yalovenko, T.M. Borikun, T.V. Sobchenko, S.O. Semak, I.V. Lukashevich, V.S. |
| citation_txt | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells / V.F. Chekhun, I.V. Zalutskii, L.A. Naleskina, N.Yu. Lukianova, T.M. Yalovenko, T.V. Borikun, S.O. Sobchenko, I.V. Semak, V.S. Lukashevich // Experimental Oncology. — 2015. — Т. 37, № 3. — С. 181-186. — Бібліогр.: 33 назв. — англ. |
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| container_title | Experimental Oncology |
| description | Aim: To assess the role of endogenous lactoferrin (LF) in the formation of the molecular phenotype of human breast cancer (BC) cell lines with varying degrees of malignancy, including cisplatin/doxorubicin resistant cell lines, and identify possible impact of exogenous LF. Materials and Methods: 5 breast cell lines of different origin — MCF-10 A, MCF-7, including doxorubicin/cisplatin resistant ones, T47D, MDA-MB-231, and MDA-MB-468. Immunocytochemistry: expression of LF, Ki-67, adhesion molecules E- and N-cadherin, CD44, CD24 rating the invasive potential of cells. Results: Expression of LF in human BC cell lines varies. It is associated with the heterogeneity of molecular profiles of cell lines in terms of adhesion. A link has been established between the level of LF expression in the resistant cell line MCF-7/CP and MCF-7/Dox, features of their molecular profile and invasive properties. Exogenous LF was shown to be capable of modifying the molecular profile and invasive properties of all the studied cell lines including resistant ones (MCF-7/CP and MCF-7/Dox). Conclusions: The sensitivity of cytostatic-resistant cell lines (MCF-7/CP and MCF-7/Dox) tends to increase under the influence of exogenous LF. It is likely that this effect is due to LF-mediated inhibition of the expression of proteins associated with drug resistance. Key Words: lactoferrin, cell lines of human breast cancer, molecular phenotype, proliferative activity, invasive potential, adhesion molecules.
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Experimental Oncology 37, 181–186, 2015 (September) 181
MODIFYING EFFECTS OF LACTOFERRIN IN VITRO ON MOLECULAR
PHENOTYPE OF HUMAN BREAST CANCER CELLS
V.F. Chekhun1, I.V. Zalutskii2, L.A. Naleskina1, N.Yu. Lukianova1,*,
T.M. Yalovenko1, T.V. Borikun1, S.O. Sobchenko1, I.V. Semak3, V.S. Lukashevich2
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv
03022, Ukraine
2State Scientific Institution “Institute of Physiology”, NAS of Belarus,
Minsk 220072, Republic of Belarus
3Institution of Education “Belarusian State University”, Minsk 220030, Republic of Belarus
Aim: To assess the role of endogenous lactoferrin (LF) in the formation of the molecular phenotype of human breast cancer (BC)
cell lines with varying degrees of malignancy, including cisplatin/doxorubicin resistant cell lines, and identify possible impact
of exogenous LF. Materials and Methods: 5 breast cell lines of different origin — MCF-10 A, MCF-7, including doxorubicin/
cisplatin resistant ones, T47D, MDA-MB-231, and MDA-MB-468. Immunocytochemistry: expression of LF, Ki-67, adhesion
molecules E- and N-cadherin, CD44, CD24 rating the invasive potential of cells. Results: Expression of LF in human BC cell lines
varies. It is associated with the heterogeneity of molecular profiles of cell lines in terms of adhesion. A link has been established
between the level of LF expression in the resistant cell line MCF-7/CP and MCF-7/Dox, features of their molecular profile and
invasive properties. Exogenous LF was shown to be capable of modifying the molecular profile and invasive properties of all the
studied cell lines including resistant ones (MCF-7/CP and MCF-7/Dox). Conclusions: The sensitivity of cytostatic-resistant cell
lines (MCF-7/CP and MCF-7/Dox) tends to increase under the influence of exogenous LF. It is likely that this effect is due
to LF-mediated inhibition of the expression of proteins associated with drug resistance.
Key Words: lactoferrin, cell lines of human breast cancer, molecular phenotype, proliferative activity, invasive potential, adhesion
molecules.
INTRODUCTION
Undoubtedly, breast cancer (BC) will soon dis-
continue being a uniform nosological form as it used
to be until recently. This is due to the fact that a vast
majority of experts in fundamental and clinical onco-
logy have recently recognized that BC is a set of dise-
ases with different “natural evolution” and clinical
features and that they should no longer be treated
in a “stereotypical manner” (in particular, this ap-
plies to using chemotherapeutic agents); instead,
tactics of individualized treatment of patients should
be applied [1]. Such a change in the views on BC —
a malignancy which belongs to the most common
ones among the women population — is due to the
fact that numerous clinical observations have proved
the presence of distinctive associative links between
certain morphological and functional characteristics,
molecular-genetic profiles of tumors and clinical mani-
festations of the tumor process in BC patients [2, 3].
It has been clearly shown that the existence of inter-
tumor and intracellular heterogeneity of populations
of malignantly transformed cells leads to a diversity
of clinical pictures of BC and oftentimes to a failure
to achieve a response after a standard treatment
in individuals with similar characteristics, such as age,
prevalence of the process, form or degree of histologi-
cal differentiation [4].
A biological phenomenon of intracellular hete-
rogeneity, which is caused by genetic instability,
is multi-dimensional and is considered to be a key
factor in determining the vector of the cancer process,
both at the onset and during the realization of various
forms of tumor progression, i.e. the aggressiveness
of the disease [5, 6]. Many researchers believe that
intracellular heterogeneity plays a crucial role in the
rate of neoplasm development, its oncogenic po-
tential, and cell survival in the conditions of dynamic
microenvironment [7–9]. The heterogeneity within the
same tumor manifests itself in the form of differences
between various groups of cells in terms of morpho-
logical structure, genetic and epigenetic status, as well
as variability in the expression of various markers,
including molecular genetic ones [10–12].
Intracellular heterogeneity makes the tumor cell
population diverse in terms of biological potential
of malignancy and helps it to expand by way of dis-
semination, colonization, metastasizing, and also
enhances its resistance to antitumor effects, primarily
to chemotherapy. There are reports showing that nu-
merous subpopulations of cells are present in a tumor
from the very onset; they feature varying functional
characteristics and differ in the degree of sensitivity
or resistance to particular medicines [13, 14]. Also,
it was established that intratumoral morphological
heterogeneity of BC is associated with the expression
of multidrug resistance (MDR) genes and the efficien-
cy of neoadjuvant chemotherapy (NChT). In particular,
it was shown that tumors featuring alveolar structures
Submitted: April 22, 2015.
*Correspondence: E-mail: lu_na_u@rambler.ru
Abbreviations used: BC — breast cancer; LF — lactoferrin; MDR —
multidrug resistance; NСhT — neoadjuvant chemotherapy.
Exp Oncol 2015
37, 3, 181–186
182 Experimental Oncology 37, 181–186, 2015 (September)
are characterized by a high level and up-regulation
of main MDR genes and, at the same time, by a poor
response to the NChT [15].
It has been proven that the parenchymatous
component of BC with the different biological ag-
gressiveness contains cells with varying phenotypic
and molecular genetic characteristics. Based on this,
methods have been developed for selective thera-
peutic interventions, which should be based on the
results of experimental studies [16, 17]. The basis for
this are numerous reports on existing relation between
the intratumoral morphological heterogeneity and
the molecular subtypes in terms of expression of ER,
PR and HER2/neu as the most intensively studied
area of clinical molecular research in BC [18, 19].
In particular, it was noted that the “triple negative BC”
more often features one single type of morphological
structures, while luminal BC mainly features 5 types
of structures: tubular, solid, trabecular, alveolar and
discrete groups of tumor cells [20]. The comparison
of the achievements of fundamental science with
the specific characteristics of the disease course
is an important link between the proposed hypotheses
about the mechanisms that may underlie the disease
development and the objective assessment of their
likelihood in practical cancer care.
Over the last decade, along with enhanced re-
search aimed to confirm the achievements of molecu-
lar genetics concerning the existence of BC heteroge-
neity in terms of molecular phenotype, as well as their
introduction into clinical practice, considerable efforts
have been made to increase our knowledge about
the role of metal-containing proteins in malignant
transformation and tumor progression, as important
components of metabolism, which are actively involved
in various processes of cells’ vital activity [21]. Our
interest is focused on fundamental research of one
of the most poorly studied among proteins of such ori-
gin — lactoferrin (LF) [22, 23] — in terms of expression
patterns in BC cells varying in phenotypic profile and
malignancy degree, and the possibilities to modify the
malignant potential of these cells when incubated with
recombinant human LF. This interest is due to the fact
that our own clinical observations of two geographical
location populations of BC patients for the first time
showed that the LF expression level is characterized
by inter-tumor heterogeneity, as it was detected only
in 48.0 and 53.6% of the cases, respectively. Fur-
thermore, it was proved that the survival rate among
women with LF-positive tumors was higher than in tu-
mors of patients without LF expression [24].
Based on the above, our aim was to conduct experi-
ments in vitro to establish LF expression patterns in cell
lines of human BC with varying malignancy potential,
including those resistant to cisplatin and doxorubi-
cin, and to check if the phenotype of these cells can
be modified by culturing them with exogenous LF.
MATERIALS AND METHODS
Cell lines and drug treatment. The studies were
conducted in an in vitro system on 5 cell lines of human
mammary of different origins: MCF-10A — immorta-
lized cells of normal human mammary; MCF-7 — in-
vasive breast ductal carcinoma; T47D — metastatic
breast ductal carcinoma; MDA-MB-231 and MDA-
MB-468 — metastatic breast adenocarcinoma.
T47D cells were cultured in RPMI-1640 medium
(Sigma) supplemented by bovine insulin (0.2 U/ml)
and 10% fetal bovine serum (FBS). MCF-7 cells were
grown in medium Dulbeccos Modified Eagles Medium
(Sigma) supplemented by recombinant human insulin
(0.01 mg/ml) and 10% FBS. MDA-MB-231 and MDA-
MB-468 cells were cultured in Leibovitz’s L-15 medium
(Sigma) supplemented by 10% FBS. MCF-10A cells
were grown in MEBM medium (Lonza) supplemented
by cholera toxin (100 ng/ml). All cultures were grown
on glass cover slips in humidified atmosphere with
5% CO2 at 37 °C. The cells were provided by the Bank
of Human and Animal Tissue Lines of the R.E. Kavetsky
Institute of Experimental Pathology, Oncology and
Radiobiology of the National Academy of Sciences
of Ukraine.
The resistant variants MCF-7/Dox and MCF-7/CP were
originated by growing initial MCF-7 cells with rai sing
concentrations of cisplatin (from 0,01 to 6 μg/ml)
or doxorubicin (from 0.1 to 32 μg/ml), respectively.
Cisplatin and doxorubicin were added twice a week after
reseeding. Every 2 months, cell survival was analyzed
by MTT assay. IC50 values for MCF-7 and MCF-7/CP cells
were 0.25 and 1 μg/ml of cisplatin, respectively, and for
MCF-7 and MCF-7/Dox cells — 0.5 and 8 μg/ml of doxo-
rubicin, respectively. Therefore, MCF-7/CP were 4 times
as much resistant to the cytotoxic effect of cisplatin
and MCF-7/DOX cells were 16 times as much resistant
to the cytotoxic effect doxorubicin as compared with
the initial MCF-7 cells.
MTT assay. Sensitivity to antitumor drugs (cis-
platin and doxorubicin) was measured every two
months using standard MTT-colorimetric test with
3-[4,5,dimethylthiasol-2–1]-2,5-diphenyltetrasolium
bromide (“Sigma”, Germany) [25].
We used a wide range of immunocytochemical
methods that allow establishing the molecular profile
of malignantly transformed cells: assessment of the
expression level of adhesion molecules such as E-cad-
herin, N-cadherin, CD44, CD24, proliferative activity
by expression of Ki-67. The LF expression in tumor cells
and their invasive properties were studied. Cells for im-
munocytochemical studies were grown on glass cover
slips, fixed in cooled mixture of methanol:acetone (1:1)
at −-20 °C for 120 min, washed in PBS and incubated
with 1% BSA for 20 min. Primary monoclonal antibod-
ies: anti-lactoferrin (ThermoScientific, USA); anti-
Кi-67 (DakoCytomation, Denmark); anti-CD24 (Neo-
Markers, USA); anti-CD44 (Diagnostic BioSystems,
USA); anti-E-cadherin (ThermoScientific, USA),
anti-CD325 ( N-Cadherin) (DakoCytomation, Denmark)
were diluted in the blocking buffer and kept at a room
Experimental Oncology 37, 181–186, 2015 (September) 183
temperature for one hour, followed by incubation with
UltraVision LP Detection System (Lab Vision, Thermo
Scientific) for 10 and 15 min; after the washing, the
immune reaction was visualized by using DAB Quanto
(Thermo Scientific). When immunocytochemical re-
actions were completed, the cells were stained with
hematoxylin by Mayer for 10–15 s and placed in Fara-
mount Aqueous Mounting Medium (DakoCytomation,
Denmark). Evaluation of the results was carried out
in 3 visual fields by light microscopy (x1000, oil immer-
sion) using the classical H-Score method:
S = 1×N1
+ + 2×N2
+ + 3×N3
+,
where S is “H-Score” index, N1
+, N2
+ and N3
+ are num-
bers of cells with low, medium or high levels of marker
expression.
It should be noted that in previous studies we have
identified biomolecular markers that characterize the
metastatic potential and invasive activity of BC cells
of abovementioned lines [18].
Invasive activity. The invasive activity was inves-
tigated using a standard method, according to manu-
facturer’s recommendations (BD Biosciences). For
this purpose, the matrigel membranes (BD BioCoat™
Matrigel™ Invasion Chamber, Bedford, MA) with pore
sizes of 8 microns were used. In the upper part of the
membrane, the cell suspension was placed in a con-
centration of 5•104 cells/well in culture medium without
the addition of bovine serum. In the lower part, the
standard culture medium supplemented with 10% FBS
was introduced. The cells were then incubated under
standard conditions at temperature of 37 °C in hu-
midified atmosphere with 5% CO2 during 48 hours.
The membranes were then fixed with methanol, fol-
lowed by staining with crystal violet B. The number
of cells on outside of membrane was evaluated using
light microscopy (×100, oil immersion).
LF preparation (obtaining). Milk of goats trans-
fected with human LF gene (joint development of SPC
on Livestock Farming of the National Academy of Sci-
ences of Belarus and the Institute of Gene Biology, Rus-
sian Academy of Sciences) was subjected to the stan-
dard procedure of casein removing. Chromatography
was performed using a medium pressure liquid chro-
matograph ACTAFPLCsystem (Amersham-Pharmacia).
Milk serum was applied to a HiPrep 16/10 SP HL column
(Amersham-Pharmacia) that was prewashed with
equilibration buffer (20 mМ phosphate Na; 0.4 М NaCl;
рН 6.5) at a rate 1 ml/min. LF was eluted with a linear
gradient 0.4 М — 1 М NaCl. Fractions containing
LF were concentrated and desalted using ultrafiltra-
tion cassettes Vivaflow 50 (Sartorius) with a molecular
weight cut-off of 30 kDa. The resulting concentrate was
lyophilized on Lyophilize ALPHA 1–4 LDplus. The purity
of obtained LF (> 99%) was assessed by denaturing gel
electrophoresis in polyacrylamide gel in the presence
of sodium dodecyl sulfate.
Recombinant human LF, which was used in ex-
periments in vitro, was diluted in PBS and added di-
rectly to the culture medium at a final concentration
of 100 μg/mL. Cells were cultured with LF during 72 h.
Statistical analysis was performed with
STATISTICA 6.0 software (StatSoft Inc., USA). The all
data were obtained in triplicate experiments. The va-
lues were expressed as means ± standard deviation
(SD). Student’s t-test was used to evaluate the sig-
nificance of the differences between groups. A value
of p < 0.05 was accepted as statistically significant.
RESULTS AND DISCUSSION
Immunocytochemical determination of LF revealed
that this metal-containing protein shows varying ex-
pression levels in the tested BC cell lines (Table 1).
The highest LF expression was observed in cell lines
T47D and MCF-7 that possess low invasive activity,
which according to the molecular phenotype is caused
by the increased adhesion properties of these cells
due to the high expression level of E-cadherin and low
expression level of CD44, in the absence of CD24 ex-
pression. Levels of N-cadherin expression were defi-
nitely moderate.
The lowest level of LF expression was seen in cell line
MDA-MB-468, that is characterized by the highest rates
of invasion activity, which according to their molecular
profile is explained by the higher expression of adhe-
sion molecules CD44 and CD24 as compared with
those in cell lines T47D and MCF-7 with a low invasive
potential. At the same time, expression of E-cadherin
has been at the values range typical for cells with
a low invasion activity. Levels of the LF expression
were slightly higher in cell line MDA-MB-231, which
by its invasive properties were inferior to cell line MDA-
MB-468, and among the tested cell lines had the lowest
level of E-cadherin expression, the highest expression
of CD44 molecules and the positive response to CD24.
Table 1. Changes in the molecular profile of BC cells with different molecular phenotype and invasive properties caused by cultivation with LF
Cell lines Level of LF expression Cells’ proliferative activity CD24 CD44 Е-cadherin N-cadherin Invasion, ×103 cells
T47D 285.0 ± 2.1 89.0 ± 1.4 0 79.0 ± 3.1 251.0 ± 5.0 53.0 ± 2.4 0.081 ± 0.002
T47D + LF − 63.0 ± 0.8* 0 43.0 ± 1.1* 263.0 ± 3.2* 49.0 ± 2.0 0.061 ± 0.005*
MCF-7 251.0 ± 2.3 291.0 ± 2.4 0 72.0 ± 2.8 268.0 ± 4.6 59.0 ± 1.6 0.087 ± 0.005
MCF-7 + LF − 170.0 ± 2.1* 0 32.0 ± 2.3* 283.0 ± 3.6* 56.0 ± 1.9 0.070 ± 0.004
MDA-MB-231 92.0 ± 1.9 268.0 ± 1.9 34.0 ± 2.6 298.0 ± 0.7 49.0 ± 1.3 31.0 ± 0.7 0.192 ± 0.021
MDA-MB-231 + LF − 130.0 ± 2.3* 34.0 ± 2.6 164.0 ± 1.7* 148.0 ± 2.9* 48.0 ± 0.9* 0.153 ± 0.014*
MDA-MB4–68 70.0 ± 1.6 249.0 ± 2.7 153.0 ± 4.0 167.0 ± 5.0 259.0 ± 2.5 18.0 ± 0.8 0.340 ± 0.011
MDA-MB-468 + LF − 119.0 ± 2.8* 110.0 ± 2.1* 96.0 ± 3.0* 293.0 ± 2.8* 31.0 ± 0.9* 0.190 ± 0.021*
MCF-10A 157.0 ± 1.8 191.0 ± 2.3 63.0 ± 1.8 174.0 ± 4.3 149.0 ± 1.1 31.0 ± 1.6 0.240 ± 0.016
MCF-10A + LF − 130.0 ± 2.7* 42.0 ± 1.6* 110.0 ± 2.3* 189.0 ± 1.9* 28.0 ± 1.1* 0.176 ± 0.009
MCF-7/CP 105.0 ± 1.7 143.0 ± 2.5 0 176.0 ± 2.1 57.0 ± 1.9 179.0 ± 1.7 0.280 ± 0.010
MCF-7/CP + LF 0 87.0 ± 1.5* 0 98.0 ± 2.2* 115.0 ± 1.6* 143.0 ± 1.8** 0.154 ± 0.013*
MCF-7/Dox 67.0 ± 1.9 95.0 ± 1.8 0 234.0 ± 2.3 28.0 ± 2.3 238.0 ± 1.9 0.352 ± 0.022
MCF-7/Dox + LF 0 62.0 ± 1.6* 0 110.0 ± 2.3* 73.0 ± 1.9* 169.0 ± 1.8* 0.142 ± 0.008*
Note: *p < 0.05 compared with cell lines without LF.
184 Experimental Oncology 37, 181–186, 2015 (September)
In immortalized cells of human mammary cell line
MCF-10A without signs of malignant transformation,
we found a high LF expression. By its invasive proper-
ties the cells of that line occupied intermediate position
between cell lines MDA-MB-468 and MDA-MB-231.
In these cells, there was a high level of CD44 and
CD24 expression and, at the same time, a fairly high ex-
pression of E-cadherin (see Table 1). It should be noted
that the proliferative activity was equally high in the
tested cell lines and did not significantly affect both the
levels of the LF expression and invasive activity. Thus,
according to our findings, the level of LF expression
in human BC cell lines of different origins is associated
with their invasive properties. At the same time, the
invasive activity of the tested cell lines is caused by the
expression of various adhesion molecules: the weak
manifestations of invasion associates with the high level
of E-cadherin expression and the lack of CD24 expres-
sion, while cells with an active invasion showed a shift
towards increased expression levels of CD44 and CD24.
The above mentioned differences are the direct evi-
dence of interlinear heterogeneity of BC cells in both the
level of LF expression and the molecular profile which
determines their invasive properties.
Along with this, we evaluated the features of LF ex-
pression in cells of the two resistant BC lines, namely
cisplatin-resistant (MCF-7/CP), and doxorubicin-
resistant (MCF-7/Dox) (see Table 1). According to our
findings, the LF expression level in cell line MCF-7/
CP was much higher than that in cell line MCF-7/
Dox. At the same time, invasive activity was more
pronounced in cell line MCF-7/Dox, which was cha-
racterized by a low expression of this metal-containing
protein. In both lines, we found low levels of E-cadherin
expression, which were significantly lower in cell line
MCF-7/Dox; high expression levels of CD44 and
N-cadherin, significantly higher in cells resistant
to doxorubicin; and absence of CD24 expression
in both cell lines. The proliferative activity was lower
in cell line MCF-7/Dox.
In the literature, there are data obtained in vivo and
in vitro systems, which show that exogenous LF can
affect the rate of tumor cells growth. Thus, in vivo
experiments showed that systemic effects of LF re-
sulted in the inhibited growth of squamous cell skin
carcinoma transplanted to normal mice; while when
the tumor was transplanted into immune-deficient
mice such effect was absent. The same tendency was
seen when metastatic colon cancer was transplanted
to normal mice and athymic animals [25]. In animals
with subcutaneously transplanted highly metastatic
colon carcinoma, the number of lung metastases de-
creased by 48.0% after LF administration. In the same
model, it was shown that the reduction of the metas-
tasis intensity was accompanied by the increased In-
terleukin 18 production and the subsequent induction
of interferon, to which the authors attribute the anti-
metastatic effect of LF. In addition, the resear chers
concluded that the activation of Interleukin 18 was
associated with the activation of caspase 1 [26]. Using
small angle X-ray scattering, light scattering and abla-
tion, it was convincingly shown that DNA, nucleotides
and oligosaccharides cause the formation of certain
LF oligomers, which differ in their antitumor activities
and influence on the immune system [27]. Finally,
in studies on clinical material received from patients
with intestinal polyposis it has been found that a long-
term LF administration in high doses was not toxic
and resulted in slower growth of polyps. The studies
have shown that after the action of digestive enzymes
the LF cleaves forming lactoferritin which retains the
biological activity of LF. In experiments conducted
on cell cultures aiming to determine the mechanisms
of LF action, it was shown that in some types of tu-
mor cells LF is capable of stimulating apoptosis and
inhi biting cell proliferation by blocking the transition
from G1 phase to S phase of the cell cycle [28, 29].
At the same time, in one of the reports it was noticed
that in human BC cell line MCF-7 LF did not inhibit
the tumor cell growth if it was cultured with this iron-
containing protein [30].
A scarce and controversial data on this issue have
prompted us to analyze whether LF really has a modi-
fying effect on the molecular profile and invasive
properties of tested BC cell lines by culturing them with
exogenous LF (see Table 1). As a result of this phase
of research, it was found that culturing all tested human
BC cell lines with LF resulted in the decrease of their
malignant potential, but the manifestations of the ac-
tion of the iron-containing protein were ambiguous.
In particular, there was a decline in proliferative activity
levels and a significant weakening of the cells’ invasive
properties due to enhanced intercellular adhesion (sig-
nificant increase in the E-cadherin expression and de-
crease in the CD44 expression). The CD24 expression
was slightly reduced, and the expression of N-cadherin
did not change substantially. It should be noted that the
most pronounced effects of changes in the molecular
profile and invasive properties under the LF influence
were observed in the BC cell lines MDA-MB-231 and
MDA-MB-468, which had the highest malignant po-
tential according to the investigated parameters that
was identified before incubating the cells with this
iron-containing protein.
Analysis of the biological effect of LF on the mo-
lecular profile of cytostatic-resistant BC cells that
characterizes their adhesive properties and invasive
activity, allowed us identifying a number of common
features and essential peculiarities in each of the
tested lines. Thus, a common feature for both resistant
lines exposed to LF was a significant inhibition of proli-
feration activity, decrease of the E-cadherin expression
level, significant decline of CD44 expression, as well
as reduction of N-cadherin expression. Directly in cell
line MCF-7/CP, the level of E-cadherin expression
increased twice. The CD44 expression decreased
by 1.7 times, as well as the index of their invasive ac-
tivity. In cell line MCF-7/Dox these indices changed
even more dramatically: the expression of E-cadherin
Experimental Oncology 37, 181–186, 2015 (September) 185
increased twice, the expression of CD44 and invasive
activity decreased by 2.5 times.
The obtained data became the basis for the study
of the LF impacts on the most important component
of molecular phenotype of resistant cells — expression
of proteins associated with MDR: namely P-gp and
GST. Immunohistochemical studies showed that
in cell line MCF-7/CP, which was not subjected to the
LF influence, P-gp expression was not detected, and
the GST expression level was 276.0 ± 2.2 (Table 2).
In cell line MCF-7/Dox, which also served as the
similar control, on the contrary, the P-gp expression
was determined (285.0 ± 1.9), while there was no ex-
pression of GST. Culturing the cells of both lines with
LF caused a substantial reduction of expression of the
tested proteins: GST halved in cell line MCF-7/CP and
P-gp halved in cell line MCF-7/Dox.
Table 2. The changes in expression of proteins associated with drug re-
sistance in BC cells with the phenotype of drug resistance caused by cul-
tivating with LF
Resistance
marker
Cell line
MCF-7/CP MCF-7/СР + LF MCF-7/Dox MCF-7/Dox + LF
P-gp 0 0 285.0 ± 1.9 120.0 ± 1.6*
GST 276.0 ± 2.2 135.0 ± 2.4* 0 0
Note: *p < 0.03 compared with cell line without LF.
The next step in the identification of the LF influ-
ence on cytostatic-resistant MCF-7 cell line was the
study of the ability of this iron-containing protein
to increase cells’ sensitivity to anticancer drugs. Our
findings showed that the doxorubicin IC50 for cell line
MCF-7/Dox after incubation with LF increased twice
as compared with the cells of that line not exposed
to LF, indicating a substantial increase in their sensiti-
vity to this cytostatic (Table 3). A similar phenomenon
of increasing the cells’ sensitivity caused by their
culturing with LF was found in cell line MCF-7/CP to-
wards cisplatin too. Less pronounced manifestations
of cells sensitivity enhancement to cytostatics in terms
of IC50 were noticed in cell line MCF-7/Dox towards
cisplatin and in MCF-7/CP towards doxorubicin. Thus,
the contact of LF with cell lines MCF-7, which is re-
sistant, among others, to doxorubicin and cisplatin,
is able of significantly improving their sensitivity to the
above-mentioned cytostatics.
Table 3. Increased sensitivity of BC cells to anticancer drugs caused by cul-
tivation with LF
Antican-
cer drug
IC50, µgM
Cell line
MCF-7 MCF-7 +
LF
MCF-7/
Dox
MCF-7/
Dox + LF
MCF-7/
СР
MCF-7/
СР + LF
Doxoru-
bicin
4.1±0.3 3.5±0.2* 23.3±2.1 12.0±0.6* 12.4±1.2 9.3±0.4*
Cisplatin 15.3±1.3 12.9±0.3* 16.0±1.0 13.0±0.4* 93.3±7.0 54.8±0.7*
Note: *p < 0.05 compared with cell line without LF.
Our findings do not contradict the results of other
studies that evidence that the manifestation of LF ef-
fects on some properties of tumor cells in vitro can
be regarded as anti-carcinogenic effect [31]. The above
gives hope for the future prospects of the prescription
of LF in the complex personalized therapy of BC pa-
tients. At the same time, our own experimental studies
indicate that, at the stage of establishing BC diagnosis,
it is needed to conduct, along with the LF expression
evaluation, also the assessment of certain molecular
markers’ expression, which can help identify the ma-
lignant potential of the neoplasm, and on this basis
to focus on the individualized prescription of LF.
The fact that LF in vitro has the ability of increasing
tumor cells’ sensitivity to cytostatics, especially resis-
tant ones, warrants further studies using exogenous
LF in vivo to confirm the effect of overcoming MDR.
A significant obstacle to large-scale research and
multi-center clinical trials is the absence of recombi-
nant human LF in the international market. At present,
the developments of genetically engineered con-
structs with gene of human LF are being carried out
actively for production of this protein on a commercial
scale [32, 33]. The successful resolution of this issue
will help approach the time when the use of LF in the
complex treatment of BC patients will become a reality.
CONCLUSIONS
1. It was shown that the tested human BC cell lines
are heterogeneous in terms of LF expression.
2. The various manifestations of LF expression
in the tested human BC cell lines are associated with
the heterogeneity of molecular profile of these cell
lines in terms of adhesion.
3. The highest level of the LF expression was ob-
served in cell lines T47D and MCF-7, which have the
lowest manifestations of invasive properties. Cell lines
MDA-MB-231 and MDA-MB-468 with highly invasive
activities feature low levels of the LF expression.
4. The relationship between the LF expression le-
vels in resistant cell lines MCF-7/CP and MCF-7/Dox
and the features of their molecular profile and invasive
properties was established.
5. The modifying effect of LF on the molecular
profile and invasive properties of all tested lines, in-
cluding resistant ones — MCF-7/CP and MCF-7/Dox,
was established.
6. The inhibitory effect of LF on the expression
of proteins associated with MDR was established
in BC cells with MDR phenotypes.
7. As a result of the cultivation with LF of initial human
BC cell lines and BC cell lines resistant to cytostatics,
their sensitivity to doxorubicin and cisplatin increased,
which was most pronounced in the resistant cell lines:
MCF-7/CP and MCF-7/Dox. This effect is due to the
inhibited expression of MDR associated proteins.
ACKNOWLEDGMENTS
The study was supported by joint scientific pro-
ject of the NAS of Ukraine and the NAS of Belarus
2015–2016 “Molecular-Biological Effects and Mecha-
nisms of Lactoferrin Action on Tumor Cells in vitro and
in vivo”.
REFERENCES
1. Polyak K, Dana-Farber. Heterogeneity in breast cancer.
J Clin Invest 2011; 121: 3786–8.
2. Albain U, Paik S, Van’t Veer L. Prediction of adjuvant
chemotherapy benefit in endocrine responsive early breast
cancer using multigene assays. The Breast 2009; 18: 141–5.
186 Experimental Oncology 37, 181–186, 2015 (September)
3. Curigliano G, Viale G, Bagnardi V, et al. Clinical re-
levance of HER2 overexpression/amplification in patients
with small tumor size and node negative breast cancer. J Clin
Oncol 2009; 27: 5693–9.
4. Semiglazov VF, Semiglazov VV. Breast cancer screening.
Prakticheskaya Onkologiya 2010; 11: 60–5 (in Russian).
5. Chekhun VF, Sherban SD, Savtsova ZD. Tumor hetero-
geneity — a dynamic state. Oncology 2012; 14: 4–12 (in Rus-
sian).
6. Yi-Hsuan Hsiao, Ming-Chih Chou, Carol Fowler,
et al. Breast cancer heterogeneity: mechanisms, proofs, and
implications. J Cancer 2010; 1: 6–13.
7. Michor F, Polyak K. The origins and implications
of intratumor heterogeneity. Cancer Prev Res 2010; 3: 1361–4.
8. Andrechek ER, Nevins JR. Mouse models of can-
cers: opportunities to address heterogeneity of human can-
cer and evaluate therapeutic strategies. J Mol Med 2010;
88: 1095–100.
9. Somasundaram R, Villanueva J, Herlyn M. Intratumoral
heterogeneity as a therapy resistance mechanism: role of mela-
noma subpopulations. Adv Pharmacol 2012; 65: 335–59.
10. Kulagina ESh. Epidemiological and molecular aspects
of breast cancer. Prakt Onkol 2010; 11: 203–16 (in Russian).
11. Nemtsova MV, Paltseva EM, Babayan AYu, et al. Mo-
lecular genetic analysis of clonal intratumoral heterogeneity
in colorectal carcinomas. Mol Biol 2008; 42: 1040–7 (in Rus-
sian).
12. Baldus SE, Schaefer KL, Engers R, et al. Prevalence
and heterogeneity of KRAS, BRAF, and PIK3CA mutations
in primary colorectal adenocarcinomas and their corres-
ponding metastases. Clin Cancer Res 2010; 16: 790–9.
13. Saunders NA, Simpson F, Thompson EW, et al. Role
of intratumoural heterogeneity in cancer drug resistance: mole-
cular and clinical perspectives. EMBO Mol Med 2012; 4: 675–84.
14. Geraschenko TS, Denisov EV, Litvyakov NV, et al.
Intratumoral heterogeneity: nature and biological significance.
Biohimiya 2013; 78: 1531–49 (in Russian).
15. Tsyganov MM, Geraschenko TS, Denisov EV, et al.
Intratumoral morphological heterogeneity of breast can-
cer: expression of multidrug resistance genes and effectiveness
of neoadjuvant antitumor chemotherapy. Sib Onkol J 2013;
(1 (Suppl)): 93–4 (in Russian).
16. Geraschenko TS, Zavyalova MV, Denisov EV, et al.
Intratumoral morphological heterogeneity of invasive ductal
breast cancer: development and molecular-genetic features.
Medi t Akad J 2012; 12: 66–8 (in Russian).
17. Perelmuter VM, Zavyalova MV, Vtorushin SV, et al.
Relations between morphological heterogeneity of infiltrative
ductal breast cancer with different forms of tumor progression.
Sib Onkol J 2007; 23: 58–63 (in Russian).
18. Onitilo AA, Engel JM, Greenlee RT, et al. Breast
cancer subtypes based on ER/PR and Her2 expression: com-
parison of clinicopathologic features and survival. Clin Med
Res 2009; 7: 4–13.
19. Guo L, Meng J, Yilamu D, et al. Significance of ERβ
expression in different molecular subtypes of breast cancer.
Diagn Pathol 2014; 9: 1–6.
20. Zavyalova MV, Denisov EV, Tashireva LA, et al.
Phenotypic drift as a cause for intratumoral morphological
he terogeneity of invasive ductal breast carcinoma not otherwise
specified. Biores Open Access 2013; 2: 148–54.
21. Chekhun VF, Shpilevaya SI. Role of endogenous iron
in development of tumor sensitivity to antitumor therapy.
Voprosy Onkologii 2010; 56: 251–61 (in Russian).
22. Berlov MN. Lactoferrin from dog neutrophils: extrac-
tion, physico-chemical and antimicrobic properties. Biohimia
2007; 72: 551–9 (in Russian).
23. Kang J-F, Li X-L, Zhou RY, et al. Bioinformatics
analysis of lactoferrin gene for several species. Biochem Ge-
netics 2008; 46: 312–22.
24. Lagutin AY, Sobchenko SA, Zadvorny TV. Evaluation
of lactofferin levels as a marker of breast cancer biological
activity. Shevchenkivska Vesna 2014; 7: 42 (in Ukrainian).
25. Hollander D, Ni J. Application of the MTT-assay
to functional studies of mouse intestinal intraepithelial lym-
phocytes. J Clin Lab Anal 1996; 10: 42–52.
26. Tsuda H, Sekine K, Takasuka N. Prevention of colon
carcinogenesis and carcinoma metastasis by orally adminis-
tered bovin lactoferrin in animals. Biofactors 2000; 12: 83–8.
27. Kuhara I, Ligo M, Itoh T. Orally administered lac-
toferrin exerts an antimetastatic effect and enhances produc-
tion of IL-18 in the intestinal epithelium. Nutr Cancer 2000;
38: 192–9.
28. Nevinskii AG, Soboleva SE, Tuzikov FV. DNA, oligo-
saccharides, and mononucleotides stimulate oligomerization
of human lactoferrin. Mol Recogn 2009; 22: 330–42.
29. Xiao Y, Monitto CL, Minhas KM, et al. Lactoferrin
down-regulates G1 cyclin-dependent kinases during growth arrest
of head and neck cancer cells. Clin Cancer Res 2004; 10: 8683–6.
30. Yang N, Stensen W, Svendsen JS, et al. Enhanced an-
titumor activity and selectivity of lactoferrin-derived peptides.
J Pept Res 2002; 60: 187–97.
31. Wolf JS, Li D, Taylor RJ, O’Malley BW Jr. Lactoferrin
inhibits growth of malignant tumors of the head and neck. ORL
J Otorhinolaryngol Relat 2003; 65: 245–9.
32. Chekhun V, Bezdenezhnykh N, Shvets J, et al. Ex-
pression of biomarkers related to cell adhesion, metastasis
and invasion of breast cancer cell lines of different molecular
subtype. Exp Oncol 2013; 35: 174–9.
33. Naroditskiy BS, Shmarov MM, Logunov DYu, et al.
Method of synthesis of recombinant human lactoferrin. Rus-
sian Patent 2008; N 2340674 (in Russian).
Copyright © Experimental Oncology, 2015
|
| id | nasplib_isofts_kiev_ua-123456789-145485 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T16:23:15Z |
| publishDate | 2015 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Chekhun, V.F. Zalutskii, I.V. Naleskina, L.A. Lukianova, N.Yu. Yalovenko, T.M. Borikun, T.V. Sobchenko, S.O. Semak, I.V. Lukashevich, V.S. 2019-01-22T12:48:41Z 2019-01-22T12:48:41Z 2015 Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells / V.F. Chekhun, I.V. Zalutskii, L.A. Naleskina, N.Yu. Lukianova, T.M. Yalovenko, T.V. Borikun, S.O. Sobchenko, I.V. Semak, V.S. Lukashevich // Experimental Oncology. — 2015. — Т. 37, № 3. — С. 181-186. — Бібліогр.: 33 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145485 Aim: To assess the role of endogenous lactoferrin (LF) in the formation of the molecular phenotype of human breast cancer (BC) cell lines with varying degrees of malignancy, including cisplatin/doxorubicin resistant cell lines, and identify possible impact of exogenous LF. Materials and Methods: 5 breast cell lines of different origin — MCF-10 A, MCF-7, including doxorubicin/cisplatin resistant ones, T47D, MDA-MB-231, and MDA-MB-468. Immunocytochemistry: expression of LF, Ki-67, adhesion molecules E- and N-cadherin, CD44, CD24 rating the invasive potential of cells. Results: Expression of LF in human BC cell lines varies. It is associated with the heterogeneity of molecular profiles of cell lines in terms of adhesion. A link has been established between the level of LF expression in the resistant cell line MCF-7/CP and MCF-7/Dox, features of their molecular profile and invasive properties. Exogenous LF was shown to be capable of modifying the molecular profile and invasive properties of all the studied cell lines including resistant ones (MCF-7/CP and MCF-7/Dox). Conclusions: The sensitivity of cytostatic-resistant cell lines (MCF-7/CP and MCF-7/Dox) tends to increase under the influence of exogenous LF. It is likely that this effect is due to LF-mediated inhibition of the expression of proteins associated with drug resistance. Key Words: lactoferrin, cell lines of human breast cancer, molecular phenotype, proliferative activity, invasive potential, adhesion molecules. The study was supported by joint scientific project of the NAS of Ukraine and the NAS of Belarus 2015–2016 “Molecular-Biological Effects and Mechanisms of Lactoferrin Action on Tumor Cells in vitro and in vivo”. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells Article published earlier |
| spellingShingle | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells Chekhun, V.F. Zalutskii, I.V. Naleskina, L.A. Lukianova, N.Yu. Yalovenko, T.M. Borikun, T.V. Sobchenko, S.O. Semak, I.V. Lukashevich, V.S. Original contributions |
| title | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells |
| title_full | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells |
| title_fullStr | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells |
| title_full_unstemmed | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells |
| title_short | Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells |
| title_sort | modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/145485 |
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