The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer
Aim: The aim of the study was to determine the phosphorylated c-Jun content and reduced and oxidized glutathione (GSH/GSSG) ratio in gastric mucosa cells during the process of gastric cancer development in rats. Materials and Methods: Gastric carcinogenesis was initiated in 80 white male rats by 10...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Zitieren: | The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer / M.O. Tymoshenko, O.O. Kravchenko, O.V. Sokur, L.I. Ostapchenko // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 191-195. — Бібліогр.: 27 назв. — англ. |
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irk-123456789-1453652019-01-21T01:23:04Z The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer Tymoshenko, M.O. Kravchenko, O.O. Sokur, O.V. Ostapchenko, L.I. Original contributions Aim: The aim of the study was to determine the phosphorylated c-Jun content and reduced and oxidized glutathione (GSH/GSSG) ratio in gastric mucosa cells during the process of gastric cancer development in rats. Materials and Methods: Gastric carcinogenesis was initiated in 80 white male rats by 10-week replacement of drinking water with 0.01% solution of N-methyl-N´-nitro-N-nitrosoguanidine, at the same time they were redefined on diet containing 5% NaCl. Then the animals were fed with standard vivarium diet till the end of 24th week. The gastric mucosa cells were examined at the end of 4th, 6th, 8th, 10th, 12th, 18th, and 24th weeks. Sandwich ELISA method was used to determine the content of phospho-c-Jun. The contents of GSH and GSSG were analyzed by spectrofluorymetric method with the use of orthophthalic aldehyde. Results: At the end of 6th, 8th, 10th weeks of MNNG and NaCl treatment the gastric mucosa cells were characterized by 4-, 6.3-, 1.9-fold higher content of phospho-c-Jun compared to the control, respectively, and 12, 18 and 24 weeks there was registered a stable increase of phospho-c-Jun content on the average at 3.6-fold compared to control values. Starting from 6th week of gastric cancer development an average decrease of GSH/GSSG was 3.4-fold compared with the control. Conclusion: During gastric carcinogenesis there was registered the decrease of GSH/GSSG ratio and increased level of phosphorylated c-Jun what points on MAP-kinase cascade activation in prooxidant conditions. Key Words: c-Jun, reduce glutathione (GSH), oxidized glutathione (GSSG), gastric cancer. 2014 Article The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer / M.O. Tymoshenko, O.O. Kravchenko, O.V. Sokur, L.I. Ostapchenko // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 191-195. — Бібліогр.: 27 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/145365 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Tymoshenko, M.O. Kravchenko, O.O. Sokur, O.V. Ostapchenko, L.I. The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer Experimental Oncology |
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Aim: The aim of the study was to determine the phosphorylated c-Jun content and reduced and oxidized glutathione (GSH/GSSG) ratio in gastric mucosa cells during the process of gastric cancer development in rats. Materials and Methods: Gastric carcinogenesis was initiated in 80 white male rats by 10-week replacement of drinking water with 0.01% solution of N-methyl-N´-nitro-N-nitrosoguanidine, at the same time they were redefined on diet containing 5% NaCl. Then the animals were fed with standard vivarium diet till the end of 24th week. The gastric mucosa cells were examined at the end of 4th, 6th, 8th, 10th, 12th, 18th, and 24th weeks. Sandwich ELISA method was used to determine the content of phospho-c-Jun. The contents of GSH and GSSG were analyzed by spectrofluorymetric method with the use of orthophthalic aldehyde. Results: At the end of 6th, 8th, 10th weeks of MNNG and NaCl treatment the gastric mucosa cells were characterized by 4-, 6.3-, 1.9-fold higher content of phospho-c-Jun compared to the control, respectively, and 12, 18 and 24 weeks there was registered a stable increase of phospho-c-Jun content on the average at 3.6-fold compared to control values. Starting from 6th week of gastric cancer development an average decrease of GSH/GSSG was 3.4-fold compared with the control. Conclusion: During gastric carcinogenesis there was registered the decrease of GSH/GSSG ratio and increased level of phosphorylated c-Jun what points on MAP-kinase cascade activation in prooxidant conditions. Key Words: c-Jun, reduce glutathione (GSH), oxidized glutathione (GSSG), gastric cancer. |
| format |
Article |
| author |
Tymoshenko, M.O. Kravchenko, O.O. Sokur, O.V. Ostapchenko, L.I. |
| author_facet |
Tymoshenko, M.O. Kravchenko, O.O. Sokur, O.V. Ostapchenko, L.I. |
| author_sort |
Tymoshenko, M.O. |
| title |
The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer |
| title_short |
The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer |
| title_full |
The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer |
| title_fullStr |
The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer |
| title_full_unstemmed |
The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer |
| title_sort |
phospho-c-jun (ser63) content in mucosa cells under experimental development of gastric cancer |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2014 |
| topic_facet |
Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/145365 |
| citation_txt |
The phospho-c-JUN (Ser63) content in mucosa cells under experimental development of gastric cancer / M.O. Tymoshenko, O.O. Kravchenko, O.V. Sokur, L.I. Ostapchenko // Experimental Oncology. — 2014. — Т. 36, № 3. — С. 191-195. — Бібліогр.: 27 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T21:30:21Z |
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2025-07-10T21:30:21Z |
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| fulltext |
Experimental Oncology 36, 191–195, 2014 (September) 191
THE PHOSPHO-c-JUN (SER63) CONTENT IN MUCOSA CELLS
UNDER EXPERIMENTAL DEVELOPMENT OF GASTRIC CANCER
M.O. Tymoshenko*, O.O. Kravchenko, O.V. Sokur, L.I. Ostapchenko
Educational and Scientific Centre “Institute of Biology”,
Taras Shevchenko National University, Kyiv 01601, Ukraine
Aim: The aim of the study was to determine the phosphorylated c-Jun content and reduced and oxidized glutathione (GSH/GSSG)
ratio in gastric mucosa cells during the process of gastric cancer development in rats. Materials and Methods: Gastric carcinoge-
nesis was initiated in 80 white male rats by 10-week replacement of drinking water with 0.01% solution of N-methyl-N´-nitro-N-
nitrosoguanidine, at the same time they were redefined on diet containing 5% NaCl. Then the animals were fed with standard vi-
varium diet till the end of 24th week. The gastric mucosa cells were examined at the end of 4th, 6th, 8th, 10th, 12th, 18th, and 24th weeks.
Sandwich ELISA method was used to determine the content of phospho-c-Jun. The contents of GSH and GSSG were analyzed
by spectrofluorymetric method with the use of orthophthalic aldehyde. Results: At the end of 6th, 8th, 10th weeks of MNNG and NaCl
treatment the gastric mucosa cells were characterized by 4-, 6.3-, 1.9-fold higher content of phospho-c-Jun compared to the con-
trol, respectively, and 12, 18 and 24 weeks there was registered a stable increase of phospho-c-Jun content on the average at 3.6-fold
compared to control values. Starting from 6th week of gastric cancer development an average decrease of GSH/GSSG was 3.4-fold
compared with the control. Conclusion: During gastric carcinogenesis there was registered the decrease of GSH/GSSG ratio and
increased level of phosphorylated c-Jun what points on MAP-kinase cascade activation in prooxidant conditions.
Key Words: c-Jun, reduce glutathione (GSH), oxidized glutathione (GSSG), gastric cancer.
Gastric cancer is the second leading cause of can-
cer deaths worldwide. Several factors are thought
to play a role in gastric carcinogenesis, including
diet, exogenous chemicals, intragastric synthesis
of carcinogens, genetic factors, infectious agents and
pathological conditions in the stomach (such as gastri-
tis) [1]. Smoked foods rich in nitrates, a high-salt diet,
and Helicobacter pylori infection seem to be major
environmental inducers of gastric cancer [2]. However,
it remains unclear which molecular signals actually
initiate the program of cell transformation.
Oxidative stress, nevertheless, appears to be an im-
portant factor in the induction of cancer [3, 4]. Reactive
oxygen species (ROS) can directly produce single-
or double-stranded DNA breaks, purine, pyrimidine,
or deoxyribose modifications, and DNA cross-links.
Persistent DNA damages can result in either arrest or in-
duction of transcription, induction of signal transduction
pathways, replication errors, and genomic instability,
which are all seen in carcinogenesis [5]. Cellular oxidative
stress can modify intercellular communication, protein
kinase activity, membrane structure and function, and
gene expression, and may result in modulation of cell
growth [3].
At the same time a vast number of cellular processes
is affected by the redox state, in which glutathione has
a pivotal role. The most prevalent non-protein thiol
in mammalian cells and the most abundant low mole-
cular-weight peptide present in eukaryotic cells [6] —
glutathione, exists in reduced (GSH) and disulfide/oxi-
dized (GSSG) forms (GSH being predominant). The re-
dox regulation affects numerous signalling pathways
through protein phosphorylation and induces selectively
a number of genes. Molecules activated in this way
include transcription factor activator protein-1 (AP-1),
responsible for the expression of a number of genes in-
cluding many cytokines, TGF-β and collagenase, AP-2,
c-Jun N-terminal kinase (JNK), protein kinase C and
tyrosine kinase [7]. The overactivity of transcription fac-
tors that are activated directly by specific phosphoryla-
tion or that became activated following phosphorylation
of other cellular proteins is an event in carcinogenesis
that increases disregulation. Redox cycling of cysteinyl
residues is one of se veral oxidant-dependent mecha-
nisms that regulate the activity of many transcription
factors, such as AP-1 [8] and particularly c-Jun,
a potent transcriptional regulator is involved in cellular
proliferation control [9, 10].
The resident nuclear protein c-Jun is one of hun-
dreds of nuclear proteins that are being targets of serine
proteinkinase cascades that are initiated in the cyto-
plasm and lead to phosphorylation and activation of this
protein [11]. c-Jun is a major component of AP-1 and
forms homodimers, or heterodimers with other Jun,
Fos or ATF proteins [12]. Although the repertoire
of AP-1 complexes varies between different cell types,
c-Jun is a dominant component in many cells [13].
It is believed, that c-Jun is a critical promoter of cellular
proliferation and dysregulated expression and activation
of its oncogene are frequently observed in many tumor
types [14] including gastric cancer [2]. But there are
a lot of evidences that c-Jun/AP-1 activation has been
implicated in various, often opposing cellular respons-
es. Although there is a consi derable evidence that c-Jun
activation can be a positive step in the events leading
Submitted: May 21, 2014.
*Correspondence: E-mail: maria.bulavka@gmail.com
Fax: +38 (044) 521-35-98
Abbreviations used: AP-1 — activator protein-1; GSH — reduce gluta-
thione; GSSG — oxidized glutathione; JNK — c-Jun N-terminal kinase;
MAP kinase — mitogen-activated protein kinase; MNNG — N-methyl-
N’-nitro-N-nitrosoguanidine; ROS — reactive oxygen species.
Exp Oncol 2014
36, 3, 191–195
192 Experimental Oncology 36, 191–195, 2014 (September)
a cell towards apoptosis, there are also many reports
stating the opposite: that under certain circumstances
c-Jun can inhibit apoptosis and promote proliferation
or differentiation. It is clear that the effects of c-Jun
on cellular responses depend strongly on the cell type
and the context of other regulatory influences that the
cell is receiving [15].
The aim of this study was to determine the changes
in phosphorylated c-Jun content and the level of GSH/
GSSG ratio in gastric mucosa cells during the process
of experimental gastric carcinogenesis using the
N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) medi-
ated gastric cancer model in rats.
MATERIALS AND METHODS
The experiments were conducted using white male
rats (n = 80) with the initial weight of 100 ± 20 g.
Gastric carcinogenesis was initiated by 10-week re-
placement of drinking water by 0.01% solution of car-
cinogen MNNG while the rats were given the feed,
containing sodium chloride (5% NaCl of dry weight).
Then the animals were fed with standard vivarium diet
till the end of the 24th week [16, 17]. The control group
of animals was fed with the standard diet during the all
experimental period. The samplings of experimental
material were taken at the end of 4th, 6th, 8th, 10th, 12th,
18th, and 24th weeks. The gastric cancer development
was verified visually and histologically.
The gastric mucosa cells were isolated by the me-
thod, based on the enzymatic disaggregation of cells
using pronase [18]. This method presupposed turning
the mucosa of the isolated stomachs outward, deliga-
tion, filling them with the pronase solution (1 mg/ml),
incubating (30 min, 37 °C) at intense stirring in the me-
dium, saturated with 95% O2 and 5% CO2, and harvest-
ing the disintegrated cells. To cytosol obtaining the
isolated cells were homogenized on ice in teflon Potter-
El vehjem homogenizer. 0.01 M formic acid was added
to the homogenate (1:1) for precipitation of proteins
[19] and the mixture was centrifuged at 20 000 g for
15 min (4 °C) at Sigma centrifuge (USA). Both GSSG
and GSH contents were measured in the supernatant.
The GSH content was registered using ortho-
phthalic aldehyde, the reaction of the latter with GSH
resulted in the formation of highly fluorescent products.
The final mixture for the analysis contained 100 μl of the
supernatant, diluted tenfold with 0.1 M phosphate
buffer with 5 mM EDTA (pH 8.0), 1.8 ml of phosphate-
EDTA buffer and 100 μl of ortho-phthalic aldehyde
(1 mg/ml in methanol). The fluorescence intensity was
measured at 420 nm with the activation of 350 nm af-
ter 15 min incubation at room temperature. The GSH
concentration was expressed as nmol/mg protein.
The GSSG content was determined similarly to fluo-
rometric method of GSH estimation with previous
incubation of supernatant with 0.04 М N-ethylmaleimid
and following substitution 0.1 M phosphate buffer with
5 mM EDTA (pH 8.0) for 0.1 N NaOH (рН = 12) [19].
The phospho-c-Jun content was revealed in gastric
mucosa cell lysate prepared with the use of Cell Lysis
Buffer (Cell Signaling Technology, USA). The phospho-
c-Jun content was measured by Sandwich ELISA
method using the assay kit PathScan Phospho-c-Jun
(Ser63) (Cell Signaling Technology, USA) and repre-
sented in conventional unit of absorbancy (λ = 450 nm)
on mg of the protein.
The protein concentration was registered by Brad-
ford’s method [20].
The experimental data were processed by the com-
mon methods of the variance analysis with 7 repeats.
The reliability of discrepancies between two samplings
was determined using Student’s criterion. The results
are presented in the values of the arithmetic mean and
standard error mean, M ± S.E.m [21].
The investigations were carried out in accordance
with the main requirements to keeping and working
with laboratory animals and to the rules of the Eu-
ropean Convention for the Protection of Vertebrate
Animals used for Experimental and other Scientific
Purposes (Strasbourg, 1986) as well as with the ethic
norms specified in the Ukrainian legislation.
RESULTS AND DISCUSSION
Histological investigations were conducted for es-
tablishment of gastric cancer progression and estima-
tion of gastric mucosa state under the gastric cancer
development. The inflammation signs, the extension
and plethora of vessels, the desquamation of epithe-
lial cells, and the increase in the production of mucus
at the end of the 6th week of experimental gastric carci-
nogenesis were shown [22]. After the effect of MNNG
for 8 weeks the gastric mucosa de monstrated the atro-
phic changes. Metaplastic modifications, sites with
hyperplastic impairments, and some cells with atypia
were observed after 10 weeks of MNNG treatment.
Some animals had adenomas and one adenocarci-
noma at the end of the 12th week of the experiment.
Such neoplasms were visualized in stomach pyloric
region in 70% rats at the end of 18th week of the ex-
perimental gastric carcinogenesis.
The role of c-Jun in cellular transformation has been
defined previously in rodent and avian model systems.
Deregulated expression of c-Jun can lead to malignant
transformation of immortalized rat fibroblasts while
transformation of primary rat embryo cells required co-
expression of an activated c-Ha-ras. In chicken embryo
fibroblasts, c-Jun can induce cellular transformation
by itself. Stable expression of a trans-activation sup-
pressing deletion mutant of c-jun in malignant mouse
epidermal cell lines inhibited tumor formation in nude
mice. In human cancer, the role of c-jun/AP-1 is less
clear. Increased constitutive levels of c-jun and c-fos
mRNA and AP-1 levels have been reported for drug-
resistant cells (such as etoposide resistant human
leukemia K562 cells) compared to drug-sensitive pa-
rental lines. In the human breast adenocarcinoma cell
line MCF-7, mitogenic stimulation by insulin or other
insulin-like growth factors leads to increased c-jun
or c-fos expression and AP-1 activity. In a study of non-
Experimental Oncology 36, 191–195, 2014 (September) 193
small cell lung primary and metastatic tumors, c-Jun
is found to be overexpressed in 31% of the cases [23].
However, c-Jun role and even its activated form
phospho-c-Jun in gastric tumorigenesis remain un-
known. The submitted studies showed the control
reference of phospho-c-Jun content at the end of 4th
week of MNNG and NaCl consumption (Fig. 1) and
morpho-histological changes at the same timepoint
weren’t observed. The gastric mucosa cells were
characterized by increased content of phospho-c-Jun
at 4.0- and 6.3-fold over the control at the end of 6th
and 8th weeks, respectively, of the MNNG and NaCl
treatment. Also it was established that the MNNG
and NaCl treatment for 10 weeks caused 1.9-fold
increase in phospho-c-Jun content compared
to the control. At the terminal stages (12th, 18th and
24th weeks) of the gastric carcinogenesis study, that
were characterized by adenocarcinomas in pyloric
region, there was a stable increase of phospho-c-Jun
content on the average at 3.6-fold in comparison with
reference values.
The odtained experimental data of high phospho-
c-Jun content in gastric mucosa cells are in agreement
with the results of the immunohistochemical analysis
that revealed activation of JNK in human gastric cancer
tissue. Also mice lacking JNK1, a major JNK isozyme,
exhibited a marked decrease in gastric carcinogenesis
induced by N-methyl-N-nitrosourea relative to their
wild-type counterparts [2].
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Control 4th 6th 8th 10th 12th 18th 24th
Ph
os
ph
o-
c-
Ju
n.
C
U/
m
g
pr
ot
ei
n
week
*
*
*
* *
*
Fig. 1. The phospho-c-Jun content in gastric mucosa cells un-
der gastric cancer development. *The difference is significant
as compared to appropriate control (p < 0.05)
c-Jun is unique in its ability to positively regulate
cell proliferation through repression of tumor sup-
pressor gene expression and function, and induction
of cyclin D1 transcription [9]. c-Jun, a specific target
of JNK [13], is phosphorylated at serines 63 and
73 and threonines 91 and 93 within the transactivation
domain [12]. Phosphorylation is the most important
regulation of c-Jun and influences the activity of a pro-
tein by affecting the DNA-binding, stability, ability
to interact with other proteins, and transactivation po-
tential [12]. This phosphorylation event was suggested
to stimulate c-Jun transcriptional activity, leading to the
autoregulatory induction of c-jun, and, consequently,
other genes [24]. JNK-mediated phosphorylation
of Ser63/73 was demonstrated to inhibit both ubiq-
uitination and degradation of c-Jun, leading to accu-
mulation of the protein and increased transcriptional
activity. Phosphorylation of c-Jun stimulates transcrip-
tional activity by recruiting co-activator CREB-binding
protein (CBP). CBP binds to the N-terminal activation
domain of c-Jun and connects the phosphorylated ac-
tivation domain to the basal transcriptional machinery.
Also c-Jun is phosphorylated at threonines
231 and 239 and serines 243 and 249 located proximal
to the DNA-binding domain in the C-terminus. These
sites are dephosphorylated during c-Jun activation
and represent inhibitory phosphate groups. Activa-
tion of c-Jun requires phosphorylation of serines
63 and 73, as well as dephosphorylation of at least
one of the C-terminal sites [12]. JNKs are also sub-
ject to inactivation. This is mediated by a specialized
group of phosphatases, called MAP Kinase Phospha-
tases [13].
Thus, the growth of the phosphorylated form
of c-Jun content from 6th to 24th weeks of gastric
cancer development was caused by its high expres-
sion, amplification of JNK-mediated phosphorylation
or inactivation of the phoshpatases. Like other Protein
Tyrosine Phosphatases, MAP Kinase Phosphatases
contain a highly reactive cysteine that mediates their
inactivation, offering a way to regulate MAP kinase
activity in response to production of ROS or exposure
to thiol-reactive compounds [13]. Besides, it has
been reported that N-nitroso compounds, which are
important gastric carcinogens and potent inducers
of cellular stress, leading to chromosomal aberrations,
point mutations, cell death, and DNA damage, induce
a specific cellular response program, which includes
the activation of JNKs [25]. ROS-dependent redox
cycling of cysteinyl thiols is also critical for the es-
tablishment of the protein-protein and protein-DNA
interactions that determine many aspects of a signal
transduction pathway. There is growing evidence that
the reversible formation of mixed disulfides between
GSH and low-pKa cysteinyl residues of proteins
( S-glutathionylation) is an important mechanism for
dynamic, posttranslational regulation of a variety
of regulatory, structural, and metabolic proteins, and
for the regulation of signaling and metabolic pathways
in intact cell systems [26]. The c-Jun protein belongs
to such molecules whose activity depends on the glu-
tathione redox state.
The major marker of intracellular redox state
is the ratio of GSH to glutathione disulfide so it has
been suggested to regulate the activation of redox-
sensitive transcriptional factors [27].
The 4th week of MNNG-stimulated carcinoge nesis
was characterised by increased GSH/GSSG ratio
(Table). Such change was the consequence of more
than twofold increased GSH content compared
with the control values while the GSSG remained
unchanged (Fig. 2). So the first carcinogenesis pe-
riod was accompanied by compensatory activation
of antioxidant cell state and there was no change
registered in phospho-c-Jun content. The GSH con-
tent remained elevated but the GSSG was increased
194 Experimental Oncology 36, 191–195, 2014 (September)
(more than 7-fold) at 6th week of MNNG treatment.
A decrease of GSH/GSSG on the average at 3.4-fold
compared with reference value was revealed since 6th
week of gastric cancer development, which proved
the prooxidant state in the gastric mucosa cells. These
indicators were caused by marked growth of GSSG
content at 6th, 8th, 10th, 12th weeks and reduction of GSH
at 8th, 12th, 18th and 24th weeks. The minimum values
of GSH/GSSG ratio and severe prooxidant state were
observed during 10th and 12th weeks of experimental
gastrocarcinogenesis. These timepoints of experiment
were characterised by the increase of phospho-c-Jun
content but the peak of this parameter was observed
at 6th and 8th weeks, when the GSH/GSSG ratio exceed
the refence value at 3.8- and 3.5-fold respectively.
Thus, there was the crucial value of intracellular redox
state when phospho-c-Jun quantity extremely grew
and then despite of amplification of prooxidant state
the phospho-c-Jun content remained consistently
high (from 12th to 24th week of gastrocarcinogenesis).
Table. The GSH/GSSG ratio in gastric mucosa cells under gastric cancer
development
Control Observation period, week
4th 6th 8th 10th 12th 18th 24th
16.1±2.6 24.4±4.3* 4.2±0.* 4.6±0.2* 1.4±0.1* 1.1±0.2* 8.2±2.0* 8.7±1.3*
Note: * the difference is significant as compared to appropriate control
(p < 0.05).
0
5
10
15
20
25
30
Control 4th 6th 8th 10th 12th 18th 24th
week
0
20
40
60
80
100
120
Control 4th 6th 8th 10th 12th 18th 24th
week
G
SH
, n
m
ol
/m
g
pr
ot
ei
n
G
SS
G
, n
m
ol
/m
g
pr
ot
ei
n
*
*
*
* * *
*
*
*
*
a
b
Fig. 2. The GSH (a) and GSSG (b) contents in gastric mucosa
cells under gastric cancer development. *The difference is sig-
nificant as compared to appropriate control (p < 0.05)
Such marked increase in the level of phospho-c-Jun
at the 8th week of MNNG-stimulated gastrocarcinogene-
sis accompanied with a moderate decrease of GSH and
increase of GSSG is a probable consequence of redox
disturbances in cell cytoplasma under ROS. It is known
that ROS activate MAPK, but the response of distinct
regulatory chains in different cell types is unequal. There
are several mechanisms of MAPK activation under ROS:
activation of redox-dependent ASK-1 as a consequence
of thioredoxin oxidation and dissociation of JNK from
the complex with GST Pi. Also inactivation of JNK-spe-
cific phosphatase by H2O2 may couse permanent JNK
activation [28]. Similar activation by ROS determined
in our study might lead to the growth of the phosphory-
lated c-Jun amount.
But if the level of oxidative stress is excessive, there
will be a direct impact of ROS on Cys residues of DNA-
binding proteins. The oxidation of SH-groups in these
residues results to reversible or irreversible (according
to the ratio ROS/antioxidants) inactivation of AP-1.
Since the promoter region of c-Jun gene has several
zones of binding of AP-1, phosphorylation and sub-
sequent activation of factor c-Jun leads to the induc-
tion of its own gene [28]. Thus the high level of GSSG
with the phospho-c-Jun amount decrease at the 10th
week may indicate to acute prooxidant conditions
that affected genome and led to an inhibition of c-Jun
expression. In spite of this the level of phospho-c-Jun
remained above the control values but less than at the
previous 8th week.
The 18th and 24th weeks were characterized by the shift
of redox-station to reduced state with the recovery
of c-Jun expression and following its phosphorylation.
Experimental evidences support an important role
of ROS in the cancer process. Oxidative stress can
occur through overproduction of reactive oxygen and
nitrogen species with the shift of GSH/GSSG ratio
to prooxidative state. Disruption of this ratio is involved
in several cellular reactions involved in signal transduc-
tion and cell cycle regulation under conditions of oxi-
dative stress. The unregulated or prolonged produc-
tion of cellular oxidants has been linked to mutation,
as well as modification of gene expression. But the ef-
fects of ROS and oxidative stress within cells appear
to be cell specific and dependent upon the form as well
as the intercellular concentration of ROS [3]. In par-
ticular, MAPK/AP-1 signal transduction pathway, in-
cluding c-Jun activation by phosphorylation, is known
to be activated by ROS, and leads to the transcription
of genes involved in cell growth regulatory pathways.
The cellular concentration of ROS appears to influ-
ence the selective activation of this transcription factor
and therefore may help explain the observation that
either cell death or cell proliferation may result from
exposure to ROS. A common effect of AP-1 activation
is an increased cell proliferation and the several lines
of evidence have demonstrated that c-fos and c-jun
are positive regulators of cell proliferation [9].
The acute oxidative injury may produce selective
cell death and a compensatory increase in cell pro-
liferation. This stimulus may result in the formation
of newly initiated preneoplastic cells and/or enhance
the selective clonal expansion of latent initiated pre-
neoplastic cells. Similarly, sublethal acute oxidative
injury may produce unrepaired DNA damage and result
in the formation of new mutations and, potentially, new
initiated cells. In contrast, sustained chronic oxidative
Experimental Oncology 36, 191–195, 2014 (September) 195
injury may lead to a nonlethal modification of normal
cellular growth control mechanisms [3].
In conclusion, the decrease of GSH/GSSG ratio and
simultaneously increased level of active phosphory lated
form of c-Jun during long period (from 6th to 24th week
of histological established progression of gastric cancer)
point on possible MAP kinase cascade activation in prooxi-
dant conditions.
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