Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics
Aim: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors of their regulation in Guerin carcinoma cells in vivo. Materials and Methods: The study was conducted on Wistar rats transplanted with wild-type Guerin carcinoma (Guerin/W...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2016
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| Cite this: | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics / V.F. Chekhun, Y.V. Lozovska, L.A. Naleskina, T.V. Borikun, A.P. Burlaka, I.N. Todor, D.V. Demash, T.M. Yalovenko, T.V. Zadvornyi, A.O. Pavlova, D.M. Storchay, N.Yu. Lukianova // Experimental Oncology. — 2016 — Т. 38, № 4. — С. 283-287. — Бібліогр.: 16 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859768972448104448 |
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| author | Chekhun, V.F. Lozovska, Y.V. Naleskina, L.A. Borikun, T.V. Burlaka, A.P. Todor, I.N. Demash, D.V. Yalovenko, T.M. Zadvornyi, T.V. Pavlova, A.O. Storchay, D.M. Lukianova, N.Yu. |
| author_facet | Chekhun, V.F. Lozovska, Y.V. Naleskina, L.A. Borikun, T.V. Burlaka, A.P. Todor, I.N. Demash, D.V. Yalovenko, T.M. Zadvornyi, T.V. Pavlova, A.O. Storchay, D.M. Lukianova, N.Yu. |
| citation_txt | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics / V.F. Chekhun, Y.V. Lozovska, L.A. Naleskina, T.V. Borikun, A.P. Burlaka, I.N. Todor, D.V. Demash, T.M. Yalovenko, T.V. Zadvornyi, A.O. Pavlova, D.M. Storchay, N.Yu. Lukianova // Experimental Oncology. — 2016 — Т. 38, № 4. — С. 283-287. — Бібліогр.: 16 назв. — англ. |
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| container_title | Experimental Oncology |
| description | Aim: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors of their regulation in Guerin carcinoma cells in vivo. Materials and Methods: The study was conducted on Wistar rats transplanted with wild-type Guerin carcinoma (Guerin/WT) and its strains resistant to cisplatin (Guerin/CP) or doxorubicin (Guerin/Dox). Animals were distributed in 6 groups treated with 5-aza and control animals without treatment. 5-Aza was injected by i.v. route (1 injection in 4 days at a dose of 2 mg/kg starting from the 4th day after tumor transplantation, 4 injections in total). Ferritin levels in blood serum and tumor tissue were measured by ELISA, transferrin and free iron complexes — by low-temperature EPR, miRNA-200b, -133a and -320a levels and promoter methylation — by real-time quantitative reverse transcription polymerase chain reaction. Results: The study has shown that 5-aza treatment caused demethylation of promoter regions of fth1 and tfr1 genes in all studied Guerin carcinoma strains. 5-Aza treatment resulted in a significant decrease of ferritin levels in tumor tissue (by 32.1% in Guerin/WT strain, by 29.8% in Guerin/Dox and by 69.1% in Guerin/CP). These events were accompanied by 3.5-fold and 2-fold increase of free iron complexes levels in tumor tissue of doxorubicin and cisplatin resistant strains, respectively. Also, 5-aza treatment resulted in significantly elevated levels of miR-200b, -133a, 320a expression in tumor tissue. After 5-aza treatment, ferritin levels in blood serum of animals with Guerin/Dox were increased by 23.9%, while in Guerin/Wt and Guerin/CP they were decreased by 17 and 16%, respectively. Conclusion: Alterations of epigenetic regulation upon in vivo treatment with 5-aza change the levels of metal-containing proteins due to DNA demethylation and altered miRNA expression profiles in Guerin carcinoma cells.
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Experimental Oncology ��� �������� ���� ��ecem�er���� �������� ���� ��ecem�er� ��ecem�er� ���
MODIFYING EFFECTS OF 5-AZACYTIDINE ON METAL-CONTAINING
PROTEINS PROFILE IN GUERIN CARCINOMA WITH DIFFERENT
SENSITIVITY TO CYTOSTATICS
V.F. Chekhun, Y.V. Lozovska*, L.A. Naleskina, T.V. Borikun, A.P. Burlaka, I.N. Todor, D.V. Demash,
T.M. Yalovenko, T.V. Zadvornyi, A.O. Pavlova, D.M. Storchay, N.Yu. Lukianova
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
Aim: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors
of their regulation in Guerin carcinoma cells in vivo. Materials and Methods: The study was conducted on Wistar rats transplanted
with wild-type Guerin carcinoma (Guerin/WT) and its strains resistant to cisplatin (Guerin/CP) or doxorubicin (Guerin/Dox).
Animals were distributed in 6 groups treated with 5-aza and control animals without treatment. 5-Aza was injected by i.v. route
(1 injection in 4 days at a dose of 2 mg/kg starting from the 4th day after tumor transplantation, 4 injections in total). Ferritin
levels in blood serum and tumor tissue were measured by ELISA, transferrin and free iron complexes — by low-temperature EPR,
miRNA-200b, -133a and -320a levels and promoter methylation — by real-time quantitative reverse transcription polymerase chain
reaction. Results: The study has shown that 5-aza treatment caused demethylation of promoter regions of fth1 and tfr1 genes in all
studied Guerin carcinoma strains. 5-Aza treatment resulted in a significant decrease of ferritin levels in tumor tissue (by 32.1%
in Guerin/WT strain, by 29.8% in Guerin/Dox and by 69.1% in Guerin/CP). These events were accompanied by 3.5-fold and
2-fold increase of free iron complexes levels in tumor tissue of doxorubicin and cisplatin resistant strains, respectively. Also, 5-aza
treatment resulted in significantly elevated levels of miR-200b, -133a, 320a expression in tumor tissue. After 5-aza treatment,
ferritin levels in blood serum of animals with Guerin/Dox were increased by 23.9%, while in Guerin/Wt and Guerin/CP they were
decreased by 17 and 16%, respectively. Conclusion: Alterations of epigenetic regulation upon in vivo treatment with 5-aza change
the levels of metal-containing proteins due to DNA demethylation and altered miRNA expression profiles in Guerin carcinoma cells.
Key Words: metal-containing proteins, 5-azacytidine, Guerin carcinoma, resistance, methylation, microRNA.
�espite recent advances in understanding tumor
nature� many questions remain unanswered. One
of them is the possi�ility of exogenous correction
of certain tumor features. Solving this pro�lem would
give us radically new approaches in cancer therapy�
which would make tumors more sensitive to cytostatic
drugs and allow to decrease toxic side effects [�].
One of the most promising targets� which could
significantly affect tumor features� is iron meta�olism
�ecause this element is a vital cofactor for many im-
portant cellular processes �energy synthesis� transcrip-
tion� cell division� oxidation reactions etc.� [�]. �uring
last few years it has �een shown that many features
of cancer cells in vitro and tumors in vivo are associ-
ated with different aspects of iron meta�olism. In our
previous works we already descri�ed the changes
in this process� in particular� altered expression of dif-
ferent metal-containing proteins in tumor cells with
different degree of malignancy and different sensitivity
to cisplatin and doxoru�icin [�]. Also� we revealed that
Walker-�5� carcinosarcoma showed gradual changes
in the levels of different metal-containing proteins at dif-
ferent stages of the development of resistance to doxo-
ru�icin. Those changes were o�served �oth in tumor
tissue and in �lood serum of the host organism [4].
Another pro�lem is the search for useful mecha-
nisms to correct mentioned changes in iron meta�o-
lism and the levels of metal-containing proteins [5].
One of the promising approaches is related to epigene-
tic mechanisms of regulation of protein expression�
such as changes in �NA methylation patterns and
miRNA expression profiles [�].
In �9�4 Czechoslovakian scientists synthesized
5-aza-�'deoxycytidine �5-azacytidine� which was
a new potential chemotherapeutic drug [�]. Later
other scientists discovered that mechanisms of cyto-
toxic action of high dose 5-azacytidine �5-aza� were
�ased on its incorporation into �NA and RNA� result-
ing in �reaks in nucleic acid chains and cell apopto-
sis� while low doses of this compound inhi�ited �NA
methyltransferase activity and caused �NA hypo-
methylation. This demethylation activity could result
in altered profile of metal-containing proteins and
changed tumor phenotype [�].
So� the aim of our study was to assess the influence
of the treatment with 5-aza on the profile of metal-
containing proteins and factors of their regulation
in Guerin carcinoma cells in vivo.
MATERIALS AND METHODS
Experimental animals. Studies were carried
out on �� Wistar female rats with su�cutaneously
transplanted Guerin carcinoma strains� wild type �WT�
or resistant to cisplatin �Guerin/CP� and doxoru�icin
Submitted: November 16, 2016.
*Correspondence: E-mail: lozovskaya.2012@mail.ru
Abbreviations used: 5-aza — 5-azacytidine; CP — cisplatin; Dox —
doxorubicin; EPR — electron-paramagnetic resonance; FTH — fer-
ritin heavy chains; MSP — methylation-specific PCR; PCR — poly-
merase chain reaction; qRT-PCR — real-time quantitative reverse
transcription PCR; TFR — transferrin receptor.
Exp Oncol ����
��� 4� �������
ORIGINAL CONTRIBUTION
��4 Experimental Oncology ��� �������� ���� ��ecem�er�
(Guerin/Dox) (2•10� cells per animal�. These tumor
strains were o�tained from National Bank of Cell Lines
and Transplanted Tumors of R.E. Kavetsky Institute
of Experimental Pathology� Oncology and Radio�io-
logy� National Academy of Sciences of Ukraine �Kyiv�
Ukraine�. The use and care of the experimental animals
have �een performed in accordance with the standard
international rules of �iologic ethics and was approved
�y Institutional Animal Care and Use Committee.
Animals were distri�uted in � groups ��� animals
per group�: �� Guerin/WT� control; �� Guerin/WT treated
with 5-aza; �� Guerin/CP� control; 4� Guerin/CP treated
with 5-aza; 5� Guerin/�ox� control; 4� Guerin/�ox
treated with 5-aza.
�� animals from each group received 5-aza �Gede-
on-Richter� Czech Repu�lic� treatment: � injection
each 4 days i/v at a dose of � mg/kg starting from the
4th day after tumor transplantation� 4 injections in total.
All of the studied parameters were measured on the
��th day after tumor transplantation.
ELISA. Ferritin levels in �lood serum and tumor
tissue were measured �y automatic �iochemical and
immunoenzyme analyzer Chem Well �9�� �USA� us-
ing ferritin ELISA kit �Uscer� China� according to the
instruction of the manufacturer. Blood serum samples
were collected and tumor homogenate was prepared
and stored at −�� °C.
Low-temperature electron-paramagnetic
resonance (EPR). Transferrin levels in �lood serum
and tumor tissue were measured using EPR method
using computerized spectrometer R-���� �Russia�
at �� � E�TA sodium salt was used as anticoagulant.
Stripe width was �5�5 G� frequency — 9.�5 GHz�
microwave power — 4� mW� amplitude modulation —
��.� G� frequency modulation — ��� kHz. g-Factor
was calculated using standard formula:
g = hν/βH,
where h is Plank’s constant� ν — frequency�
β — Bohr’s magneton� H — external magnetic field
in resonance.
Apoptosis and necrosis rates were estimated
using classical Annexin V/PI method as previously
descri�ed [5] with the use of BeckmanCoulter EPICS®
XL Flow cytofluorimeter.
Total DNA isolation. Total �NA extraction was
performed with “�NA-Sor� B” �NA Isolation Kit �Am-
plisens� Russia� according to the manual� provided
�y manufacturer.
Methylation-specific polymerase chain reac-
tion (PCR) (MSP). Bisulfite conversion involves the
deamination of unmodified cytosine residues to uracil
under the influence of hydrosulfite ion from water solu-
tion of sodium �isulfite. Such treatment doesn’t affect
5-mC and in further amplification uracils are amplified
as thymines� whereas 5-mC residues get amplified
as cytosines. Bisulfite conversion was performed
using EZ �NA Methylation Gold-Kit �Zymo Research�
USA� according to manufacturer’s protocol. Aliquots
of �isulfite-modified �NA were stored at −�� ˚C and
were used for MSP. MSP was performed using the
standard protocols; primer sequences are availa�le
in Ta�le �. TSH�B and GAP�H were used as reference
control genes [9].
Table 1. Primer sequences, used in MSP
Gene Primer sequence Size of resulting
product
FTH1_M F: 5' — cga ggg ttt tta gcg gtc — 3' 128 bpR: 5' — atc tct tat aac cgc gtc gac — 3'
FTH1_U F: 5' — gtg agg gtt ttt agt ggtt — 3' 128 bpR: 5' — aat ctc tta taa cca cat caa c — 3'
TFR1_M F: 5' — gta gtt ggg att ata ggc gc — 3' 180 bpR: 5' — taa tta cca aac gcg ata act c-3'
TFR1_U F: 5' — tga gta gtt ggg att ata ggt gt — 3' 180 bpR: 5' — taa tta cca aac aca ata act cac — 3'
Notes: FTH1_M — ferritin heavy chains, methylated promoter; FTH1_U —
ferritin heavy chains, unmethylated promoter; TFR1_M — transferrin re-
ceptor 1, methylated promoter; TFR1_U — transferrin receptor 1, unmethy-
lated promoter.
PCR products were analyzed �y agarose gel elec-
trophoresis in �.�% agarose “Low EEO� Type �-A”
�Sigma� USA�. After electrophoresis the results was
visualized �y ethidium �romide� photographed under
UV light and evaluated �y a computer program Total-
La� v�.��.
Total RNA isolation. Total RNA extraction was
performed with “Ri�ozol” RNA Isolation Kit �Ampli-
sens� Russia�. RNA concentration was measured
with Nano�rop ����c Spectrophotometer �Thermo
Scientific� USA�. Purity of isolated RNA was controlled
�y analyzing the ratio of O� at ���/��� nm. RNA was
dissolved in TE �uffer and stored at −�� °С.
Single-stranded c�NA was synthesized from
��� ng of total RNA� using TaqMan® MicroRNA Kit for
reverse transcription.
Real-time quantitative reverse transcription
PCR (qRT-PCR). Reaction mix for reverse transcrip-
tion was prepared according to manufacturer’s proto-
col. Reverse transcription was performed with thermal
cycler “Tertsik” �“�NA Tehnologіya”� Russian Federa-
tion�. qRT-PCR was performed on Applied Biosystems
�9��HT Fast Real-Time PCR System using TaqMan®
MicroRNA primers and manufacturer’s protocol.
Small nucleolar RNA RNU4� was used as an en-
dogenous control for normalization of miRNA expres-
sion. Relative expression of the studied miRNAs was
identified �y comparative Ct method [�]. Experiments
were performed in triplicates for each line� and PCR
was performed three times for each sample. Expres-
sion differences �etween the studied miRNA levels
relative to control was calculated �y the formula:
Fold change = 2−ΔΔCt [8],
where ΔCt �target — control� is equal to the diffe-
rence �etween threshold cycles for miRNA �target� and
the threshold cycle for RNU4� �control�:
ΔCt (target — control) = Ct (target) — Ct(control);
ΔΔCt = ΔCt (experiment) − ΔCt (control).
Statistical analysis. Experimental data were ana-
lyzed using the Student’s t-test. p-Values less than
�.�5 were considered statistically significant. Statisti-
cal analysis of the o�tained data was performed using
the STATISTICA �.� software.
Experimental Oncology ��� �������� ���� ��ecem�er���� �������� ���� ��ecem�er� ��ecem�er� ��5
RESULTS AND DISCUSSION
It is known that demethylating agents� such
as 5-aza� enhance the expression of oncosuppressive
genes [��]. In addition to affecting methylation status
of promoter CpG islands� 5-aza targets are transcrip-
tion factors that are involved in the cell cycle regula-
tion. In particular� c-Myc activates expression of the
transferrin receptor � �TfR��� ferritin and iron-regulated
protein � �IRP��. These proteins are important regula-
tors of iron meta�olism in the cell� and are critical for
proliferation and division of tumor cells [��].
We� resources �https://www.e�i.ac.uk/Tools/
seqstats/em�oss_cpgplot and https://genome.
ucsc.edu� were used to analyze �NA segments
at a distance of �.5 k� in up- and downstream regions
from the transcription start sites. We found regula-
tory CpG-islands in promoter regions of transferrin
receptor � �tfr1� and ferritin heavy chains �fth1� genes�
as well as miRNA-���a� -����� and -���a.
Thus� we decided to investigate the changes
in methylation status of CpG-islands in tfr1 and
fth1 gene promoter areas in Guerin carcinoma strains�
wild-type and resistant to doxoru�icin and cisplatin� af-
ter the session of 5-aza-�ased therapy. We esta�lished
that the development of resistance to doxoru�icin and
cisplatin is accompanied �y hypomethylation of stu-
died CpG areas �Ta�le ��. After 5-aza treatment� levels
of the fth1 and tfr1 promoter methylation in all strains
were almost identical pointing on their demethylation.
The level of promoter methylation of transferrin recep-
tor � gene in the sensitive and resistant to doxoru�icin
strains after 5-aza treatment were identical� while
in cisplatin-resistant strain it was two times higher
�see Ta�le ��.
However� the level of ferritin protein in the tumor
tissue under the influence of 5-aza was reduced
in all strains �Fig. ��� indicating that additional regula-
tory mechanisms are involved in response to genome
methylation changes.
Another epigenetic mechanism� responsi�le for
protein expression regulation is RNA interference�
in which miRNAs play the major role. miRNAs are
small noncoding RNAs� which regulate the expression
of proteins at post-transcriptional level. For example�
the targets of oncosuppressor miRNA-���� and -���a
are mRNA of heavy �FTH�� and light �FTL� chains of fer-
ritin molecule. Together with CpG methylation of these
genes� miRNA-���� and -���a regulate the presence
of ferritin in the cells [��].
It was recently shown �on heme �inding protein
�GCR�� that the changes in iron �alance in cells play
a key role in the maturation of different miRNAs [��].
Iron-�ound transferrin �inds to transferrin recep-
tors � and � �TfR�� �� on a cell surface and enters the
cell via endocytosis. Then divalent metal transpor-
ter � ��MT�� releases the iron into the cytoplasm�
forming the “la�ile iron pool”� which can �e stored
in ferritin or used �y mitochondria to synthesize heme.
Heme is critical for the maturation of pre-miRNA tran-
scripts �ecause it �inds to the processing complex�
which consists of �GCR� and �rosha proteins. An-
other way of iron-dependent regulation is pre-miRNAs
export from the nucleus to the cytoplasm� where�
Table 2. Changes of fth1 and tfr1 promoter methylation levels in Guerin carcinoma cells after 5-aza treatment
Genes
Level of promoter methylation (r. u.)
Guerin/WT Guerin/Dox Guerin/CP
Control + 5-aza Control + 5-aza Control + 5-aza
fth1 7.15 ± 0.24 2.40 ± 0.31* 4.50 ± 0.20# 2.30 ± 0.10*# 5.34 ± 0.30# 2.70 ± 0.60*#
tfr1 1.63 ± 0.01 1.27 ± 0.02* 2.25 ± 0.18# 1.21 ± 0.12*# 3.50 ± 0.10# 2.60 ± 0.11*#
Note: *p < 0.05 compared with control group; #p < 0.05 compared with control WT strain.
Table 3. Changes in expression of miRNA-133a, -200b and -320a in Guerin carcinoma tumor tissue after 5-aza treatment
ΔCt
Guerin carcinoma strain
WT Guerin/Dox Guerin/CP
Control + 5-aza Control + 5-aza Control + 5-aza
miR-133a 0.81 ± 0.01 1.72 ± 0.02* 0.41 ± 0.02# 1.33 ± 0.01*# 0.62 ± 0.01# 1.32 ± 0.01*#
miR-200b 0.70 ± 1.25 4.60 ± 0.61* 0.42 ± 0.05# 0.60 ± 0.01* 0.35 ± 0.25# 9.70 ± 0.15*#
miR-320a 0.29 ± 0.19 0.46 ± 0.08* 0.05 ± 0.04# 0.35 ±0.03* 0.11 ± 0.03# 0.39 ± 0.01*
Note: *p < 0.05 compared with control group; #p < 0.05 compared with control WT strain.
0
5
10
15
20
25
30
Guerin/WT Guerin/Dox Guerin/CP
Fe
rr
iti
n
le
ve
ls
(n
g/
m
g
of
ti
ss
ue
)
Control 5-aza
*
#
*#
#
*#
Fig. 1. Changes of ferritin levels in tumor tissue of Guerin carci-
noma sensitive and resistant to cytostatics after 5-aza treatment.
*p < �.�5 compared with control group; #p < �.�5 compared with
control WT strain
Guerin/WT Guerin/Dox Guerin/CP
Control 5-aza
*
#
*#
#
*#
0
0.5
1
1.5
2
2.5
Fr
ee
ir
on
c
om
pl
ex
es
(r
.u
.)
Fig. 2. Levels of free iron complexes in Guerin carcinoma tissue
after 5-aza treatment. *p < �.�5 compared with control group;
#p < �.�5 compared with control WT strain
��� Experimental Oncology ��� �������� ���� ��ecem�er�
depending on iron levels� C-domain-�inding protein
PCBP� forms active dimers. �imerized PCBP� �inds
�ICER resulting in miRNA maturation. Increase of iron
levels in a cell leads to PCBP� monomerization and its
transition into inactive form [�4].
Measurement of free iron complexes �y EPR
spectroscopy �Fig. �� showed that 5-aza treatment
caused no changes in Guerin/WT cells. On the other
hand� levels of free iron complexes in �oth resistant
strains significantly elevated after 5-aza treatment and
�ecame close to those of the sensitive strain.
Next� we have studied the effects of 5-aza on the
levels of miRNA� which regulate different metal-con-
taining proteins. We found that 5-aza treatment caused
the changes in the levels of miRNA-���a� -���� and
���a in tumor cells of all studied Guerin carcinoma
strains. Changes of the expression in miR-���a after
demethylating agent exposition had unidirectional
nature in sensitive and resistant Guerin carcinoma
cells �Ta�le ��.
We determined that 5-aza treatment resulted in the
increase of miR-���a and -���a levels in all three
strains. Levels of miR-���� also increased in all stu-
died strains� �ut were the most pronounced in Guerin/
CP cells. O�tained data on miRNA-���a and -����
expression were in good accordance with the data
on ferritin levels in tumor tissue� mentioned a�ove. So�
despite hypomethylation of its promoter� fth1 expres-
sion is silenced �y miRNAs on the post-transctriptional
level.
It is known that tfr1 and mdr1 mRNAs are direct
targets of miRNA-���a. Overexpression of �oth pro-
teins is o�served in many cancer cells� resistant to dif-
ferent cytostatics. So� we speculate that the increase
of microRNA-���a expression caused �y 5-aza� could
sensitize malignant cells to anticancer drugs [�5].
�ue to constant �inding of �NMT enzymes� 5-aza
causes apoptotic effect on poorly differentiated stem
cells� �ut the exact mechanism of this influence is still
unclear [��]. We assessed the apoptotic and necrotic
rates of Guerin carcinoma cells� and it was found that
in Guerin/WT the treatment with 5-aza resulted in the
increased num�er of cells in a state of necrosis and
apoptosis — �y ��.� and 49%� respectively� com-
pared to untreated control. In Guerin/CP and Guerin/
�ox cells under the influence of 5-aza� the num�er
of cells in a state of necrosis did not change� and the
num�er of cells in a state of apoptosis increased twice
�Ta�le 4�.
So� we can conclude that 5-aza treatment resulted
in unidirectional changes in all studied Guerin carcino-
ma strains. Particularly� we o�served hypomethylation
of fth1 and tfr1 promoters� and� as a result� elevation
of free iron levels in tumor cells. This caused significant
increase of expression of miRNAs involved in regu-
lation of iron levels� resulting in silencing of ferritin
expression and the loss of resistance to cytostatics.
These changes in tumor �ehavior were confirmed
on organism level. After 5-aza treatment� in �lood
serum of animals with Guerin/�ox ferritin levels were
increased �y ��.9%� while in Guerin/WT and Guerin/
CP — decreased �y �� and ��%� respectively. Also�
after 5-aza treatment we o�served lowering of ceru-
loplasmin activity �see Ta�le 4�. These results indicate
that organism’s iron stores are depleted �in accor-
dance with the data on increase of free iron in tumor
tissue�. �ecrease of ceruloplasmin activity in �lood
serum �as we showed in our previous studies� is a non-
tumor marker of tumor sensitivity to chemotherapy.
So� we can conclude that of 5-aza can �e used
as an exogenous modifier of the content of metal-
containing proteins �oth in tumor and in an organism.
Moreover� 5-aza was capa�le to change some features
of drug-resistant tumors� making them more sensi-
tive to cytostatics �ecause of mdr1 deregulation and
decrease of ceruloplasmin activity in �lood serum.
Our results demonstrated that 5-aza could �e con-
sidered as a promising agent� which can �e used in low
doses in com�ination with convenient chemotherapy
�especially cisplatin and doxoru�icin� to increase
antitumor effects.
ACKNOWLEDGEMENT
The study was performed in terms of “Functional
Genomics and Meta�olomics in System Biology”
Program of Scientific Research of Biochemistry�
Physiology and Molecular Biology �epartment of NAS
of Ukraine.
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Table 4. Indices of metal-containing proteins in blood serum of animals with Guerin carcinoma after 5-aza treatment
Proteins
Guerin carcinoma strain
WT Guerin/Dox Guerin/CP
Control + 5-aza Control + 5-aza Control + 5-aza
Ferritin, ng/ml 13.43 ± 1.19 11.20 ± 1.05# 12.11 ± 1.23 15.90 ± 1.13* 12.50 ± 1.21 10.50 ± 1.10*#
Transferrin, r. u. 0.17 ± 0.02 0.23 ± 0.01# 0.27 ± 0.01 0.30 ± 0.01*# 0.22 ± 0.09 0.34 ± 0.01*
Ceruloplasmin, r. u. 5.12 ± 0.11 4.10 ± 0.23# 2.30 ± 0.01# 1.80 ± 0.03* 2.60 ± 0.01*# 2.0 ± 0.02*#
Note: *p < 0.05 compared with untreated strain; #p < 0.05 compared with untreated sensitive strain.
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| id | nasplib_isofts_kiev_ua-123456789-137697 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-02T06:17:39Z |
| publishDate | 2016 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Chekhun, V.F. Lozovska, Y.V. Naleskina, L.A. Borikun, T.V. Burlaka, A.P. Todor, I.N. Demash, D.V. Yalovenko, T.M. Zadvornyi, T.V. Pavlova, A.O. Storchay, D.M. Lukianova, N.Yu. 2018-06-17T15:11:58Z 2018-06-17T15:11:58Z 2016 Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics / V.F. Chekhun, Y.V. Lozovska, L.A. Naleskina, T.V. Borikun, A.P. Burlaka, I.N. Todor, D.V. Demash, T.M. Yalovenko, T.V. Zadvornyi, A.O. Pavlova, D.M. Storchay, N.Yu. Lukianova // Experimental Oncology. — 2016 — Т. 38, № 4. — С. 283-287. — Бібліогр.: 16 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/137697 Aim: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors of their regulation in Guerin carcinoma cells in vivo. Materials and Methods: The study was conducted on Wistar rats transplanted with wild-type Guerin carcinoma (Guerin/WT) and its strains resistant to cisplatin (Guerin/CP) or doxorubicin (Guerin/Dox). Animals were distributed in 6 groups treated with 5-aza and control animals without treatment. 5-Aza was injected by i.v. route (1 injection in 4 days at a dose of 2 mg/kg starting from the 4th day after tumor transplantation, 4 injections in total). Ferritin levels in blood serum and tumor tissue were measured by ELISA, transferrin and free iron complexes — by low-temperature EPR, miRNA-200b, -133a and -320a levels and promoter methylation — by real-time quantitative reverse transcription polymerase chain reaction. Results: The study has shown that 5-aza treatment caused demethylation of promoter regions of fth1 and tfr1 genes in all studied Guerin carcinoma strains. 5-Aza treatment resulted in a significant decrease of ferritin levels in tumor tissue (by 32.1% in Guerin/WT strain, by 29.8% in Guerin/Dox and by 69.1% in Guerin/CP). These events were accompanied by 3.5-fold and 2-fold increase of free iron complexes levels in tumor tissue of doxorubicin and cisplatin resistant strains, respectively. Also, 5-aza treatment resulted in significantly elevated levels of miR-200b, -133a, 320a expression in tumor tissue. After 5-aza treatment, ferritin levels in blood serum of animals with Guerin/Dox were increased by 23.9%, while in Guerin/Wt and Guerin/CP they were decreased by 17 and 16%, respectively. Conclusion: Alterations of epigenetic regulation upon in vivo treatment with 5-aza change the levels of metal-containing proteins due to DNA demethylation and altered miRNA expression profiles in Guerin carcinoma cells. The study was performed in terms of “Functional Genomics and Metabolomics in System Biology” Program of Scientific Research of Biochemistry, Physiology and Molecular Biology Department of NAS of Ukraine. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics Article published earlier |
| spellingShingle | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics Chekhun, V.F. Lozovska, Y.V. Naleskina, L.A. Borikun, T.V. Burlaka, A.P. Todor, I.N. Demash, D.V. Yalovenko, T.M. Zadvornyi, T.V. Pavlova, A.O. Storchay, D.M. Lukianova, N.Yu. Original contributions |
| title | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics |
| title_full | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics |
| title_fullStr | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics |
| title_full_unstemmed | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics |
| title_short | Modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics |
| title_sort | modifying effects of 5-azacytidine on metal-containing proteins profile in guerin carcinoma with different sensitivity to cytostatics |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/137697 |
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