1208onc
Redox state of gastric cancer: Relationship with tumor progression
A.P. Burlaka1, I.I. Ganusevich1, M.R. Gafurov2, S.M. Lukin1, E.P. Sidorik1
1
R.E. Kavetsky Institute of Experimental Pathology, Oncology and
Radiobiology NAS of Ukraine, Kyiv
2
Institute of Physics of Kazan Federal University, Kazan, Russia,
Summary. High levels of reactive oxygen species generation rate permanently
alter vital functions of cells, acting as the main messenger modulator series of
intracellular signaling cascades that lead to cancer progression. It is known that
superoxide radicals by acting on the intracellular signaling pathways responsible
for the regulation of extracellular matrix degradation, in particular - the
regulation of the activity of gelatinases (matrix metalloproteinase-2 and -9 (MMP2 and MMP-9)). Purpose: To investigate the relationship levels generate
superoxide radicals, NO, concentrations of the active forms of MMP-2 and -9 in
tumor and adjacent tissue, the relationship of these parameters with
clinicopathological parameters patients with GC - stage of the disease categories
N and M. Methods: Electrons spin resonance (ESR) in liquid nitrogen
temperature, zymography in polyacrylamide gels. Results: Indicators of activity of
MMP-2 and MMP-9 in tissue GC correlated with the generation rate of superoxide
and nitric oxide levels (r = 0.48 ÷ 0.67, p <0.05). Furthermore, all of the above
figures are in direct positive depending on the disease stage (r = 0.59 ÷ 0.71, p
<0.05). MMP-2 and -9 in a tumor, as well as the speed of generation of superoxide
levels and NO, positively correlates with regional metastases (r = 0.45 and 0.37,
respectively; p <0.05), but in contrast to (Figure 3), the activity of MMP-2 is
inversely related to the level of distant metastasis GC (r = 0.58; p <0.05), the same
trend is observed for MMP-9. Conclusions: The tumor cells are characterized
reprogrammed metabolism of mitochondria, a high level of cellular hypoxia,
defective functioning of the redox system and unregulated levels of molecules,
forming an aggressive tumor phenotype. Evaluation of the disease in patients with
GC by determining the generation rate of superoxide radicals, NO levels and
activity of MMP-2 and -9, give additional opportunity to monitor the effectiveness
of anticancer therapy, adjust treatment regimens and improve the survival rates of
patients.
Keywords: superoxide radicals, matrix metalloproteinases, gastric cancer,
metastasis, diagnostics.
INTRODUCTION
Superoxide radicals can induce a wide spectrum of cell responses that
depend on their rate of generation, the exposure duration, location and nature of
the metabolites, which can be formed [1]. Low level of the superoxide radicals
excites mitogenic effect and initiate cells poliferation and cells survival rate. At
the same time, the intermidate levels produce short-time or total stop cells cycle
and induction of cells differentiation [2]. At high speed to generate superoxide
radicals can easily react with membrane lipids, causing a change in membrane
permeability, oxidation-induced genome instability functioning, due to its
oxidative modification and oxidative modification of proteins, resulting in a
change or loss of catalytic activity of enzymes or altered sensitivity proteins to
proteolytic degradation. In this case, superoxide radicals initiate apoptosis or
necrosis [3, 4]. On the other hand, when the rate of generation of oxygen radicals
such that irreversibly changed cells activity, they can act as a primary messenger,
modulating some intracellular signaling cascades that lead to the progression of
cancer.
Indeed,
it
was
mitogenproteinkinase
shown
(MARK),
that
superoxide-dependent
phosphatidylinositol-3-kinase
activation
(PI3K/Akt),
phospholipase C-G1 (PLCg1), protein kinase C, nuclear transcription factor - κ B
(NF-κB) and Jak/Stat leads to cancer progression [2 - 6].
Yes, there is some evidence that superoxide radicals by influencing
intracellular signaling pathways engaged in the regulation of extracellular matrix
degradation, in particular - the regulation of the activity of proteolytic enzymes
[7, 8].
Among the proteases, which activated by superoxide radicals, matrix MMP2 and -9 and also A and B play an important role. They are ferments from the Zndepended endopeptidase family and they are produced by tumor cells and
adjacent tissues, endothelium and immunocompetent cells and their precursors.
They also realize extracellular matrix degradation in process of tumor invasion
and metastasis [13, 14]. In that way, in modern investigation it is given that
extracellular matrix destruction suppression, stabilization and fortification are
critical characteristic of the malignant progress [13]. It is also given that
expression levels and/or gelatinases activities are connected with general survival
rate and metastasis level of cancer carrier, especially, gastric cancer (GC) carrier
[15 - 18].
In our days, among general nosological forms of malignant tumors GC takes
the second place in the world. The indexes of 2011 year shows, that GC takes
fourth place between men and seventh between woman, the second place between
man and third – between woman in structure of mortality in Ukraine [19]. It is
known that the therapy effectiveness and survival indexes can be improved by
using the prognosis of oncological clinical course and suitable correction of the
therapy plan. In particular, when GC using a number of prognostic indicators,
among which the most common indicators of TNM [20], in particular, the
categories N and M. However, data on the complex relationship of these
prognostic indicators based on levels generate superoxide radicals, NO and MMP
activity, very few and poorly documented.
The purpose of the work is to examine the levels of interdependence
generate superoxide radicals, NO, concentration of active forms of MMP-2 and -9
in tumor and adjacent tissue, the relationship of these indicators with clinical and
pathological parameters of patients with GC - stage of disease, N and M
categories.
MATERIALS AND METHODS
The examples of tumor and adjoining tissue (which was taken at a distance
of 1.5 and 2.5 cm from the tumor) of 58 GC patients (T1-4, N0-2, M0-1, G1 - G4),
who were treated at Kiev municipal clinical oncological center. All the patients
were informed that the examples of their tumors, which was removed on
operation, would be used for research purposes and they gave a permission. The
average age of the patients was 58 ± 10.5 year. There were 33 men and 15
women, 8 patients of I stage of disease, 12 - II, 17 - III and 11 – IV. The 24
(50%) patients had a metastasis in lymph nodes, 6 (12.5%) had a metastasis
distance. Patients were divided according to the degree of malignancy of tumors
as follows: G1 – 4, G2 – 9, G3 – 22 and G4 – 13 patients. All the patients had a
diagnosis, stage of disease and metastasis presence, which was verified in
concordance with evidence-based medicine (in the appropriate clinical and
instrumental examinations, morphologically).
The tissue examples for analysis were frozen in special press-form in liquid
nitrogen in 77K, they were extracted just before the investigation, the ESRspectrums were registered on computerized radiospectrometer ESR RE-1307 in
temperature of liquid nitrogen. The concentration of active and latent forms
MMP-2 and -9 were measured by using a zymography in polyacrylamide gel
(with adding some gelatin as a substrate) based on protein SDS-electrophoresis
[21].
RESULTS AND DISCUSSION
Figure 1 shows the ESR spectra of gastric tissue samples, taken at different
distance from the tumor. The closer to the tumor, the brighter its significant impact
on healthy tissue. The cells of gastric tissue at a distance of 5 cm from the tumor
are characteristic of healthy tissue kit ESR signals (1). At a distance of 2.5 cm from
the tumor (2) the intensity of the ESR signal with g = 1.94, which characterizes the
activity of FeS-protein N-2 in NAD∙H-ubiquinonoxidoreduktases electron
Figure1. Electrontransport chain functioning changes of the gastric tissue
cells examples, which was taken on the different distances from the tumor.
ESR spectrums:
1 – stomach wall tissues (5 cm from the tumor);
2 – stomach wall tissues (2.5 cm from the tumor);
3 – stomach wall tissues (1.5 cm from the tumor).
transport complex of the respiratory chain of mitochondria is reduced by 3.5 times
in compared with healthy tissue (1). Also, increased levels of ubisemiquinon (ESR
signal with g = 2.00) and significantly (6.7 times) increases the level of complexes
NO with FeS-protein N-type. Not registered ESR signal with g = 2.42 and g =
2.25, characterizing the cytochrome P-450 detoxifying system cells. Surrounding
tissue is characterized by a loss of mitochondrial cell functional activity related to a
combination of oxidation phosphorylation. ESR spectra of tissue cells similar ESR
spectra fabric GC stage I disease (Figure 2). The level of speed generation of
superoxide radicals by mitochondria of cells in tissue samples 2 and 3 depends on
the distance from the tumor and determined in the range of 1.9 ± 0.23 nmol/g wet
tissue∙min. to 5.0 ± 0.38 nmol/g wet tissue∙min. (rate of 0.25 ± 0.18 nmol/g wet
tissue∙min.). The level of NO in these tissues is also dependent on the distance
from the tumor and is in the range of 2.0 ± 0.15 nmol/g wet tissue to 6.0 ± 0.49
nmol/g wet tissue at a rate of 1.5 ± 0.15 nmol/g wet tissue.
There are GC tissue ESR-spectrums on different stages of disease,
appropriating the different invasion levels and tumor metastasis. Analyzing ESRspectrum in Figure.2 we found the following. There is not ESR-signal with g =
2.15 and 2.42 in the tumor tissue, which characterizing functioning in cells detox
catalytic system cycle Р-450 cytochromes activity. Was revealed a intension
decrease of the signal g = 1.94 depended on the stage of disease, characterizing by
FeS–protein
N-2
in
НАД∙Н-ubiquinonoxidoreduktases
respiratory
chain
electrontransport complex of mitochondria. It brings to increase of ESR-signal
with g = 2.03 intention. This signal characterizes NO and N-type FeS-protein
complexes generation, which is increasing in III stage GC in regard to I and II
stages and forming triplet ESR-signal with g = 2.007. On the latest stages of
disease the ESR-signal with g = 1.94 intention, the breath and phosphorylation
coupling level reduce, and glycolysis and cells hypoxia levels rises. At the same
time, the velocity of superoxide radicals generation by GC cells mitochondrias
rises with the tumor progress stage increase ranging from 0.65 ± 0.19 nmol/g wet
tissue∙min. to 2.5 ± 0.26 nmol/g wet tissue∙min. (rate of 0.25 ± 0.18 nmol/g wet
tissue∙min.) and the level of NO increases from 2.1 ± 0.22 nmol/g wet tissue in the
tumors of patients with stage I to 3.5 ± 0.31 nmol/g wet tissue in tumors of patients
with stage IV disease (norm 1.5 ± 0.15 nmol/g wet tissue).
Figure 2. Mitochondria electrontransport chain functioning and detox GC cells
system depended on the states of disease changes.
ESR-spectrums:
1 – stomach tissues (5 cm from the tumor)
2 – GC in I stage
3 – GC in II stage
4 – GC in III stage
∙
Figure 3. The velocity of superoxide radical (O2 ) generation and NO level with
GC metastasis.
There is also shown that the tumors of the patients with metastasis (N0-2М1-2
categories) characterized by superoxide radical generation velocity indexes, which
are in 1.3 times higher (p < 0.05) than the same indexes in the tumors of patients
with metastasis in regional lymph nodes, but without an individual metastasis
(N1-2М0 categories), and in 2 times higher than the patients without metastasis
(N0М0 categories). The same trend is observed for NO, the level of which fabric
GC with distant metastases and metastases to regional lymph nodes only
significantly higher than levels in tumor tissues of patients without metastases (p
<0.05). It was established that the rate of generation of superoxide radicals and
nitric oxide levels positively correlate with regional (r = 0.63 and 0.69,
respectively; p <0.05) and distant metastases (r = 0.72 and 0.43, respectively; p
<0.05) GC.
We investigated MMP-2 and -9 activity level in GC tissues depended on the
stage of disease and metastasis level, we also revealed the correlation between this
indexes and superoxide radical generation velocity and NO levels in the tumor.
Indicators concentrations of active MMP-2 forms varied in the range 0,1 ÷
52.8 µg/g tissue, the average is 8.2 ± 4.9 µg/g tissue. Indicators of MMP-9 ranged
from 0.05 to 28.8 µg/g tissue, mean is 8.3 ± 5.9 µg/g tissue. The activity of both
gelatinases did not differ considerably and/or authentically depending on sex, age
and category T of the patients. But in the tumors with G2 differentiation level
MMP-2 concentration were in 6 times higher than the same index of GC with G1
differentiation level (p<0.05). MMP-2 activity considerably rises from II stage
(p<0.05), amount to the maximum values on II (p < 0.05) and saves the high levels
on the IV stage of disease (Figure.4). MMP-9 activity rises regularly from I to IV
stages in 1.7 times, but this difference is not authentically (p > 0.05).
Figure 4. MMP-2 activity level in the GC depend on the stage of disease.
On the whole there is shown that MMP-2 and MMP-9 activity indexes in
GC tissues correlate with superoxide radical generation velocity an NO levels (r =
0.48 ÷ 0.67, p < 0.05). Furthermore, all this indexes positively depend on the stage
of disease (r = 0.59 ÷ 0.71, p < 0.05).
The connection between gelatinases activity level in the GC and his
metastasis was analyzed. In spite of gelatinases active form concentration in the
tumor in N1-2 category being a few higher than the same indexes in N0 category,
the authentically difference between them was not revealed (p > 0.05) (Figure.5).
Thus, the concentration of the active form of MMP-2 is in inverse proportion to the
category M, i.e. patients without distant metastasis activity of MMP-2 is almost 2
times higher than that in patients with metastases (p <0.05). MMP-2 latent form
maximum activation in the tumors testifies that the extracellular matrix complex
destruction in that stage of the tumor progress raise, when the individual metastasis
did not revealed clinically, in other words, there was, probably, their forming
and/or the tumor cells dissemination. The results are correlated with data on the
ability of the tumor through the relevant signaling pathways in advance (pre-)
create a favorable microenvironment in the so-called premetastatic niches [22, 23].
Figure 5. Levels of gelatinases activity in tumor and GC metastasis.
Thus, the activity of MMP-2 and -9 in the tumor, as well as the rate of
generation of superoxide radicals and levels of NO, positively correlate with
regional metastasis (r = 0.45 and 0.37, respectively; p <0.05), but unlike them
(Figure 3), the activity of MMP-2 is in inverse proportion to the level of distant
metastases GC (r = 0.58; p <0.05), the same trend is typical for MMP-9. That is,
the presence of distant metastasis for tumors characterized by high rates of speed to
generate superoxide radicals and nitric oxide levels, but low values of activity of
MMP-2 and -9. ''Down-regulation'' MMP can be explained by the fact that at very
high speeds the generation of superoxide radicals and increased oxidative
processes that can be observed at the terminal stage of cancer with secondary
metastasis, is the disintegration of signaling pathways and regulatory relations at
the level of the genome and postsynthetic stage of formation of MMP activity.
Results of the our investigation shows that the tumor cells are characterizing
by reprogrammed mitochondria metabolism, the high level of cells hypoxia, defect
redox-system functioning and unregulated molecular level, which forms the
aggressive phenotype of the tumor. It becomes apparent in the unregulated
proliferation, migration and the tumor cells intrusion, correlate with the disease and
can be used in diagnostics like a helping objective test. Thus, the assessment of the
disease in patients with GC by determination of the redox status of the tumor, and
among them the speed to generate superoxide radicals, NO levels and activity of
MMP-2 and -9, give more opportunity to monitor the effectiveness of anticancer
therapy, adjust treatment regimens and improve survival rates of patients.
CONCLUSION
1. MMP-2 and -9 activity indexes in the tumor tissue correlate with superoxide
radical generation velocity and NO levels (r = 0.48 ÷ 0.67, p < 0.05).
Furthermore, all this indexes positively depend on the stage of disease (r =
0.59 ÷ 0.71, p < 0.05).
2. MMP-2 and -9 activity in the tumor, like a superoxide radical generation
velocity and NO levels correlate with regional metastasis positively(r = 0.45
и 0.37, respectively, p < 0.05) , but unlike of them MMP-2 and -9 activity
depends of distant metastases GC level reversibly (r = 0.58; p < 0.05).;
3. The tumor cells are characterized by reprogrammed mitochondria
metabolism, the high level of cells hypoxia, defect redox-system functioning
and unregulated molecular level, which forms the aggressive phenotype of
the tumor. It becomes apparent in the unregulated proliferation, migration
and the tumor cells intrusion, correlated with the disease and can be used in
diagnostics like a helping objective test.
4. Assessment of the disease in patients with GC by determination of the redox
status of the tumor, and among them the generation rate of superoxide
radicals, NO levels and activity of MMP-2 and -9, give an additional
opportunity to monitor the effectiveness of anticancer therapy, adjust
treatment
regimens
and
improve
survival
rates
of
patients.
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