Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin
Aim : To study in vivo the peculiarities of changes of iron metabolism and antioxidant system in dynamics of growth of Guerin carcinoma with different sensitivity to cisplatin. Materials and Methods: In order to evaluate the content of metallothionein-1 (MT-1) in tumor homogenates and blood serum of...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Cite this: | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin / V.F. Chekhun, Y.V. Lozovska, A.P. Burlaka, N.Y. Lukyanova, I.N. Todor, L.A. Naleskina // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 196-201. — Бібліогр.: 26 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859668589248774144 |
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| author | Chekhun, V.F. Lozovska, Y.V. Burlaka, A.P. Lukyanova, N.Y. Todor, I.N. Naleskina, L.A. |
| author_facet | Chekhun, V.F. Lozovska, Y.V. Burlaka, A.P. Lukyanova, N.Y. Todor, I.N. Naleskina, L.A. |
| citation_txt | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin / V.F. Chekhun, Y.V. Lozovska, A.P. Burlaka, N.Y. Lukyanova, I.N. Todor, L.A. Naleskina // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 196-201. — Бібліогр.: 26 назв. — англ. |
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| description | Aim : To study in vivo the peculiarities of changes of iron metabolism and antioxidant system in dynamics of growth of Guerin carcinoma with different sensitivity to cisplatin. Materials and Methods: In order to evaluate the content of metallothionein-1 (MT-1) in tumor homogenates and blood serum of rats with cisplatin-sensitive and cisplatin-resistant Guerin carcinoma the immunoenzyme method was used. The evaluation of ceruloplasmin activity, content of “free iron” complexes, superoxide and NO-generating activity of NADPH-oxidase and iNOS activity in neutrophils, blood serum and tumor homogenates was measured by EPR-spectroscopy. Results: Maximal accumulation of MT-1 in blood serum and tumor, more pronounced in resistant strain, at the border of latent and exponential phase of growth has been shown that is the evidence of protective role of this protein in the respect to the generation of free radical compounds. It has been determined that in animals with cisplatin-resistant strain of Guerin carcinoma, increase of “free iron” complexes is more apparent both on the level of tumor and organism on the background on increase of CP/TR ratio that is the consequence of organism antioxidant protection system disorder. Conclutions: Mentioned changes in metabolism of iron with its accumulation in tumor and further reprogramming of mitochondria metabolism and activity of NADPH-oxidase for non-transformed cells are favorable conditions for the formation of oxidative phenotype of tumor. Key Words: Guerin carcinoma, metallothionein-1, antioxidant system, transferrin, ceruloplasmin, “free iron” complexes, activity of NADPH-oxidase and iNOS neutrophils.
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196 Experimental Oncology 36, 196–201, 2014 (September)
PECULIARITIES OF ANTIOXIDANT SYSTEM AND IRON
METABOLISM IN ORGANISM DURING DEVELOPMENT OF TUMOR
RESISTANCE TO CISPLATIN
V.F. Chekhun, Y.V. Lozovska, A.P. Burlaka, N.Y. Lukyanova*, I.N. Todor, L.A. Naleskina
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine
Aim: To study in vivo the peculiarities of changes of iron metabolism and antioxidant system in dynamics of growth of Guerin car-
cinoma with different sensitivity to cisplatin. Materials and Methods: In order to evaluate the content of metallothionein-1 (MT-1)
in tumor homogenates and blood serum of rats with cisplatin-sensitive and cisplatin-resistant Guerin carcinoma the immunoenzyme
method was used. The evaluation of ceruloplasmin activity, content of “free iron” complexes, superoxide and NO-generating acti-
vity of NADPH-oxidase and iNOS activity in neutrophils, blood serum and tumor homogenates was measured by EPR-spectro-
scopy. Results: Maximal accumulation of MT-1 in blood serum and tumor, more pronounced in resistant strain, at the border
of latent and exponential phase of growth has been shown that is the evidence of protective role of this protein in the respect
to the generation of free radical compounds. It has been determined that in animals with cisplatin-resistant strain of Guerin car-
cinoma, increase of “free iron” complexes is more apparent both on the level of tumor and organism on the background on increase
of CP/TR ratio that is the consequence of organism antioxidant protection system disorder. Conclutions: Mentioned changes
in metabolism of iron with its accumulation in tumor and further reprogramming of mitochondria metabolism and activity of NADPH-
oxidase for non-transformed cells are favorable conditions for the formation of oxidative phenotype of tumor.
Key Words: Guerin carcinoma, metallothionein-1, antioxidant system, transferrin, ceruloplasmin, “free iron” complexes, activity
of NADPH-oxidase and iNOS neutrophils.
Today, condition of antioxidant system (AOS) has
significant place in the chain of insufficiently studied
rearrangements occurring in the organism of host
in the process of development of neoplasm. It has been
proved that activation of oxidative processes, which cause
disintegration of cellular membranes and destabilization
of many elements of metabolism underlie pathological
changes associated with malignant transformation of cell.
The increase of oxidation of lipids, proteins and DNA
becomes more intensive during progression of tumor
process. It has been determined that tumor progression
is accompanied with development of oxidative stress and
this consequently requires effective mechanisms of pro-
tection from damaging impact of reactive oxygen species
(ROS) and other free radical compounds. The system in-
activating free radical compounds is AOS. By localization,
antioxidant protection system is divided into the intra- and
extracellular antioxidants. To the intracellular antioxidants
are referred glutathione, α-tocopherol, Zn/ Mg-SOD,
catalase, glutathione peroxidase, ferritin and other.
System of extracellular antioxidants consists of low-
molecular substances (ascorbic and uric acids, bilirubin)
and high-molecular compounds (extracellular superoxide
dismutase, SH-groups of proteins, ceruloplasmin —
CP and transferrin — TR). Development of tumors causes
the activation of AOS of blood, in particular, involvement
of CP and TR [1–4]. CP is known to be multifunctional
copper-containing protein — oxidase of α2-globulin
fraction of blood plasma of mammals, which performs
non-specific protection at exo- and endogenous da-
mages. Also, it is connected with hematopoietic system
playing role of the catalyst in transformation of Fe2+
in Fe3+ with its followed including in apo-transferrin.
It has been determined that development of malignant
process causes accumulation in organism of “free iron”
(Fe2+), which activates processes of lipid peroxidation
(LPO) that causes in turn increase of antioxidant acti-
vity of blood serum, exactly activity of CP and TR [6–8].
By its origin, TR is single chain iron-containing glyco-
protein of β-globulin fraction of blood serum, which
plays the main role in transfer and regulation of iron
metabolism in tissues and also impacts the antioxidant
potential of blood. It should be mentioned that CP along
with TR forms molecular system able to regulate content
of iron ions (Fe2+), total antioxidant activity of blood serum
and oxidation of Fe2+ by CP. It has been showed that CP/
TR (lactoferrin) ratio increases in cancer patients com-
pared with indices of healthy people that is the evidence
of significant content of Fe2+ as a pro-oxidant marker [4,
6–11]. Thus, complicated system of interaction between
CP and TR, which is fully connected with transformation
and redistribution of iron in organism with its further
involvement in the processes of generation of ROS,
proves once again the importance of this microelement
in occurrence and development of malignant neoplasm.
In recent time, significant attention was given to one
more little-studied component of AOS of organism —
metallothionein (MT). For instance, it has been showed
that MT owing to significant content of SH-bonds
is capable to change redox-condition and act as ac-
tive interceptor of superoxide radicals [12–14]. For
this reason, evaluation and analysis of the processes,
which allow cells to regulate reaction to exo- and
Submitted: July 16, 2014.
*Correspondence: E-mail: oncom@onconet.kiev.ua
Abbreviations used: AOS — antioxidant system; CP — ceruloplasmin;
EPR — electron paramagnetic resonance; iNOS — inducible NO-syn-
tase; LPO — lipid peroxidation; MT — metallothionein; NADPH — nico-
tinamide adenine dinucleotide phosphate reduced; ROS — reactive
oxygen species; TNF — tumor necrosis factor; TR — transferrin.
Exp Oncol 2014
36, 3, 196–201
Experimental Oncology 36, 196–201, 2014 (September) 197
endo genous impacts at tumor process, have very
important practical significance. On the example
of antioxidant protection in development of malignant
neoplasms in organism, conception of “tumor-host”
interaction is confirmed. Study of AOS of organism
(exactly chelators of metal ions with variable atomicity,
MT-1) and parallel determination of “free iron” com-
plexes and generation of ROS, especially taking into
account sensitivity of malignant neoplasm to antitumor
drugs, is insufficiently studied direction in oncology.
Taking into account mentioned above, the aim
of our study was to analyze the peculiarities of iron
metabolism and AOS in organism of host in dynamics
of tumor drug resistance development.
MATERIALS AND METHODS
Animals and experimental model. Object of our
study included 80 male rats with sensitive and resistant
to cisplatin Guerin carcinoma. Guerin carcinoma was
transplanted subcutaneously (2 × 106 cells/rat). Resistant
strain was obtained through subsequent transplantations
of Guerin carcinoma after carrying out of chemotherapy
course with cisplatin to the rats. To obtain resistant tumor,
10 courses of chemotherapy were carried out. All animals
were decapitated with adhering current bioethical norms.
According with the aim of study, change in the level
of metal-containing proteins in blood of animals was
determined in different periods of growth of Guerin
carcinoma — 1, 3, 5, 7, 17 and 23 days after transplanta-
tion. By conventional bioethics, 1–5 day of development
of tumor is considered as latent growth period, 6–14 day
is referred to the phase of exponential growth, and
23 day — to the terminal phase of tumor growth.
In blood serum and tumor tissue of experimental
animals, content of the following metal-containing pro-
teins was studied: MT-1 and TR [4]. Also, in blood and
tumors of these animals, “free iron” complexes were
determined [2]. Moreover, in blood of experimental
rats, activity of CP and superoxide- and NO-generating
activity of NADPH-oxidase, iNOS neutrophils was de-
termined. In tumor tissue, level of superoxide radicals
in mitochondria was registered. Mentioned above
indices in tumor were studied on 7th, 14th and 23rd days
after its transplantation, exactly in those periods, when
changes may obtain essential significance.
Evaluation of the content of MT-1 in blood se-
rum and tumor homogenates of rats with Guerin
carcinoma by immunoenzyme method. Content
of MT-1 in blood serum and tumor homogenate was
determined by immunoenzyme method — IEM (ELISA)
using appropriate kits of reagents: USCN (China) with
the help of automatic biochemical and immunoenzyme
analyzer Chem Well 2990. Samples of blood serum for
IEM were obtained according with the recommenda-
tions indicated in instructions to the kits. All samples
of blood serum had no signs of hemolysis. Homo-
genate of tumor tissue was obtained in physiological
solution in the ratio of ⅓.
E v a l u a t i o n o f C P a c t i v i t y , c o n t e n t
of TR, “free iron” complexes and superoxide-
and NO-gene rating activity of NADPH-oxidase,
iNOS neutrophils in blood of experimental animals
by EPR. In blood of experimental animals, activity
of CP and content of TR, quantity of “free iron” com-
plexes and super oxide- and NO-generating activity
of NADPH-oxidase, iNOS neutrophils were analyzed
by method of electron paramagnetic resonance (EPR)
at the temperature of liquid nitrogen 77 K. As antico-
agulant, trilon B was used. Samples for the study with
computerized spectrometer were prepared using spe-
cial press-form with followed freezing in liquid nitrogen.
Determination of content of TR, “free iron”
complexes and levels of superoxide radicals in tu-
mor tissue of experimental animals by EPR. To de-
termine these indices, 0.5 g of tumor tissue was placed
in special press-forms and frozen in liquid nitrogen. Af-
ter this, EPR was applied using spin trap — 1-hydroxy-
2,2,6,6-tetramethyl-4-oxo-piperidine hydrochloride
(TEMPONE-H) [4].
Statistical analysis. Significance of differences
between indices of different groups was evaluated
using Student’s t-criterion. Differences were con-
sidered significant at р<0.05. Statistical processing
of data was carried out using software STATISTICA 6.0.
RESULTS AND DISCUSSION
Studies showed that from 1st to 7th day after trans-
plantation of tumor, gradual increase of MT-1 was ob-
served in blood serum of animals. Maximal value of this
index both in tumor and blood serum was registered
on 7th day. It means that results of our studies prove
that this metal-containing protein plays key role exactly
in the end of latent and beginning of exponential stage
of tumor growth protecting malignant neoplasm from
reactive oxygen forms. Also, it has been confirmed
by our data concerning generation of free radical com-
pounds exactly in mentioned period of tumor growth
that will be discussed below. At the peak of exponential
tumor growth (14 days after transplantation), gradual
decrease of MT-1 in tumor tissue and blood serum was
observed. Mentioned changes were more pronounced
in development of resistant tumor (Table 1).
These data give ground for assumption that
MT-1 plays important role in forming of resistant
phenotype in animals with Guerin carcinoma. Our view
coincides with the results of some studies, which have
showed that due to metallothioneins, cells are ca-
pable to acquire resistance, in particular, to the tumor
necrosis factor (TNF) [15, 16]. Presence in molecule
of MT sulfhydryl groups with nucleophilic properties
gives them opportunity to bind not only metal ions, but
also ROS [13, 14]. It has been determined that high
levels of metallothioneins are found in the places of in-
flammation and malignant transformation of tissue.
Data of literature show that MT plays significant role
in occurrence and development of tumor process [16,
17]. On the example of patients with hepatocellular
carcinoma, it has been shown that high expres-
sion of MT-1 in tumors correlates with low efficacy
of chemotherapy with carboplatin [18]. It has been
198 Experimental Oncology 36, 196–201, 2014 (September)
shown that hyperexpression of MT is antiapoptotic
and stimulates increase the proliferative activity of tu-
mor cells. MT activates and specifically interacts with
p50 (subunit of NFkB transcription factor) that causes
the transactivation of the latter [14].
Table 1. Content of MT-1 in tumor tissue and blood serum of animals
with Guerin carcinoma in different stages of its growth
Groups
of animals
by periods
after trans-
plantation
Content of МТ-1, pg/ml
Sensitive strain Resistant strain
serum tumor serum tumor
Control
(intact ani-
mals)
282.40±9.73 − 279.40±8.23 −
1 day 319.41±14.23 − 320.10±15.14 −
3 days 334.22±17.13* − 350.12±19.80* −
5 days 535.10±34.50* − 750.34±21.18* −
7 days 634.20±31.23* 740.33±23.14 820.10±32.14* 920.43±38.12*
14 days 424.12±9.24* 510.18±28.10* 506.92±15.52* 512.31±21.18*
23 days 423.64±13.51* 390.14±16.13* 399.61±10.21* 490.12±31.21*
Note: *р < 0.05 — significant regarding control values.
Till this day, condition of extracellular high-molecular
AOS of blood at development of malignant neoplasm
remains insufficiently studied. As stated above, exactly
such proteins, as TR and CP in blood plasma, form AOS
that regulates concentration of renewed iron ions [4,
18]. The main role in antioxidant protection of orga nism
is performed by these proteins due to the inhibition
of iron-induced peroxidation of the components of ex-
tracellular environment (i.e., due to the oxidase activity
of CP and iron-binding and transport function of TR).
First of all, we draw attention to the one of the proteins,
which participates in iron metabolism, exactly — TR. Ac-
cording with data of li terature, decrease of TR in dyna-
mics of tumor growth in 2.5–3 times on the background
of simultaneous increase of CP in 1.5–2 times occurs
in blood plasma of animals [19].
Results of our studies of TR content in blood of ex-
perimental animals with Guerin carcinoma (sensitive
and resistant strains) showed that almost on the first
day of tumor growth, its level in blood starts gradually
decreasing compared with control (Table 2). In tumor
tissue of sensitive to cisplatin strain of Guerin carci-
noma, level of TR remained on the same level both
in latent and exponential growth phases. In the end
of terminal growth phase, insignificant decrease
of this index was observed that may be connected with
the processes of necrobiotic changes and necrosis
in tumor. The same tendencies concerning content
of TR were observed in blood serum and tumor of re-
sistant strain.
Table 2. Content of TR in tumor tissue and blood of animals with Guerin
carcinoma in different stages of its growth
Groups of animals
by periods of trans-
plantation
TR level (RU)
Sensitive strain Resistant strain
serum tumor serum tumor
Control (intact animals) 0.93±0.07 − 0.88±0.1 −
1 day 0.67±0.06 − 0.82±0.21 −
3 days 0.55±0.04* − 0.64±0.12 −
5 days 0.48±0.12* − 0.58±0.10 −
7 days 0.35±0.03* 0.15±0.05 0.47±0.12* 0.16±0.05
14 days 0.24±0.05* 0.15±0.09 0.39±0.20* 0.14±0.12
23 days 0.16±0.07* 0.10±0.05* 0.21±0.04* 0.12±0.05*
Note: *р < 0.05 — significant regarding control values.
It should be mentioned that recent publications
on mechanisms of metal metabolism in organism
point at significant role of TR in transfer and accumu-
lation of iron. For instance, on the example of cell line
of small cell lung carcinoma NCL-H510, expression
of this protein on their surface was detected by method
of immune fluorescence [20]. Also, it has been de-
termined that synthesis of TR 10 times increases
during cell proliferation. Thus, it has been proved
in study that hyperexpression of TR induces cellular
proliferation, but fully depends on regulatory system
of iron metabolism (auto crine mechanism of regula-
tion) [20]. Other studies in vivo showed that at pre-
sence in organism of malignant process, decrease
of content of TR and decrease of iron concentration
in blood serum occurs [21–23]. It results in disorder
of iron transfer from blood serum to ferritin in tumor
cells that is the evidence of accumulation and storage
of iron ions in tumor cells. Since another one metal-
containing protein, which is a part of extracellular high-
molecular AOS of organism and indirectly participates
in the processes of recovery and oxidation of iron
in blood, is CP, we decided to study activity of this
protein in host blood, as well as to analyze CP/ TR ra-
tio in dynamics of tumor growth as marker of reserve
possibility of AOS (Table 3).
Table 3. Activity of CP and CP/TR ratio in blood of animals with Guerin
carcinoma in different stages of its growth
Groups of animals
by periods of trans-
plantation
CP activity (RU)
Sensitive strain Resistant strain
CP CP/TR CP CP/TR
Control (intact animals) 0.86 ± 0.09 0.92 0.88 ± 0.1 0.85
1 day 1.94 ± 0.11 1.49* 0.48 ± 0.06 0.58*
3 days 5.51 ± 0.09* 10.01* 0.50 ± 0.04 0.78
5 days 7.21 ± 0.16* 15.02* 0.69 ± 0.09 1.18
7 days 7.03 ± 0.10* 20.08* 1.13 ± 0.25* 2.40
14 days 5.79 ± 0.12* 24.12* 1.8 ± 0.18* 4.61
23 days 5.99 ± 0.15* 37.43* 2.76 ± 0.31 13.14
Note: *р < 0.05 — significant regarding control values.
Obtained results prove that along with decrease
of content of TR (Table 2), increase of CP activity oc-
curs (Table 3) in blood of animals with sensitive and
resistant Guerin carcinoma that is the evidence of dis-
order of iron transport in host organism. After all, ratio
CP/TR increases, and it may cause the increase of “free
iron” concentration. Thus, as activity of CP and TR al-
ters, disorder of balance oxidant/antioxidant occurs
with further development of oxidative stress. However,
values of CP/TR in different stages of tumor growth
were lower in blood of animals with resistant strain,
than in blood of animals with sensitive strain. It can
be explained by the fact that as resistance of cisplatin
forms, changes in transportation of the component
of CP, copper, occurs due to the series of proteins-
transporters. Moreover, according to the data of lit-
erature, key protein-transporter of copper CTR1 plays
important role not only in transporting of copper, but
also cisplatin. Authors of these studies showed that,
on the one hand, copper is required for transporta-
tion of platinum inside cell, and, on the other hand,
it competes with platinum for the sites of binding [24].
Series of studies on cellular lines of breast and ovarian
Experimental Oncology 36, 196–201, 2014 (September) 199
cancer resistant to cisplatin have showed alteration
in expression of series of proteins-transporters of cop-
per: CTR1, ATP7A, ATP7B, which also are involved
in transportation of cisplatin and its derivatives. Direct
correlation between delivery of copper and cisplatin
in cells and decrease of transportation of these met-
als in tumor with phenotype resistant to cisplatin has
been determined [25]. Clinical studies showed direct
correlation between increase of concentrations of cop-
per and CP in blood serum of patients with breast,
ovarian, lung, and intestine cancers and progression
of tumor process [26]. For this reason, we have made
an assumption that low levels of CP activity in blood
of animals with resistant to cisplatin strain (regarding
indices of sensitive strain) are connected with disorder
of transportation of copper as a result of decrease
of expression of proteins-transporters CTR1, АТР7А,
АТР7В.
It has been determined that at malignant trans-
formation, which is accompanied with disorder
of iron metabolism in organism, misbalance of system
of antioxidant protection with involvement of metal-
containing proteins CP and TR is observed. Total
antioxidant activity of serum concerning Fe2+-induced
LPO is mainly depended on content of these proteins.
It should be mentioned that exactly TR binds iron ions
Fe3+ that prevents their renewal and participation
in Fenton/Haber — Weiss reaction, which indirectly
causes release of free radical compounds with further
damage of many metabolic processes [9, 10]. It has
been shown that interaction between metals (first
of all iron) and hydrogen peroxide results in forma-
tion of the most toxic form of ROS. It damages DNA,
RNA as well as carbohydrates, amino acids and lipids.
Moreover, presence of ions of these metals may cause
spontaneous and induced oxide modification of pro-
teins. It is known that these changes acquire higher
sensitivity to proteolysis that causes invasion of tumor
in surrounding tissues [10].
For the full understanding of changes of CP and
TR as metal-containing proteins, as well as important
components of AOS, we have studied quantity of “free
iron” complexes in dynamics of tumor growth both
in tumor tissue and peripheral blood.
Obtained results showed that increase of “free
iron” complexes in tumor tissue of both strains in dy-
namics of development of malignant neoplasms was
accompanied with simultaneous increase of the rate
of generation of superoxide radicals (Table 4).
However, stronger increase of level of superoxide
radicals was observed in resistant tumors, especially
23 days after transplantation. It demonstrates that for
resistant tumors, more active generating of superoxide
radicals is typical compared with sensitive tumors that
in turn causes more apparent destabilization of iron-
containing proteins. Moreover, we have determined
direct correlation between increase of rate of ge-
nerating of superoxide radicals and number of “free
iron” complexes in sensitive (r = 0.98) and resistant
(r = 0.79) tumors.
Table 4. Number of “free iron” complexes and rate of generating of su-
peroxide radicals in tumor tissue of Guerin carcinoma in different stages
of its growth
Groups
of ani mals
by periods af-
ter transplan-
tation
“Free iron” complexes (RU)
Rate of generating of su-
peroxide radicals in tumor
tissue (nmol/g of fresh
tissue·min)
Sensitive
strain
Resistant
strain
Sensitive
strain
Resistant
strain
7 days 0.19 ± 0.08 0.21 ± 0.07 0.50 ± 0.1 0.51 ± 0.23
14 days 0.61 ± 0.05* 0.68 ± 0.04* 0.51 ± 0.1 0.63 ± 0.18
23 days 1.95 ± 0.08* 2.00 ± 0.08* 0.56 ± 0.09 0.64 ± 0.11*
Note: *р < 0.05 — significant regarding control values.
At the same time, number of “free iron” complexes
in peripheral blood of the same animals in dynamics
of malignant process was studied. It is well-known that
emergence of “free iron” in blood is stipulated by acti-
vation of LPO and damage of membranes, hemolysis,
destruction of iron-containing proteins, transformation
of Fe3+-transferrin in apo-transferrin: Fe3+-TR е-→ Fe2+ +
apo-TR, as well as release of deposited iron from ferritin.
It is also known that ions Fe2+ play important role in LPO,
and iron “overload” of organism causes damage of cel-
lular structures. Low-molecular iron complexes are pro-
moters of free radical reactions, and emergence of “free
iron” pool in blood causes the formation of ·ОН and О2
..
Our studies have determined increase of “free iron”
complexes in peripheral blood of animals in dynamics
of growth of both tumor strains (Figure).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1 3 5 7 14 23
Days after tumor transplantation
Re
la
tiv
e
un
its
Sensitive strain
Resistant strain
Figure. Number of “free iron” complexes in peripheral blood
of rats with sensitive and resistant Guerin carcinoma in dyna-
mics of tumor growth
It should be mentioned that in peripheral blood
of intact animals, “free iron” complexes were not
detected. Mentioned increase of level of superoxide
radicals and “free iron” complexes in experimental
animals are the evidence of development of oxidative
stress. Moreover, we have studied rate of generating
of superoxide radicals by NADPH-oxidase and activity
of iNOS neutrophils depending on phases of growth
of Guerin carcinoma of both strains. Gradual increase
of these indices from the beginning of tumor growth
compared with their values in corresponding controls
has been determined (Table 5). As seen from Table 5,
changes of activity of NADPH-oxidase and iNOS
neutrophils in blood of animals with Guerin carcinoma
in dynamics of growth of both sensitive and resistant
strains are unidirectional. However, in blood of animals
with resistant strain, these indices are slightly higher,
than in animals with sensitive tumor strain.
200 Experimental Oncology 36, 196–201, 2014 (September)
Table 5. Rate of generating of superoxide radicals by NADPH-oxidase and
iNOS neutrophils of rat blood in dynamics of Guerin carcinoma growth
Groups
of animals
by periods af-
ter transplan-
tation
Activity of NADPH-
oxidase of neutrophils
(nmol/103 cells·min)
Activity of iNOS neutrophils
(nmol/103 cells·min)
Sensitive
strain
Resistant
strain
Sensitive
strain
Resistant
strain
Control (in-
tact animals)
0.28 ± 0.05 0.29 ± 0.04 1.60 ± 0.02 1.76 ± 0.04
1 day 0.70 ± 0.03* 0.77 ± 0.02* 2.01 ± 0.07 2.04 ± 0.05
3 days 0.98 ± 0.09* 0.96 ± 0.09* 2.15 ± 0.11* 2.11 ± 0.12*
5 days 1.08 ± 0.09* 1.12 ± 0.08* 2.95 ± 0.15* 3.55 ± 0.15*
7 days 2.12 ± 0.03* 2.09 ± 0.01* 3.71 ± 0.20* 3.80 ± 0.16*
14 days 1.95 ± 0.02* 2.35 ± 0.18* 4.12 ± 0.10* 3.95 ± 0.15*
23 days 1.98 ± 0.05* 2.46 ± 0.19* 3.79 ± 0.06* 4.16 ± 0.21*
Note: *р < 0.05 — significant regarding control values.
To determine connection between indices of iron
metabolism and indices of oxidative stress, we have
carried out study using correlation analysis. Results have
showed that increase of “free iron” complexes in periphe-
ral blood is accompanied by activation of generating
superoxide radicals by NADPH-oxidase with prevalence
in resistant tumors (r = 0.59 at growth of sensitive and r
= 0.70 — resistant tumor). This fact points at the most
essential disorders in iron metabolism in organism of tu-
mor host at development of resistant tumor. It has been
determined that as number of “free iron” complexes
in peripheral blood of animals increases, activity of iNOS
neutrophils also significantly increases (r = 0.50 at sensi-
tive and r = 0.76 at resistant tumor). Also, we have proved
direct correlation between content of powerful antioxi-
dant MT-1 and activity of NADPH-oxidase of neutrophils
of sensitive (r = 0.67) and resistant (r = 0.51) tumor. Simi-
larly, direct correlation between level of MT-1 and activity
of iNOS of neutrophils of sensitive (r = 0.67) and resistant
(r = 0.65) strains has been determined. There is an idea
in literature that MT-1 performs protective role from dam-
aging action of superoxide radicals due to the SH-bonds
and cysteine as components of this metal-containing
protein [17]. On the example of Guerin carcinoma, it has
been shown that increase of content of MT-1 in tumor
tissue of resistant strain directly correlates with high level
of generation of superoxide radicals in it (r = 0.72). This
coefficient was lesser in sensitive tumor and equaled r
= 0.59. Obtained results demonstrate that MT-1 in the
process of Guerin carcinoma growth plays important
role in antioxidant protection of transformed cells from
destructive impact of free radical compounds.
Thus, in animals with sensitive and resistant to cis-
platin Guerin carcinoma, change of condition of AOS
components was determined in dynamics of tumor
growth: content of MT-1, TR and activity of CP that
points at disorder of iron metabolism in host organism.
In conclusion, on the model of Guerin carcinoma
with sensitive and resistant to cisplatin strains, misba-
lance of regulatory proteins of iron metabolism has been
shown: MT-1, TR and CP in dynamics of deve lopment
of tumor process on the level of complicated system
of interaction between tumor and organism. It has been
determined that in the initial stages of development
of neoplasm, changes in two functionally coupled com-
ponents of pro- and AOS takes place in blood serum
and tumor tissue of animals: significant increase of ge-
nerating superoxide radicals and simultaneous increase
of MT-1. It has been determined that in dynamics of tu-
mor growth in animals of both strains, increase of “free
iron” complexes both in tumor tissue and blood serum
takes place on the background of significant increase
of CP/TR ratio that shows disorder of functioning of host
AOS which participates in the recovery processes and
iron oxidation. In animals with strain resistant to cispla-
tin, mentioned changes were more pronounced that
was manifested by increase of number of “free iron”
complexes in tumor and blood of animals with further
activation of generating superoxide radicals. It has been
shown that tumor cells are capable to alter metabolism
of iron via its accumulation in malignant neoplasm with
followed reprogramming of mitochondria metabolism
and activity of NADPH-oxidase of non-transformed cells
contributing the increase of generating superoxide radi-
cals. Mentioned changes are favorable for the formation
of oxidative phenotype and appropriate conditions for
the progression of tumor lesion.
ACKNOWLEDGEMENT
The study is performed in terms of the topic
№ 2.2.5.361 “Molecular epidemiology of metal-
containing proteins in tumor-host interactions”
of the target research program “Functional genomics
and metabolomics in system biology” (2012–2016).
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Copyright © Experimental Oncology, 2014
|
| id | nasplib_isofts_kiev_ua-123456789-145366 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-11-30T12:16:37Z |
| publishDate | 2014 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Chekhun, V.F. Lozovska, Y.V. Burlaka, A.P. Lukyanova, N.Y. Todor, I.N. Naleskina, L.A. 2019-01-21T08:16:18Z 2019-01-21T08:16:18Z 2014 Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin / V.F. Chekhun, Y.V. Lozovska, A.P. Burlaka, N.Y. Lukyanova, I.N. Todor, L.A. Naleskina // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 196-201. — Бібліогр.: 26 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145366 Aim : To study in vivo the peculiarities of changes of iron metabolism and antioxidant system in dynamics of growth of Guerin carcinoma with different sensitivity to cisplatin. Materials and Methods: In order to evaluate the content of metallothionein-1 (MT-1) in tumor homogenates and blood serum of rats with cisplatin-sensitive and cisplatin-resistant Guerin carcinoma the immunoenzyme method was used. The evaluation of ceruloplasmin activity, content of “free iron” complexes, superoxide and NO-generating activity of NADPH-oxidase and iNOS activity in neutrophils, blood serum and tumor homogenates was measured by EPR-spectroscopy. Results: Maximal accumulation of MT-1 in blood serum and tumor, more pronounced in resistant strain, at the border of latent and exponential phase of growth has been shown that is the evidence of protective role of this protein in the respect to the generation of free radical compounds. It has been determined that in animals with cisplatin-resistant strain of Guerin carcinoma, increase of “free iron” complexes is more apparent both on the level of tumor and organism on the background on increase of CP/TR ratio that is the consequence of organism antioxidant protection system disorder. Conclutions: Mentioned changes in metabolism of iron with its accumulation in tumor and further reprogramming of mitochondria metabolism and activity of NADPH-oxidase for non-transformed cells are favorable conditions for the formation of oxidative phenotype of tumor. Key Words: Guerin carcinoma, metallothionein-1, antioxidant system, transferrin, ceruloplasmin, “free iron” complexes, activity of NADPH-oxidase and iNOS neutrophils. The study is performed in terms of the topic № 2.2.5.361 “Molecular epidemiology of metal-containing proteins in tumor-host interactions” of the target research program “Functional genomics and metabolomics in system biology” (2012–2016). en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin Article published earlier |
| spellingShingle | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin Chekhun, V.F. Lozovska, Y.V. Burlaka, A.P. Lukyanova, N.Y. Todor, I.N. Naleskina, L.A. Original contributions |
| title | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin |
| title_full | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin |
| title_fullStr | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin |
| title_full_unstemmed | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin |
| title_short | Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin |
| title_sort | peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/145366 |
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