Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀
Aim: To study the redox-dependent mechanism of antiradical, antitumor and antimetastatic action of L-arginine hydrochloride (L-Arg) and coenzyme Q₁₀ (CoQ₁₀) in vivo. Materials and Methods: The study was performed on С57Вl mice with transplanted Lewis lung carcinoma treated by intraperitoneal injecti...
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nasplib_isofts_kiev_ua-123456789-1379882025-02-09T12:55:12Z Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ Burlaka, A.P. Ganusevich, I.I. Golotiuk, V.V. Vovk, A.V. Lukin, S.M. Original contributions Aim: To study the redox-dependent mechanism of antiradical, antitumor and antimetastatic action of L-arginine hydrochloride (L-Arg) and coenzyme Q₁₀ (CoQ₁₀) in vivo. Materials and Methods: The study was performed on С57Вl mice with transplanted Lewis lung carcinoma treated by intraperitoneal injections of L-Arg at low or high doses (60 and 360 mg/kg body weight), CoQ₁₀ (0.2 and 1.2 mg/kg body weight) or their combinations. Electron paramagnetic resonance was applied for analysis of mitochondrial electron transport chain, СoQ₁₀ levels, free iron (FI), the level of NO, and the rate of superoxide radical generation. The activity of matrix metalloproteinase (MMP)-2 and -9 in tumor tissue was determined by zymography method in polyacrylamide gel. Results: Administration of L-Arg at high doses caused an inhibition of tumor growth by 48 ± 8.0%, increase of superoxide radical generation rate and NO levels to a value of 1.23 ± 0.14 аnd 2.26 ± 0.31 nm/g tissue · min, and decreased activity of MMP-2 and -9 (3.55 ± 0.8 and 4.8 ± 1.0 r.u., respectively). Treatment with L-Arg at low doses stimulated tumor growth and increased the levels of MMP-2 and -9 activities (8.44 ± 2.7 and 9.8 ± 3.1 r.u., respectively). Administration of СoQ₁₀ at high doses significantly decreased superoxide radical generation rate to the values of 0.44 ± 0.09 nm/g tissue · min, levels of free iron and NO, and caused tumor growth inhibition by 54 ± 11.3%. The combined use of L-Arg and СoQ₁₀ at high doses caused tumor growth inhibition by 51 ± 7.4% compared to Lewis lung carcinoma-bearing untreated animals (р < 0.05). Conclusions: Administration of L-Arg and СoQ₁₀ caused the dose-dependent effect on the rate of generation of superoxide radicals, level of ubisemyquinone, complexes NOFeS-proteins, levels of FI and NO. L-Arg at low doses positively modulated MMP-9 activity that promoted tumor progression. Upon combined use of L-Arg and СoQ₁₀, superoxide radicals and NO form the redox state that causes decrease of MMP-2, -9 activities with consequent inhibition of tumor invasion and metastasis. 2016 Article Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ / A.P. Burlaka, I.І. Ganusevich, V.V. Golotiuk, A.V. Vovk, S.М. Lukin // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 31–35. — Бібліогр.: 26 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/137988 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Burlaka, A.P. Ganusevich, I.I. Golotiuk, V.V. Vovk, A.V. Lukin, S.M. Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ Experimental Oncology |
| description |
Aim: To study the redox-dependent mechanism of antiradical, antitumor and antimetastatic action of L-arginine hydrochloride (L-Arg) and coenzyme Q₁₀ (CoQ₁₀) in vivo. Materials and Methods: The study was performed on С57Вl mice with transplanted Lewis lung carcinoma treated by intraperitoneal injections of L-Arg at low or high doses (60 and 360 mg/kg body weight), CoQ₁₀ (0.2 and 1.2 mg/kg body weight) or their combinations. Electron paramagnetic resonance was applied for analysis of mitochondrial electron transport chain, СoQ₁₀ levels, free iron (FI), the level of NO, and the rate of superoxide radical generation. The activity of matrix metalloproteinase (MMP)-2 and -9 in tumor tissue was determined by zymography method in polyacrylamide gel. Results: Administration of L-Arg at high doses caused an inhibition of tumor growth by 48 ± 8.0%, increase of superoxide radical generation rate and NO levels to a value of 1.23 ± 0.14 аnd 2.26 ± 0.31 nm/g tissue · min, and decreased activity of MMP-2 and -9 (3.55 ± 0.8 and 4.8 ± 1.0 r.u., respectively). Treatment with L-Arg at low doses stimulated tumor growth and increased the levels of MMP-2 and -9 activities (8.44 ± 2.7 and 9.8 ± 3.1 r.u., respectively). Administration of СoQ₁₀ at high doses significantly decreased superoxide radical generation rate to the values of 0.44 ± 0.09 nm/g tissue · min, levels of free iron and NO, and caused tumor growth inhibition by 54 ± 11.3%. The combined use of L-Arg and СoQ₁₀ at high doses caused tumor growth inhibition by 51 ± 7.4% compared to Lewis lung carcinoma-bearing untreated animals (р < 0.05). Conclusions: Administration of L-Arg and СoQ₁₀ caused the dose-dependent effect on the rate of generation of superoxide radicals, level of ubisemyquinone, complexes NOFeS-proteins, levels of FI and NO. L-Arg at low doses positively modulated MMP-9 activity that promoted tumor progression. Upon combined use of L-Arg and СoQ₁₀, superoxide radicals and NO form the redox state that causes decrease of MMP-2, -9 activities with consequent inhibition of tumor invasion and metastasis. |
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Article |
| author |
Burlaka, A.P. Ganusevich, I.I. Golotiuk, V.V. Vovk, A.V. Lukin, S.M. |
| author_facet |
Burlaka, A.P. Ganusevich, I.I. Golotiuk, V.V. Vovk, A.V. Lukin, S.M. |
| author_sort |
Burlaka, A.P. |
| title |
Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ |
| title_short |
Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ |
| title_full |
Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ |
| title_fullStr |
Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ |
| title_full_unstemmed |
Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ |
| title_sort |
superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of l-arginine hydrochloride and coenzyme q₁₀ |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2016 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/137988 |
| citation_txt |
Superoxide- and no-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q₁₀ / A.P. Burlaka, I.І. Ganusevich, V.V. Golotiuk, A.V. Vovk, S.М. Lukin // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 31–35. — Бібліогр.: 26 назв. — англ. |
| series |
Experimental Oncology |
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2025-11-26T00:19:06Z |
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2025-11-26T00:19:06Z |
| _version_ |
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| fulltext |
Experimental Oncology 38, 31–35, 2016 (March) 31
Superoxide- and no-dependent mechaniSmS
of antitumor and antimetaStatic effect of L-arginine
hydrochLoride and coenzyme Q10
A.P. Burlaka1,*, I.І. Ganusevich1, V.V. Golotiuk2, A.V. Vovk1, S.М. Lukin1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
2Ivano-Frankivsk National Medical University, Ivano-Frankivsk 76018, Ukraine
Aim: To study the redox-dependent mechanism of antiradical, antitumor and antimetastatic action of L-arginine hydrochloride
(L-Arg) and coenzyme Q10 (CoQ10) in vivo. Materials and Methods: The study was performed on С57Вl mice with transplanted
Lewis lung carcinoma treated by intraperitoneal injections of L-Arg at low or high doses (60 and 360 mg/kg body weight),
CoQ10 (0.2 and 1.2 mg/kg body weight) or their combinations. Electron paramagnetic resonance was applied for analysis of mito-
chondrial electron transport chain, СoQ10 levels, free iron (FI), the level of NO, and the rate of superoxide radical generation. The
activity of matrix metalloproteinase (MMP)-2 and -9 in tumor tissue was determined by zymography method in polyacrylamide
gel. Results: Administration of L-Arg at high doses caused an inhibition of tumor growth by 48 ± 8.0%, increase of superoxide
radical generation rate and NO levels to a value of 1.23 ± 0.14 аnd 2.26 ± 0.31 nm/g tissue · min, and decreased activity
of MMP-2 and -9 (3.55 ± 0.8 and 4.8 ± 1.0 r.u., respectively). Treatment with L-Arg at low doses stimulated tumor growth and
increased the levels of MMP-2 and -9 activities (8.44 ± 2.7 and 9.8 ± 3.1 r.u., respectively). Administration of СoQ10 at high
doses significantly decreased superoxide radical generation rate to the values of 0.44 ± 0.09 nm/g tissue · min, levels of free iron
and NO, and caused tumor growth inhibition by 54 ± 11.3%. The combined use of L-Arg and СoQ10 at high doses caused tumor
growth inhibition by 51 ± 7.4% compared to Lewis lung carcinoma-bearing untreated animals (р < 0.05). Conclusions: Administra-
tion of L-Arg and СoQ10 caused the dose-dependent effect on the rate of generation of superoxide radicals, level of ubisemyquinone,
complexes NOFeS-proteins, levels of FI and NO. L-Arg at low doses positively modulated MMP-9 activity that promoted tumor
progression. Upon combined use of L-Arg and СoQ10, superoxide radicals and NO form the redox state that causes decrease
of MMP-2, -9 activities with consequent inhibition of tumor invasion and metastasis.
Key Words: L-arginine, coenzyme Q10, superoxide radical, nitric oxide, matrix metalloproteinase, Lewis lung carcinoma.
One of the approaches to search for innovative anti-
cancer agents with fundamentally different mechanism
of action is the use of compounds — donors of nitric oxide
(NO), which can modulate the levels of NO and other
regulators of transport rate of electrons in the electron
transport chain in mitochondria. Normally functioning
mitochondrial metabolism is a process of fine-adjustable
dynamic balance of thousands of anabolic and catabolic
reactions and cell signaling systems. Since NO is the key
signaling molecule in tumor-induced angiogenesis, its
antitumor activity can be manifested at least due to its
ability to regulate angiogenic pathway [1–5].
Coenzyme Q10 (CoQ10) plays the key role in the
mechanism of functioning of the power gene rating
cell system and in the formation of redox potential
in mitochondria through the regulation of superoxide
radical generation to control gene transcription and
cellular and extracellular signaling factors. Physiologi-
cal levels of CoQ10 in plasma are 0.68–1.1 µmol/l and
are supported mainly by endogenous synthesis and
to a lesser extent by pro ducts of exogenous origin. Ac-
tivity of CoQ10 decreases with aging as well as in various
pathologies, including diabetes, chronic heart failure,
myocardial infarction and cancer [6]. There is a growing
amount of evidence in favor of the use of food additives
with CoQ10 for prevention and treatment of pathological
conditions, NO donors and other regulators of electron
transport in mitochondria, including CoQ10 are explored
as anticancer agents [7, 8]. However, mechanisms
of their action have been studied insufficiently. The
matrix metalloproteinases (MMPs), which play a role
in tumor invasion and metastasis [9, 10], are redox-
dependent enzymes [11–13], therefore, they may
be involved in the realization of anti-tumor effects
of compounds-regulators of electron transport [14, 15].
The aim of the study was to investigate the redox-
dependent mechanism of anti-radical, antitumor and
antimetastatic action of L-Arg and CoQ10 in vivo.
materiaLS and methodS
The study was performed on 84 С57Вl male
mice weighting 22.4 ± 1.12 g bred in animal facility
of R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology of the NAS of Ukraine. The
animals were kept on a standard diet. Animal study
protocols and operation procedures were carried out
in accordance with the main requirements to keeping
and working with laboratory animals and to the rules
of local Bioethics Committee.
Lewis lung carcinoma (LLC) was used as a tu-
mor model. LLC cells (5 • 105 cells per animal) were
transplanted subcutaneously [16]. The compounds
(L-Arginine hydrocloride (L-Arg) (Sigma, USA),
Submitted: February 16, 2016.
*Correspodence: E-mail: apburlaka@gmail.com
Abbreviations used: СоQ10 — coenzyme Q10; EPR — electron para-
magnetic resonance; FI — free iron; L-Arg — L-arginine hydrochlo-
ride; LLC — Lewis lung carcinoma; MMP — matrix metalloprotein-
ase; NO — nitric oxide; О2
· — superoxide radical.
Exp Oncol 2016
38, 1, 31–35
32 Experimental Oncology 38, 31–35, 2016 (March)
CoQ10 (Sigma, USA)) were administered intraperi-
toneally daily for 10 days at low or high doses: L-
Arg — 60 and 360 mg/kg body weight; CoQ10 — 0.2 and
1.2 mg/kg body weight. The animals were distri buted
into 8 experimental groups; І — intact animals (n = 10);
ІІ — tumor control (n = 11); ІІІ and ІV — animals with
tumors treated with high (n = 12) and low (n = 10)
doses of L-Arg, respectively; V та VІ — animals with
tumors treated with high (n = 10) and low (n = 10) doses
of СоQ10, respectively; VІІ and VІІІ — animals with tu-
mors treated with high (n = 10) and low (n = 10) doses
of L-Arg and СоQ10, respectively. Animals of group II re-
ceived daily intraperitoneal injections of physiological
saline at corresponding volume for 10 days and served
as a positive control.
The research of the electron transport chain in mito-
chondria, СoQ10 levels in mitochondria and free iron (FI)
in tumor cells was performed by electron paramagnetic
resonance (EPR) method with compu terized spectrom-
eter RE-1307 using the technology of low-temperature
stabilization of biological material (77 K). The rate of su-
peroxide radical generation by mitochondria of cells was
determined by EPR method using a spin trap (2,2,6,6,-tet-
rametyl-4-oxypiperidine) at the room temperature
in a special paramagnetic pure quartz cuvette. The level
of NO in the tumor tissue was investigated by EPR using
the Spin Traps techno logy (spin trap — diethyl dithiocar-
bamates (Sigma)) at the temperature of 77 K [13].
The activity of MMP-2 and -9 in tumor tissue was
determined by zymography method in polyacrylamide
gel (with addition of gelatin as a substrate) based
on SDS-electrophoresis of proteins [17].
The tumor volume was determined by the formula 1:
V = (π/6) · D1 · D2 · D3, (1)
where V — tumor volume (mm3); D1, D2, D3 — tu-
mor length, width, and height.
The number of metastases was counted, and their
volume — by the formula 1.
The antitumor activity of the studied compounds
was evaluated by tumor growth inhibition:
G% = (vc−ve)/mk · 100%, (2)
where G% — percentage of tumor growth inhibition
by volume; vc — average tumor volume in the control;
ve — average tumor volume in the study group.
Statistical analysis of the obtained data was
performed with the use of Origin 7.0 program and
Student’s t-criterion. The data were presented as the
mean with the standard deviation (M ± SD). The differ-
ences were considered significant at p < 0.05.
reSuLtS and diScuSSion
The results of EPR study of LLC are shown in Fig. 1.
In tumor cells of group II (EPR spectrum 1) there
were registered the low levels of free radical form
of СoQ10 — ubisemiquinone (g = 2.00) — 0.15 ±
0.02 r.u., Fe-S-protein N-2 (g = 1.94) responsible for
coupling of oxidation and phosphorylation — 0.14 ±
0.01 r.u. Also, there was noted an accumulation of high
levels of FI (g = 2.20–2.40) — 0.86 ± 0.08 r.u., the level
of NOFe-S-protein complexes was 0.18 ± 0.02 r.u.
Magnetic field
g-factor2.40
1
2
3
4
2.20 2.00
2.007 1.94
fig. 1. Effect of L-Arg and CoQ10 at high doses on mitochon-
drial electron transport chain in LLC cells: 1 — tumor of mice
in group II; 2 — tumor of mice in group III; 3 — tumor of mice
in group V; 4 — tumor of mice in group VII
In animals injected with L-Arg at high doses,
nanomolar levels of NO in the tumor tissue were
generated, what caused 2.3-fold decrease of FI le vel
compared to that in group II (р < 0.05). Under these
conditions, there were recorded significant increase
of the levels of ubisemyquinone up to 0.21 ± 0.02 r.u.,
and NOFeS-proteins complexes in the mitochondrial
electron transport chain (up to 0.48 ± 0.02 r.u.), while
activity of FeS-protein N-2 remained unaltered (0.14 ±
0.013 r.u.).
Administration of СoQ10 resulted in the decreased
FI content in tumor tissue of animals (0.21 ± 0.02 r.u.)
compared to groups II and III (р < 0.05), and increased
formation of NO-FeS-proteins complex (0.58 ±
0.04 r.u.). The level of ubisemyquinone in the I com-
plex of mitochondrial respiratory chain was 0.22 ±
0.01 r.u. what was significantly lower compared with
group II; p < 0.05); activity of FeS-protein N-2 was
0.11 ± 0.02 r.u. CoQ10, providing mitochondria with
ubiquinone (NADH-ubiquinone-oxidoreductase and
succinate dehydrogenase complexes), promotes the
restoration of oxidative phosphorylation via its eleva-
tion in the electron transport chain.
The combined therapy with L-Arg and СoQ10 resulted
in the decrease of FI level — 0.28 ± 0.03 r.u. compared
to group II (p < 0.05), ubisemyquinone (0.15 ± 0.01 r.u.)
and NOFeS-proteins complexes (0.38 ± 0.04 r.u.)
Experimental Oncology 38, 31–35, 2016 (March) 33
compared to groups ІІІ and IV (p < 0.05), increase
of FeS-protein N-2 activity — 0.14 ± 0.02 (compared
to group IV; p < 0.05).
Fig. 2 shows the data on the rate of generation
of superoxide radicals by LLC cells. In the tumors
of group II the value was 0.75 ± 0.13 nm/g tissue • min,
while in the lungs of intact animals it was equal to 0.19 ±
0.03 nm/g tissue • min. In the case of administration
of high doses of L-Arg, the rate of superoxide radical
generation was 1.23 ± 0.14 nm/g tissue • min, that
was significantly higher than corresponding values
of group II (р < 0.05). Therapy with L-Arg at low doses
did not influence on this index (0.78 ± 0.22 nm/g tis-
sue • min). The use of high doses of СoQ10 caused
nearly 2-fold decrease in levels of superoxide radi-
cals (0.44 ± 0.09 nm/g tissue • min) in mitochondria
of tumor cells compared to group II (p < 0.05). After
administration of low СoQ10 doses this index did not
change (0.62 ± 0.08 nm/g tissue • min). Combined
administration of L-Arg and СoQ10 at low doses reduced
the superoxide radical generation rate in mitochondria
of tumor cells (0.32 ± 0.04 nm/g tissue • min) com-
pared with groups II, III, IV, VI (p < 0.05), where as high
doses of this compounds reduced this index to 0.65 ±
0.09 nm/g tissue • min what was lower than in group III
(p < 0.05). Thus, the regulators of the electron transport
rate in mitochondrial respiratory chain decreased the
rate of superoxide radical generation to corresponding
levels in a dose dependent manner.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2 3 4 5 6 7 8
Groups of animals
О
2• -
ge
ne
ra
tio
n
ra
te
, n
m
/g
ti
ss
ue
•
m
in
fig. 2. The rate of superoxide generation (O2
·) in the mitochon-
dria of cells: 1 — lungs of intact mice; 2 — tumor control; 3 —
tumors of mice that administered high doses of L-Arg; 4 — low
doses of L-Arg; 5 — tumors of mice treated with high doses
of CoQ10; 6 — low doses of CoQ10; 7 — tumors of mice treated with
high doses of L-Arg and СoQ10 and 8 — L-Arg and СoQ10 at low
doses. p < 0.05 compared to tumor control (group II)
Fig. 3 shows the results of the research of NO con-
tent in tumor cells. In the tumor bearing animals this
rate was 1.95 ± 0.21 nm/g tissue • min, which ex-
ceeded the rate in the group of intact animals — 1.45 ±
0.18 nm/g tissue • min (p < 0.05). NO level in the tumor
after administration of high doses of L-Arg increased
up to 2.26 ± 0.31 nm/g tissue • min versus 1.58 ±
0.19 nm/g tissue • min in the case of low dose L-Arg
treatment, and the difference between these indexes
is statistically significant. The levels of NO is the cases
of administration of high and low doses of СoQ10 were
1.51 ± 0.12 and 1.62 ± 0.23 nm/g tissue • min, respec-
tively. Combined therapy with L-Arg and СoQ10 at high
doses resulted in the increased level of NO up to the
values of 2.2 ± 0.3 nm/g tissue • min compared
with groups IV, V, VI (р < 0.05), whereas in the case
of low dose combined treatment this index (1.47 ±
0.09 nm/g tissue • min) was significantly lower than
in groups II, III and VII (р < 0.05). Therefore, the NO do-
nor and its combined use with СoQ10 at high doses
cause the significant increase of NO levels in tumor
cells, which is a positive factor in the antitumor and
antimetastatic therapy.
0
0.5
1
1.5
2
2.5
3
1 2 3 4 5 6 7 8
Groups of animals
NO
le
ve
l,
nm
/g
ti
ss
ue
•
m
in
fig. 3. The level of NO in the mitochondria of cells: 1 — lungs
of intact mice; 2 — tumor control; 3 — tumors of mice treated
with high doses of L-Arg; 4 — low doses of L-Arg; 5 — tumors
of mice treated with high doses of CoQ10; 6 — low doses of CoQ10;
7 — tumors of mice treated with high doses of L-Arg and СoQ10;
8 — L-Arg and СoQ10 at low doses. p < 0.05 compared with tumor
control (group II)
Administration of L-Arg at high doses causes
an increase in NO level, augments mitochondrial dam-
age, intensificates generation of superoxide radicals,
increases the rate of oxidation-induced mutations
of mtDNA, and induces tumor cell apoptosis [8].
NO at low doses promotes tumor invasiveness, angio-
genesis and immune tolerance [1, 7]. Administration
of СoQ10 at high doses to animals with LLC significantly
reduced NO levels in tumor cells compared to control
animals (see Fig. 3). Combined use of L-Arg and
СoQ10 at low doses caused an increase of NO levels
in tumor cells compared to group II (р < 0.05).
Summarizing the obtained information, one may
suppose that high doses of regulators of the electron
transport rate based on NO and СoQ10 donors cause
correlating effects at the level of mitochondria. These
compounds influence regulation of superoxide radical
generation rate and NO level, and subsequently modu-
late MMP-2 and -9 activities. Activity of MMP-2 and
-9 in tumor tissue of the control animals greatly
exceeded these indices in the lungs of the intact ani-
mals and correlated with the values of the superoxide
radicals generation rate by tumor cells in these groups
of animals (Fig. 2, 4, 5). In tumors of animals injected
with high doses of L-Arg a significant (almost 2 times;
p < 0.05) decrease in activity of both gelatinases was
observed compared with the control group, which
could be a consequence of the ultrahigh rate of super-
oxide radical generation. On the contrary, low doses
of L-Arg caused an insignificant increase in activity
of MMP-2 in tumor tissue compared to the control
that was due to the regulatory impact of the increase
34 Experimental Oncology 38, 31–35, 2016 (March)
in rate of superoxide radical generation in tumor cells
of this group of animals (see Fig. 2, 4, 5). Treatment
with both high and low doses of СоQ10 led to the sig-
nificant superoxide regulated decrease (1.7–2.3 times;
p < 0.05) in activity of both gelatinases, compared
to group II (see Fig. 2, 4, 5).
0
2
4
6
8
10
12
1 2 3 4 5 6 7 8
Groups of animals
M
M
P-
2
ac
tiv
ity
, r
. u
.
fig. 4. Activity of MMP-2 (r.u.): 1 — lungs of intact mice;
2 — tumor control; 3 — tumors of mice treated with high doses
of L-Arg; 4 — low doses of L-Arg; 5 — tumors of mice treated
with high doses of CoQ10; 6 — low doses of CoQ10; 7 — tumors
of mice treated with high doses of L-Arg and СoQ10; 8 — L-Arg and
СoQ10 at low doses. p < 0.05 compared to tumor control (group II)
0
2
4
6
8
10
12
14
1 2 3 4 5 6 7 8
Groups of animals
M
M
P-
9
ac
tiv
ity
, r
. u
.
fig. 5. Activity of MMP-9 (r.u.): 1 — lungs of intact mice; 2 —
tumor control; 3 — tumors of mice treated with high doses
of L-Arg; 4 — low doses of L-Arg; 5 — tumors of mice treated
with high doses of CoQ10; 6 — low doses of CoQ10; 7 — tumors
of mice treated with high doses of L-Arg and СoQ10; 8 — L-Arg and
СoQ10 at low doses. p < 0.05 compared to tumor control (group II)
Activity of MMP-2 and -9 in tumors of animals
injected by both substances at high or low doses
was significantly below the control values (p < 0.05).
In this case (Table 1, 2) low gelatinase activity levels
corresponded to slower primary tumor growth and
decreased metastasis in experimental animals. In the
control group and group of animals injected with low
doses of L-Arg, high gelatinase activity levels were
observed in tumor tissue along with accelerated LLC
growth and metastasis.
Tables 1 and 2 shows the data on antitumor and
antimetastatic activity of L-Arg and СоQ10 administered
at low and high doses. As one may see, the antitumor
and antimetastatic activity of L-Arg differed significantly
depending on the level of NO in tumor cells (p < 0.05).
Thus, high doses of the compound caused a decrease
in the tumor volume by 48.0 ± 8.0%, while low doses
stimulated its growth, number of metastases and their
volume (p < 0.05). Treatment with СoQ10 at high and low
doses resulted in inhibition of the tumor growth by 54.0 ±
11.3 and 39.0 ± 5.4%, respectively, the difference be-
tween these indexes was insignificant. Combined ef-
fect of both compounds at high doses caused tumor
growth inhibition by 51.0 ± 7.4%. Thus, a NO donor
and СoQ10 showed the antitumor and antimetastatic
activity, and the pro- and anti-tumor effect of NO de-
pended on its dose. These data were in accordance
with those of other authors who demonstrated the dual
effect of NO donors [1, 7, 8]. Superoxide regulated
MMP-2 and -9 mediate anti-tumor effects of L-Arg
and СoQ10 at the level of intercellular matrix proteolysis,
because reduced activity of gelatinases contributes
to inhibition of migration, dissemination of tumor cells
and formation of metastatic centers.
Table 1. Antitumor activity of L-Arg and СoQ10
Groups
of animals
Doses
High Low
Tumor vo-
lume, mm3
Inhibition
of tumor
growth, %
Tumor vo-
lume, mm3
Inhibition
of tumor
growth, %
Tumor control 1600 ± 95 – 1600 ± 95 –
L-Arg 832 ± 1151 48 ± 8.0 1840 ± 1251 15 ± 2.6
СоQ10 736 ± 1371 54 ± 11.3 970 ± 1121,2 39 ± 5.4
L-Arg and СоQ10 784 ± 1041 51 ± 7.4 1410 ± 992,3 12 ± 3.1
Note: 1p < 0.05 compared to tumor control (group II); 2p < 0.05 compared
to animals treated with L-Arg; 3p < 0.05 compared to animals treated with
CoQ10.
Table 2. Antimetastatic activity of L-Arg and СoQ10
Groups
of animals
Doses
High Low
Number
of meta-
stases
Volume
of metasta-
ses, mm3
Number
of meta-
stases
Volume
of metasta-
ses, mm3
Tumor control 6.8 ± 3.1 139 ± 54 6.8 ± 3.1 139 ± 54
L-Arg 3.6 ± 1.1 79 ± 21 10.8 ± 1.1 212 ± 33
СоQ10 3.1 ± 2.3 76 ± 13 4.7 ± 2.31 89 ± 131
L-Arg and СоQ10 3.2 ± 1.5 78 ± 18 4.0 ± 2.51,2 99 ± 181
Note: 1p < 0.05 compared to animals treated with L-Arg; 2p < 0.05 compared
to animals treated with CoQ10.
Reprogramming of mitochondria metabolism, dis-
orders of redox homeostasis in mitochondria electron
transport chain are characteristic for malignant tumor
cells. That is accompanied by the replacement of four-
to single-electron restoration of oxygen molecule with
subsequent formation of superoxide radicals. Increased
production of superoxide radicals in tumor cells
promotes cancer progression through the activation
of signaling pathways that regulate metabolic changes,
proliferation, angiogenesis and metastasis [18, 19].
CoQ10 plays the role of electron transporter in com-
plexes I, II and III electron transport chain of mitochon-
dria. Reduced form of CoQ10 protects the membrane,
proteins and mitochondrial DNA from oxidative agents,
including superoxide radicals. We have found that
CoQ10 realizes its protective function restoring the
electron transport process and reducing the level
of generation of superoxide radicals by mitochondria
in LLC cells. These data are in agreement with the
data on protective effect of CoQ10 toward UV irradiated
astrocytes [20] and fibroblasts [21].
Recent studies have shown that NO takes part in the
regulation of tumor cell proliferation, in particular, in its in-
hibition and induction of apoptosis, which is possible due
Experimental Oncology 38, 31–35, 2016 (March) 35
to the ability of NO to inactivate iron-containing enzyme
involved in the synthesis of ATP and DNA replication.
Activation of NO synthase (NOS) and increased levels
of NO generation can have both antitumor effect, and
promote the initiation and progression of tumor [2].
The use of different doses of NO may allow to regulate
phenotypic responses through molecular mechanisms
that form pro- and antitumor effects.
Our results showed a dose-dependent action
of L-Arg on redox-dependent processes in LLC. Thus,
the use of L-Arg at high doses leads to increased levels
of NO and superoxide radicals in mitochondria of LLC
cells, increasing their cytotoxic effect by formation
of peroxynitrite, which can cause DNA damage and
initiation of apoptosis. Similar results were obtained
in the study of dose-depending action of NO donors
on proliferation and apoptosis in Ehrlich ascites
carcinoma [22]. It should be noted that the use
of NO donors at low doses enhances the functional
activity of mitochondria, stimulates the formation
of superoxide radicals in physiological range and,
conversely, at high doses — depresses mitochodrial
respiration, inhibits the incorporation of arachidonic
acid and activates its output.
In modern studies of suppression, stabilization
or enhancement of the degradation of extracellular
matrix are considered critical characteristics
of malignant progression [9] as well as expression
level of gelatinases is thought to be associated with the
metastasis and overall survival of cancer patients [10].
Known synthetic inhibitors inhibit MMP activity in in vitro
experiments and in animal models, but failed in clinical
trials [23]. On the other hand, some stu dies have shown
that antioxidants [15, 24], inclu ding CoQ10 [25, 26],
suppress tumor growth by inhibition of MMP activity via
intracellular regulation of reactive oxygen species. Our
results showed that MMP-2 and -9 mediate antitumor
effects of L-Arg and CoQ10 at the level of intercellular
matrix proteolysis. Redox-regula ted decrease of gela-
tinases activity promotes inhibition of tumor cell migra-
tion, dissemination and formation of metastatic niches.
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