Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors
Aim: The aim of the present study was to investigate the dynamics of nitric oxide derivative (NOD) formation in mice with transplanted tumors and to analize whether synthetic NO-synthase inhibitors, NO-donors and natural compounds could modulate NOD synthesis. Materials and Methods: In the study F1(...
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irk-123456789-1387092018-06-20T03:06:33Z Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors Deryagina, V.P. Ryzhova, N.I. Golubkina, N.A. Original contributions Aim: The aim of the present study was to investigate the dynamics of nitric oxide derivative (NOD) formation in mice with transplanted tumors and to analize whether synthetic NO-synthase inhibitors, NO-donors and natural compounds could modulate NOD synthesis. Materials and Methods: In the study F1(C57BlxCBA), CBA/Lac, BDF and Balb/c mice were used. Endogenous NOD synthesis in mice with transplanted Ehrlich carcinoma (EC) and Lewis lung carcinoma (LLC) was estimated by measuring urine nitrates (NA) and nitrites (NI) excretion and their concentration in tumor tissue determined by cadmium-reduction method. Results: It is shown that EC development is accompanied by increased endogenous NOD formation whereas LLC growth — by its decrease. Total NOD excretion with urine in EC-bearing mice during tumor development was in the range of 1.1x10–7-7.0x10–6 mol/kg body weight that was 1.7–6.8 times higher than that in LLC-bearing mice. Treatment of EC-bearing animals with Nώ-nitro-L-arginine and aminoguanidine resulted in decreased NOD formation causing moderate tumor growth retardation. Effect of treatment with nitroprusside was shown to be dependent on the rout of its administration and dosage. Treatment of EC-bearing mice with picnogenol, tannic acid, spirulina and paprika enriched with selenium resulted in tumor growth inhibition at the early stage of EC growth accompanied by stimulation of endogenous NOD formation. Conclusion: Regulation of endogenous NOD formation towards normal physiological levels or hyperproduction of these compounds may result in tumor growth suppression. 2012 Article Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors / V.P. Deryagina, N.I. Ryzhova, N.A. Golubkina // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 29-33. — Бібліогр.: 23 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138709 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Deryagina, V.P. Ryzhova, N.I. Golubkina, N.A. Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors Experimental Oncology |
| description |
Aim: The aim of the present study was to investigate the dynamics of nitric oxide derivative (NOD) formation in mice with transplanted tumors and to analize whether synthetic NO-synthase inhibitors, NO-donors and natural compounds could modulate NOD synthesis. Materials and Methods: In the study F1(C57BlxCBA), CBA/Lac, BDF and Balb/c mice were used. Endogenous NOD synthesis in mice with transplanted Ehrlich carcinoma (EC) and Lewis lung carcinoma (LLC) was estimated by measuring urine nitrates (NA) and nitrites (NI) excretion and their concentration in tumor tissue determined by cadmium-reduction method. Results: It is shown that EC development is accompanied by increased endogenous NOD formation whereas LLC growth — by its decrease. Total NOD excretion with urine in EC-bearing mice during tumor development was in the range of 1.1x10–7-7.0x10–6 mol/kg body weight that was 1.7–6.8 times higher than that in LLC-bearing mice. Treatment of EC-bearing animals with Nώ-nitro-L-arginine and aminoguanidine resulted in decreased NOD formation causing moderate tumor growth retardation. Effect of treatment with nitroprusside was shown to be dependent on the rout of its administration and dosage. Treatment of EC-bearing mice with picnogenol, tannic acid, spirulina and paprika enriched with selenium resulted in tumor growth inhibition at the early stage of EC growth accompanied by stimulation of endogenous NOD formation. Conclusion: Regulation of endogenous NOD formation towards normal physiological levels or hyperproduction of these compounds may result in tumor growth suppression. |
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| author |
Deryagina, V.P. Ryzhova, N.I. Golubkina, N.A. |
| author_facet |
Deryagina, V.P. Ryzhova, N.I. Golubkina, N.A. |
| author_sort |
Deryagina, V.P. |
| title |
Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors |
| title_short |
Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors |
| title_full |
Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors |
| title_fullStr |
Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors |
| title_full_unstemmed |
Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors |
| title_sort |
production of nitrogen oxide derivatives under the influence of no-synthase inhibitors and natural compounds in mice with transplanted tumors |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2012 |
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Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/138709 |
| citation_txt |
Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors / V.P. Deryagina, N.I. Ryzhova, N.A. Golubkina // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 29-33. — Бібліогр.: 23 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT deryaginavp productionofnitrogenoxidederivativesundertheinfluenceofnosynthaseinhibitorsandnaturalcompoundsinmicewithtransplantedtumors AT ryzhovani productionofnitrogenoxidederivativesundertheinfluenceofnosynthaseinhibitorsandnaturalcompoundsinmicewithtransplantedtumors AT golubkinana productionofnitrogenoxidederivativesundertheinfluenceofnosynthaseinhibitorsandnaturalcompoundsinmicewithtransplantedtumors |
| first_indexed |
2025-07-10T06:24:49Z |
| last_indexed |
2025-07-10T06:24:49Z |
| _version_ |
1837240098684403712 |
| fulltext |
Experimental Oncology ��� ������ ���� ��arc����� ������ ���� ��arc�� ��arc�� ��
PRODUCTION OF NITROGEN OXIDE DERIVATIVES UNDER THE
INFLUENCE OF NO-SYNTHASE INHIBITORS AND NATURAL
COMPOUNDS IN MICE WITH TRANSPLANTED TUMORS
V.P. Deryagina1,*, N.I. Ryzhova1, N.A. Golubkina2
1Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center of RAMS, Moscow 115478, Russia
2Institute of Nutrition of RAMS, Moscow 109240, Russia
Aim: The aim of the present study was to investigate the dynamics of nitric oxide derivative (NOD) formation in mice with trans-
planted tumors and to analize whether synthetic NO-synthase inhibitors, NO-donors and natural compounds could modulate NOD
synthesis. Materials and Methods: In the study F1(C57BlxCBA), CBA/Lac, BDF and Balb/c mice were used. Endogenous NOD
synthesis in mice with transplanted Ehrlich carcinoma (EC) and Lewis lung carcinoma (LLC) was estimated by measuring urine
nitrates (NA) and nitrites (NI) excretion and their concentration in tumor tissue determined by cadmium-reduction method. Results:
It is shown that EC development is accompanied by increased endogenous NOD formation whereas LLC growth — by its decrease.
Total NOD excretion with urine in EC-bearing mice during tumor development was in the range of 1.1x10–7-7.0x10–6 mol/kg body
weight that was 1.7–6.8 times higher than that in LLC-bearing mice. Treatment of EC-bearing animals with Nώ-nitro-L-arginine
and aminoguanidine resulted in decreased NOD formation causing moderate tumor growth retardation. Effect of treatment with
nitroprusside was shown to be dependent on the rout of its administration and dosage. Treatment of EC-bearing mice with picno-
genol, tannic acid, spirulina and paprika enriched with selenium resulted in tumor growth inhibition at the early stage of EC growth
accompanied by stimulation of endogenous NOD formation. Conclusion: Regulation of endogenous NOD formation towards
normal physiological levels or hyperproduction of these compounds may result in tumor growth suppression.
Key Words: nitric oxide derivatives, NO-synthase inhibitors, natural antioxidants, tumors.
T�e role of nitric oxide in tumor biology is am-
biguous and is studied insufficiently. NO-production
in �uman and animal organisms is based on enzymatic
NO-synt�ase transformation of L-Arg-guanidine frag-
ment in t�e reaction wit� oxygen [�� �]. Long duration
of NO-biosynt�esis ������� fold longer t�an basic level�
results in genotoxicity effect� induces dose-dependant
DNA-destruction etc� leading to tumor development.
�ultifold increase in NO-production is considered
to be a consequence of inducible NO-synt�ase acti-
vation �iNOS�� being expressed in different cell types
in normal state and pat�ology� including macrop�ages�
microglyal cells� keratinocytes� �epatocytes� astro-
cytes� endot�elial cells of blood vessels� epit�elial cells
and a variety of �uman tumor cells affected by cyto-
kines: interleukin-� and �� interferon-γ or t�eir com-
bination wit� tumor necrosis factor-α or -β� etc [��7].
Direction of NO effect is defined by many factors:
concentration� exposition� reaction products wit� key
reagents �oxygen and its active forms� carbon dioxide�
etc� and targets �metals� t�iol containing aminoacids�
proteins� etc�. Clinical investigations s�ow t�at NOS
expression in many tumor tissues is often disturbed.
T�at is true for tumors of central nervous system�
stomac�� colon and mammary gland� and melanoma�
w�ere NOS activity is found to be elevated. A direct
correlation is s�own between iNOS expression in tis-
sues of t�ese tumors and t�e following parameters:
stage of a disease� vessel development in tumor� �ig�
frequency of metastases� w�at result in poor prognosis
[8���]. Animal experiments revealed t�at during iNOS
activation� utilization of enzyme selective in�ibitors
resulted in tumor growt� suppression [��� ��]. Exoge-
nous NO is s�own to in�ibit endogenous NO synt�esis.
�ec�anisms of feedback include direct NOS inactiva-
tion via NO binding by �em-containing enzyme group
and in�ibition of m-RNA iNOS expression [��].
At t�e same time NO plays a positive role in defense
reactions of an organism. T�us� NO secreted by non-
specific immunity cells� macrop�ages and neutrop�ils�
s�ows oxidizing and antimicrobial properties and is re-
sponsible for p�agocytes cytostatic and cytotoxic po-
tential wit� respect to tumor cells [�5]. T�ere is anot�er
significant property of NO: apoptosis initiation� inclu-
ding t�at in transformed cells� due to violation of mi-
toc�ondrial oxidative p�osp�orylation� ribonucleotide
reductase metabolism� etc [�6]. In some instances�
w�en NO cell production is low �w�at promotes survival
of transformed cells�� it is proposed t�at t�e activity
of iNOS s�ould be restored wit� t�e use of medicinal
agents or wit� gene t�erapy [7]. NO concentration
in biological fluids of organism� tumor tissues� tumor
microenvironment is s�own to predict t�e activity
of key proteins for carcinogenesis — suc� as p5��
extracellular signal-regulated kinase �ERK�� �ypoxic
inducible factor �HIF�� Akt etc. [�7]. Taking into account
t�e above information a special scientific and practical
interest will include investigations of NO biosynt�esis
Received: October 24, 2011.
*Correspondence: E-mail: Derygina@inbox.ru
Abbreviations used: AG — aminoguanidine; ASA — acetylsalicylic
acid; CZ — L-carnozine; Dq — Diquertin; EC — Ehrlich carcinoma;
GA — gallic acid; I-3-C — indol-3-carbinol; iNOS — inducible
NO-synthase; LLC — Lewis lung carcinoma; L-NMMA — Ng-mono-
methyl-L-arginine; L-NNA — Nώ-nitro-L-arginine; NA — nitrates;
NI — nitrites; NOD — nitric oxide derivatives; NP — nitroprusside;
Pg — pycnogenol; Se-P — sweet pepper; Se-Sp — spirulina;TA —
tannic acid; VS — vine stone.
Exp Oncol ����
��� �� �����
�� Experimental Oncology ��� ������ ���� ��arc��
levels upon t�e influence of NOS modulators and natu-
ral antioxidants in different models of tumor growt�.
T�e aim of t�e present study was to investigate t�e
dynamics of nitric oxide derivatives �NOD� formation
in mice wit� transplanted tumors and to analize w�et�-
er synt�etic NO-synt�ase in�ibitors� NO-donors and
natural compounds could modulate NOD synt�esis.
MATERIALS AND METHODS
��� male F��C57BlxCBA�� CBA/Lac� BDF and
Balb/c mice weig�ting ����� g were used in t�e work.
Animals were fed wit� briquetted feed wit� constant
access to water. All experimental procedures were con-
ducted following t�e normative rules of bioet�ics.E�rlic�
carcinoma �EC� and Lewis lung carcinoma �LLC� tumor
strains were received from Russian Oncologic Scientific
Centre of RA�S ��oscow� Russia�. LLC and EC cells
�5x��6 or ��6 cells per mice� were inoculated subcutane-
ously� in rig�t armpit and rig�t inguinal region.
T�e following reagents were used: Ng-monomet�yl-
L-arginine �L-N��A�� Nώ-nitro-L-arginine �L-NNA��
N-[[�-�aminomet�yl�p�enyl]met�yl]-et�animidamide
di�ydroc�loride �����W�� aminoguanidine �AG� �Sig-
ma-RBI�; L-carnozine �CZ� �BioC�emika�. Ot�er re-
agents: nitroprusside �NP�� acetylsalicylic acid �ASA��
etc. were of Russian production. W�ile c�oosing NOS
modulators� c�emical properties of compounds were
taken into consideration [�].
Endogenous NOD formation in t�e organism
was evaluated on two tumor growt� models — LLC
�6� mice� and EC ��� mice� by analysis of nitrate �NA�
and nitrite �NI� excretion wit� urine per day and t�eir
concentration in tumor tissue.
Gat�ering of diurnal urine for NA and NI analysis
of mice F��C57BlxCBA� wit� LLC was ac�ieved before
tumor transplantation and after it on t�e �d� �t�� �6t��
��st and ��t� day. NA and NI concentrations in tumor
tissue were determined on t�e ��t�� ��st� �8t� and ��nd
day of tumor growt�.
Gat�ering of diurnal urine of Balb/c-mice wit�
EC was performed before EC transplantation and after
it on t�e �t�� ��t�� �7t�� ��t� and ��nd day of tumor growt�.
NA and NI concentrations in tumor tissue of BDF mice
wit� EC transplanted tumor were determined on t�e
��nd day of tumor growt�.
Investigation of synt�etic NOS in�ibitors and
NO donors effect on EC growt� was carried out
on ��� F��C57BlxCBA� and BDF mice. 7 groups of �y-
brid mice ����� animals per group� were formed. T�e
control group was composed from �6 mice. BDF mice
were divided into � groups� �� mice per group. EC was
transplanted to all mice subcutaneously. T�e intra-
peritoneal introduction was used for administration
of an active compound solution� �.� ml� 5 days per
week �except special cases discussed later� to ani-
mals from t�e ��8t� groups beginning from t�e 8t� day
after EC transplantation. In total� eac� animal received
�� doses. T�ere were formed t�e following groups:
�� control� �� treatment wit� 5� mg/kg L-N��A� ��
��� mg/kg L-NNA� �� ��� mg/kg AG� 5� combined
treatment wit� ��� mg/kg AG and �� mg/kg ASA. T�e
6 group of animals received � mg/kg of NP on t�e �nd
and �d day after EC transplantation and later sub-
cutaneously �� µg/kg of NP. T�e 7t� group received
subcutaneously 8� µg/kg of NP on t�e 8t� day after
EC transplantation. T�e 8t�-group of mice received
���� mg/kg of CZ wit� water since t�e second day
after transplantation and during all t�e experiment.
T�e effect of selective in�ibitor iNOS-����W
on EC growt� was studied on �� BDF mice wit� trans-
planted EC �group �� control� n=��� and group ��� n=���
treated subcutaneously wit� �� mg/kg ����W in�ibitor
solution from day � after tumor transplantation. In total�
�� ����W administrations per animal were performed.
Investigation of effect of natural biologically ac-
tive compounds was analyzed in CBA/Lac �n=5���
F��C57BlxCBA� �n=5�� and Balb/c �n=��� male mice using
t�e following preparations: pycnogenol �Pg�� containing
>=6�% of procyanidins �Biolandes� France�� diquertin
�Dq� wit�>��% of di�ydroquercetin �Flavir� Russia��
indol-�-carbinol �I-�-C� �Sigma�� p�enolic acids: gallic
�GA�� tannic �TA� and also Se-enric�ed paprika �Se-P�
� mg Se/kg �ayak cultivar grown on sodium selenate
containing �.��% NPK-fertilizers� and algae spirulina-
Arthospira platensis �Se-Sp� � g Se/kg; production of
“Agro-Victoria” corporation� Russia�. T�e preparations
were given per os as water suspensions ��� weeks
before EC transplantation and after it up to t�e end of ex-
periment 5 times per week. T�e eut�anasia of animals
was performed under t�e lig�t ester anest�etic.
Tumor growt� in�ibition �TGI� under t�e effect
of tested compounds was estimated in tumor growt�
dynamics using mass and volume parameters in com-
parison wit� t�e control group according to [�8].
For diural urine sampling animals were placed in ex-
c�ange cages �5 per cage� for �� � wit�out feeding
and wit� free water access. To exclude NI oxidation
�.� ml of ��% sodium �ydroxide solution was used.
Removal of protein-carbo�ydrate component in urine
water extract was ac�ieved using ZnS�� and potassium
ferricyanide.
On t�e ��t�� ��st� �8t� days after LLC transplantation
�or ��nd day for EC� t�e eut�anasia of animals under
t�e lig�t ester anest�etic was performed. Tumors were
removed� �omogenized and t�e samples of tissue ex-
tracts were obtained as follows: ��� g of �omogenized
tumor tissue were placed in a calibrated vessel� bidis-
tilled water was added �tissue/water ratio= �:��� and
t�e resulting mixture was �eated at 6�° С during �5 min.
To prevent NI destruction� pH of water suspension was
adjusted to 7.��7.� using buffer solution. T�en t�e
mixure was cooled to room temperature� protein-car-
bo�ydrate component was removed by centrifugation
for �5 min at 5�� g. NI and NA content in all reagents�
water and materials was controlled.
NI and NA concentration in urine and tumors was
determined by spectrop�otometric met�od using
Griss reagent wit� prereduction of NA to NI by porous
cadmium [��]. Optical density of colored solutions was
determined on spectrop�otometer SF-�6.
Experimental Oncology ��� ������ ���� ��arc����� ������ ���� ��arc�� ��arc�� ��
Statistical analysis of t�e data �mean value±SD�
was performed by standard met�ods using Stu-
dent’s t-test.
RESULTS AND DISCUSSION
EC growt� in Balb/c mice was accompanied
by increase of NA and NI excretion wit� urine
�Table ��. By t�e end of experiment �day ��� urine
NI and NA excretion increased by �.6 �p<�.��� and
67.� �p<�.��� times respectively compared to t�ese
parameters of animals wit� t�e initial tumor nods �day
��. NI excretion in EC-bearing mice was in t�e range
�.6х���7—�.�х���6 mol/kg bw� and NA excretion —
8.5х���8—5.7х���6 mol/kg bw. Total nitro compound
excretion during EC growt� increased by �5.5 fold
�in N��
- equivalents�. During t�is period mean tumor
volume increased by 67.� times. A positive correlation
between EC volume and diurnal total NI and NA excre-
tion wit� urine was revealed �r=�.���.
NOD excretion by F��C57BlxCBA� mice wit� trans-
planted LLC demonstrated certain peculiarities
�Table ��. One day after LLC inoculation maximal
excretion values of NI and NA were �.� times �ig�er
t�an t�e respective parameters of �ealt�y animals� and
were constantly �ig�er by ��.��7�.5% versus control
at all time points during LLC growt�.
NI concentration in LLC tumor tissue �Table ��
was in t�e range of �.8��.6x���6 mol/kg� increasing
monotonously during tumor development w�ereas
NA concentration in t�e majority of samples was neg-
ligible. Total NA+NI content in tumor tissue did not
exceed 5.�x���6 mol/kg and did not depend on tumor
development.
Total amount of NI and NA in EC tumor tissue iso-
lated from BDF mice on day �� was 5.5 times �ig�er
�p<�.��� t�an t�eir maximal total content in LLC tumor
tissue �Table ��.
Investigation of t�e effect of synt�etic compounds
capable to modulate NOS activity �as s�own t�at in-
traperitoneal administration of L-NNA ���� mg/kg�� AG
���� mg/kg�� AG ���� mg/kg�+ASA ��� mg/kg�� to �y-
bride mice resulted in statistically significant EC growt�
in�ibition �Table ��. T�e earliest and stable TGI was
demonstrated for NNA ���%� p<�.���. AG caused
low TGI ������%� p<�.�5� but in combination wit�
ASA it in�ibited EC growt� more efficiently ������%�
p<�.�5�. NP effect depended on t�e dose and rout
of administration: subcutaneous administration
Table 1. Urine NI and NA excretion in dynamics of Ehrlich carcinoma growth in Balb/c micea)
Parameter Day of tumor growth
0 4 10 17 24 32
Tumor volume, mm3 No tumor 49±21 720±117 1480±197 2189±809 3300±462
N02
- excretion 0 (3.6 ± 2.6)×10-7 (4.5 ± 1.09)×10-7 (5.8 ± 4.64)×10-7 (1.0 ± 0.53)×10-6 (1.3 ± 0.55)×10-6
N03
- excretion 1.1 ± 0.44×10-7 (8.5 ± 9.9)×10-8 (8.9 ± 3.0)×10-7 (3.3 ± 1.1)×10-6 (3.1 ± 0.3)×10-6 (5.7 ± 2.97)×10-6
Total NI and NA excre-
tion (mol/kg bw)
(1.1 ± 0.44)×10-7 (4.45 ± 3.29)× 10-7 (1.34 ± 0.38)×10-6 (3.88 ± 1.41)×10-6 (4.1 ± 0.73)×10-6 (7.0 ± 3.63)×10-6
Note. a) mean values ± SD (n=10).
Table 2. Urine NI and NA excretion of F1(C57BlxCBA) mice bearing Lewis lung carcinoma
Days of LLC growth / Group
of animals
NI and NA excretiona), mol/kg bw Comparison with controlb)
N02
- N03
- Total NI and NA
2 (2.94 ± 1.12) ×10-7 (3.42 ± 0.15)×10-6, p<0.01 (3.71 ± 0.20)×10-6, p<0.01 +91.2
9 0.0 (7.67 ± 2.31)×10-7, p<0.01 (7.67 ± 2.31)×10-7, p<0.01 -60.5
6 (5.93 ± 7.9)×10-8 (5.11 ± 4.0)×10-7, p<0.01 (5.7 ± 4.14)×10-7, p<0.01 -70.5
21 0.0 (1.18 ± 0.36)×10-6 (1.18 ± 0.36)×10-6 -39.2
30 0.0 (1.51 ± 0.43)×10-6 (1.51 ± 0.43)×10-6 -22.2
Control (healthy animals) (2.6 ± 1.04)×10-7 (1.68 ± 0.85)×10-6 (1.94 ± 1.12)×10-6 0.0
Note. a) mean values ± SD (n=10); compared to the control. b) (-) — % of NOD decrease; (+) — % of NOD increase.
Table 3. NI and NA concentration in tumor tissue of mice bearing LLC or EC
Mice (days of tumor growth)a) NI and NA concentration, mol/kg tissue
N02
- N03
- Total NI and NA
F1 mice with LLC (14) (2.8 ± 1.1) ×10-6 0.0 (2.8 ± 1.1)×10-6
F1 mice with LLC (21) (3.70 ± 1.5)×10-6 (1.6 ± 0.6)×10-6 (5.3 ± 1.98)×10-6
F1 mice with LLC (28) (4.6 ± 1.5)×10-6 0.0 (4.6 ± 1.5)×10-6
BDF mice with EC (32) (1.19 ± 0.37)×10-6 (1.72 ± 0.38)×10-5 (2.91 ± 0.42)×10-5
Note. a) mean values + SD (for 10 animals).
Table 4. Effect of NO-synthase inhibitors on Ehrlich carcinoma growth and endogenous production of NOD derivatives in F1(C57BlxCBA) and BDF mice
Groups of EC-bea-
ring mice, compounds
(dose, mg/kg bw)
Tumor growth inhibition
(TGI), %*
NO2
- excretion,
mol/kg bw* NO3
- excretion, mol/kg bw* Total NI and NA, mol/kg bw
(days of EC growth)
Effect on NOD
excretion, %
1. Control 0 (0.68 ± 0.25)×10-6 (6.8 ± 2.24) × 10-6 (7.48 ± 2.57)×10-6 (22) 0
2. L-NMMA (50) 0 (1.5 ± 0.62)×10-6 p<0.01 (5.5 ± 1.59)×10-6 (7.0 ± 2.02)×10-6 (22) -6
3. L-NNA (100) 18–39 p<0.05; p<0.01 (0.79 ± 0.33)×10-6 (2.71 ± 1.03)×10-6 p<0.01 (3.50 ± 1.27)×10-6 p<0.01 (22) -53
4. AG (140) 18–22 p<0.05 (1.02 ± 0.36)×10-6 (4.53 ± 1.81)×10-6 p<0.05 (5.55 ± 2.19)×10-6 (22) -26
5. AG (140) + ASA (10) 20–42 p<0.05; p<0.01 ** **
6. EC + NP (4) +(28–124) p<0.05; p<0.01 (0.47 ± 0.15) × 10-6 (1.12 ± 0.33)×10-5 p<0.01 (1.17 ± 0.36)×10-5 p<0.01 (22) +56
7. NP (0.08) 21–26 p<0.05; p<0.01 ** **
8. CZ (130) 21–29 p<0.05; p<0.01 (1.78 ± 1.0)×10-6 (2.92 ± 1.17)×10-6 p<0.01 (4.70 ± 1.86)×10-6 p<0.05 (22) -37
9. Control 0 (0.35 ± 0.17)×10-6 (4.83 ± 2.12)×10-6 (5.18 ± 2.35)×10-6 (22) 0
10. 1400W (13.0) 0 (0.14 ± 0.13)×10-6 p<0.01 (3.74 ± 1.35)×10-6 (3.88 ± 1.49)×10-6 (22) -25
Note. *- compared with respective the control; +( )- increase in tumor growth; **-not determined; Ng-monomethyl-L-arginine (L-NMMA); Nώ-nitro-L-arginine (L-
NNA); AG — aminoguanidine; ASA — acetylsalicylic acid; NP — nitroprusside; CZ — L-carnozine. 1–8 groups — mice F1(C57BlxCBA). 9–10 groups — mice BDF.
�� Experimental Oncology ��� ������ ���� ��arc��
of 8� µg/kg of NP resulted in �6% EC growt� in�ibition
�p<�.�5� but intraperitoneal administration of �ig�er
dose �� mg/kg� on days � and � after EC transplanta-
tion stimulated tumor growt� up to ���% �p<�.���.
In mice treated wit� L-NNA and AG tumor growt�
in�ibition by 5�% �p<�.��� and a decrease of NOD
formation by �6% were recorded.
It s�ould be pointed out t�at L-N��A often used
in investigations as iNOS in�ibitor �5� mg/kg� did not
affect EC growt� w�ile N�D excretion wit� urine was
close to t�at of control animals. Subcutaneous admin-
istration of ����W� selective iNOS in�ibitor ��� mg/kg��
did not cause statistically significant EC tumor depres-
sion t�oug� NOD excretion was decreased by �5.�%.
Investigation of natural preparation effect
on EC growt� �as s�own t�at prolonged per os con-
sumption of �5� mg PG/kg and 75 mg I-�-C/kg caused
statistically significant TGI by 5�% and 56% respec-
tively �p<�.���. During t�e w�ole experiment Pg effect
was stable and resulted in ��% decrease of tumor
mass �p<�.���. GA ���� mg/kg�� TA ���� mg/kg� and
Se-Sp �� g/kg� statistically significantly increased t�e
latent period of tumor nod formation by �.���.6 times
and in�ibited tumor growt� during two weeks after
EC transplantation. At t�e early stages of EC develop-
ment TGI was equal to ��% �GA�� 78% �TA�� 75% �Sp��
8�% �Se-Sp� and 65% �Se-P� �p<�.�5�.
Total NI and NA excretion detected on days ���
�� or �� of EC growt� in control group of animals was
6.�x���6 mol/kg bw �F� mice�; �.��x���5 mol/kg bw
�CBA/Lac mice� and 6.��x���6 mol/kg bw �Balb/c mice�
�Table 5�. Pg� Dq� TA� Sp and Se-P administration
resulted in increase of NOD excretion by ��.�%
�p<�.�5�� �7.�% �p<�.�5�� �7.7%� 65.6% �p<�.���
and ���.6% �p<�.��� accordingly compared to t�e
control group of animals. Only GA in�ibited NO forma-
tion by ��% �p<�.�5� on day �� of EC growt� w�ereas
I-�-C and Se-Sp did not affect NO biosynt�esis.
T�us� t�e results �ave s�own t�at EC growt� is ac-
companied by increased NOD formation w�ereas LLC
tumor growt� is c�aracterized by depression of NOD
biosynt�esis. It s�ould be noted t�at NO endogenous
synt�esis determination via NOD urine excretion is an in-
tegral parameter including also t�e amount of NOD� be-
ing formed from nitrogen oxide� necessary for normal
function of cardiovascular� immune� endocrine and ner-
vous systems. NO synt�esis by constitutive NO-synt�ase
isoforms is considered not to be accompanied by toxic
effects of NO and its derivatives as concentration levels
capable to cause necessary p�ysiological reactions are
in nano-to micromolar range [��]. T�ere are few data
on NO levels in animal tumor tissues. Study of �uman
breast cancer �CF7 cells �as s�own t�at t�e activity
of several key proteins �HIF-�α� ERK� and p5� � is regu-
lated by distinct t�res�old concentrations of nitric oxide.
At low steady-state concentrations of NO �<5� nmol��
ERK p�osp�orylation was induced via a guanylate
cyclase-dependent mec�anism. HIF-�α accumulation
was associated wit� an intermediate amount of NO
�>�.�x���7 ��� w�ereas p5� serine �5 p�osp�orylation
occurred at considerably �ig�er levels �>�.�x���7 �� [�7].
In our experiment endogenous formation of NOD
in EC mice beginning from t�e �t� to t�e ��t� day of tumor
growt� was in t�e range �.�x���7�7.�x���6 mol/kg bw and
for LLC-mice — 5.7x���7��.7�x���6 mol/kg bw. At t�e
same time t�e total NI and NA concentration in tumor
tissue was �.��x���5 mol/kg� w�ile NOD concentration
in LLC tumor did not exceed 5.�x���6 mol/kg tissue.
It is obvious t�at mean NOD concentration in EC and
LLC tumors is significantly �ig�er t�an concentrations
necessary for activation of proteins responsible for cells
proliferation and apoptosis.
One can assume t�at t�e ability of NOS in�ibitors
�L-NNA and AG� to suppress tumor growt� is related
to t�eir ability to depress iNOS activity [��]. Also it �as
been s�own t�at NP could �ave oppositely directed
effects and stimulate tumor growt� at t�e relatively
�ig� doses �� mg/kg� and suppress it at lower doses
�8� µg/kg�. T�ere are t�e data demonstrating t�at
in tumor microenvironment wit� low content of glucose
and oxygen tumor cells may be more sensitive to �ig�
concentrations of NO and peroxinitrite t�at normal
ones. T�at is w�y in several cases NO donors are con-
sidered to be effective for promotion of c�emo- and
radiot�erapy efficiency [�6]. However� we �ave s�own
t�at EC �as been developing against t�e augmented
NOD biosynt�esis and double intra-peritoneal injection
of a �ig� NP dosage t�e next day after EC inoculation�
w�ic� only stimulated tumor growt�.
Table 5. Effect of natural antioxidants on Erlich carcinoma growth and endogenous production of NO derivatives (NOD) in CBA/Lac, F1(C57BlхСВА) and Balb/c mice
Groups EC mice,
compounds (dose,
mg/kg bw)
Parameters
Tumor growth inhibition
(TGI), or increase in tu-
mor growth (+), %*
NO2
- excretion,
mol/kg bw* NO3
- excretion, mol/kg bw* Total NI and NA mol/kg bw,
(days of EC growth)
Effect on NOD
excretion, %
CBA/Lac (control) 0 (2.3 ± 0.9)×10-6 (0.88 ± 0.24)×10-5 (1.11 ± 0.30)×10-5 (21) 0
CBA/Lac Pg (150) 31–52 p<0.01 (3.2 ± 1.1)×10-6 (1.28 ± 0.56)×10-5 p<0.05 (1.6 ± 0.67)×10-5 p<0.05 (21) +44.2
CBA/Lac VS (300) ( + ) 45 p<0.05 (2.1 ± 0.8)×10-6 (1.06 ± 0.32)×10-5 (1.27 ± 0.38)×10-5 (21) +14.4
CBA/Lac Dq (150) 0 (3.2 ± 1.2)×10-6 (1.32 ± 0.51)×10-5 p<0.05 (1.64 ± 0.61)×10-5 p<0.05 (21) +47.8
CBA/Lac I-C (75) 27–56 p<0.01 (2.3 ± 0.9)×10-6 (0.92 ± 0.33)×10-5 (1.15 ± 0.42)×10-5 (21) +3.6
F1 (control) 0 (0.94 ± 0.38)×10-6 (5.46 ± 1.69)×10-6 (6.4 ± 1,9)×10-6 (14) 0
F1 GA (100) 27–91 p<0.05; p<0.01 (1.02 ± 0.44)×10-6 (3.33 ± 1.17)×10-6 p<0.01 (4.35 ± 1.48)×10-6 p<0.05 (14) -32.0
F1 TA (100) 24–78 p<0.01 (1.34 ± 0.50)×10-6 (6.83 ± 2.73)×10-6 (8.17 ± 3.04)×10-6 (14) +27.7
F1 Sp (1000) 41–75 p<0.05; p<0.01 (1.5 ± 0.5)×10-6 p<0.05 (0.91 ± 0.25)×10-5 p<0.01 (1.06 ± 0.28)×10-5 p<0.01 (14) +65.6
F1 Se-Sp (1000) 41–89 p<0.05; p<0.01 (1.42 ± 0.58)×10-6 (4.43 ± 1.9) ×10-6 (5.85 ± 2.35) × 10-6 (14) -8.6
Balb/c (control) 0 (0.87 ± 0.30)×10-6 (5.42 ± 1.52)×10-6 (6.29 ± 1.73)×10-6 (23) 0
Balb/c Se-P (1000) 38–65 p<0.05; p<0.01 (0.9 ± 0.4)×10-6 (1.26 ± 0.54)×10-5 p<0.01 (1.35 ± 0.57)×10-5 p<0.01 (23) +114.6
Note: Each group contained 10 mice. Natural antioxidants: pycnogenol (Pg), diquertin (Dq), indol-carbinol (I-C), phenolic acids: gallic acid (GA) and tannic
acid (TA), vine stone (VS) and Se-enriches plants: sweet pepper (Se-P), spirulina (Se-Sp); * — compared with respective control.
Experimental Oncology ��� ������ ���� ��arc����� ������ ���� ��arc�� ��arc�� ��
Lack of L-N��A-dependent in�ibition of NOD
endogenous formation can be explained by peculiari-
ties of its metabolism. It is known t�at NOS converts
L-N��A to N-�ydroxy-N-met�yl-L-agrinine� w�ic�
in�ibits t�e enzyme irreversibly. But in certain tissues
and cells especially in �ypoxic conditions� L-N��A
metabolism may be accompanied by t�e formation
of L-arginine or may possess ot�er peculiarities [�].
Taking into account t�e NOD endogenous forma-
tion in a model of EC tumor growt�� its �5�5�% reduc-
tion upoin t�e use of L-NNA� AG and ���W appears
to be insufficient to attain its normal p�ysiological level.
In conditions of developing tumor acceptable iNOS
blocking will not be able to produce stable defense
response of an organism� but may retard tumor growt�.
At t�e same time significant depression of iNOS activity
may �ave also negative consequences. It is known t�at
N� is formed in t�e oxidation reaction of L-arginine�
w�ic� is also a precursor of endogenous synt�esis
of polyamines. T�is supposes t�e existence of an inte-
grated mec�anism of nitric oxide and polyamine syn-
t�esis regulation. Polyamines are known to stimulate
mammalian cells proliferation and potentiate carcino-
genesis. T�e results s�ow t�at blocking of NO-synt�ase
may cause pretumor c�anges in intestine due to t�e
decreased NO release and induction of ornitine-de-
carboxylase — an enzyme of polyamine synt�esis [��].
A study of t�e effect of natural compounds wit�
pronounced antioxidant properties on tumor growt�
and endogenous NOD formation �as revealed t�at
t�e use of � from � tested compounds resulted in tu-
mor suppression on t�e background of stimulation
of NOD formation. One can suppose t�at suc� effect
could be related not only to direct reactions wit� free
radicals so typical for antioxidants but also to ot�er
mec�anisms involved in stimulation of detoxification
defense systems t�roug� antioxidant responsive
elements present in t�e promoter region of genes
inducible upon oxidative and c�emical stresses [��].
In conclusion� our study �as demonstrated t�at
EC development is accompanied by increased endo-
genous NOD formation w�ile LLC growt� — by depres-
sion of NOD synt�esis. �odulators of NO-synt�ase
activity �L-NNA and AG� decreased NOD formation�
inducing simultaneously moderate� but stable de-
crease of tumor development in EC-bearing mice. T�e
use of NP �ad oppositely directed effects on EC growt�
dependent on t�e dose and t�e rout of administration.
�ost of natural antioxidants tested demonstrated
antitumor activity at t�e early stage of EC growt�
stimulating endogenous NOD formation.
REFERENCES
1. Granik VG, Grigoriev NB. Nitrogen oxide. Mos-
cow: Vuzovskaya Kniga, 2004. 360 pp. (In Russian).
2. Hibbs JBJ. Synthesis of nitric oxide from L-arginine: a re-
cently discovered pathway induced by cytokines with antitumour
and antimicrobial activity. Res Immunol 1991; 142: 565–9.
3. Lala PK, Chakraborty C. Role of nitric oxide in carcino-
genesis and tumour progression. Lancet Oncol 2001; 2: 149–56.
4. Fukumura D, Kashiwagi S, Jain RK. The role of nitric
oxide in tumor progression. Nat Rev Cancer 2006; 6: 521–34.
5. Proskuryakov SJ, Konoplyannikov AI, Ivannikov AI,
et al. Nitrogen oxide in neoplastic prossess. Voprosy Oncologii
2001; 47: 257–69 (In Russian).
6. Ridnour LA, Thomas DF, Switzer C, et al. Molecular
mechanisms for discrete nitric oxide levels in cancer. Nitric
Oxide 2008; 19: 73–6.
7. Fitzpatrick B, Mehibel M, Cowen RL, Stratford IJ.
iNOS as therapeutic target for treatment of human tumors.
Nitric Oxide 2008; 19: 217–24.
8. Gallo O, Masini E, Morbidelli L, et al. Role of nitric
oxide in angiogenesis and tumor progression in head and neck
cancer. J Natl Cancer Inst 1998; 90: 587–96.
9. Swana HS, Smith SD, Perrotta PL, et al. Inducible nitric
oxide synthase with transitional cell carcinoma of the bladder.
J Urol 1999; 161: 630–4.
10. Song ZJ, Gong P, Wu YE. Relationship between the
expression of iNOS, VEGF, tumor angiogenesis and gastric
cancer. World J Gastroenterol 2002; 8: 591–5.
11. Lagares-Garcia JA, Moore RA, Collier B, et al. Nitric
oxide synthase as a marker in colorectal carcinoma. Am Surg
2001; 67: 709–13.
12. Chen YK, Huse SS, Lin LM. Inhibitory effect of in-
ducible nitric oxide synthase inhibitors on DMBA-induced
hamster buccal-pouch squamous-cell carcinogenesis. Nitric
Oxide 2005; 13: 232–9.
13. Singh RP, Agarwal R. Inducible nitric oxide synthase-
vascular endothelial growth factor axis: a potential target
to inhibit tumor angiogenesis by dietary agents. Curr Cancer
Drug Targets 2007; 7: 475–83.
14. Colasanti M, Persichini T, Menegazzi M, et al. Induc-
tion of nitric oxide synthase mRNA expression. Suppression
by exogenous nitric oxide. J Biol Chem 1995; 270: 26731–3.
15. Stuehr DJ, Nathan CF. A macrophage product respon-
sible for cytostasis and respiratory inhibition in tumor target
cells. J Exp Med 1989; 169: 1543–55.
16. Hirst DG, Robson T. Nitrosative stress as mediator
of apoptosis: implications for cancer therapy. Curr Pharm
Des 2010; 16: 45–55.
17. Thomas DD, Espey MG, Ridnour LA, et al. Hipoxic
inducible factor 1α, extracellular signal-regulated kinase, and
p53 are regulated by distinct threshold concentrations of nitric
oxide. J List 2004; 101: 8894–9.
18. Methods of pre clinical investigation of new pharmaco-
logical compounds. 2nd ed. Khabriev RU, ed. Moscow: Medi-
cine Press, 2005; 637–51 (In Russian).
19. Cortas NK, Wakid NW. Determination of inorganig
nitrate in serum and urine by a kinetic cadmium-reduction
method. Clin Chem 1990; 36: 1440–3.
20. Hirst DG, Robson T. Nitrosative stress as mediator
of apoptosis: implications for cancer therapy. Curr Pharm
Des 2010; 16: 45–55.
21. Rao CV, Indranie C, Simi B, et al. Chemopreventive
properties of a selective inducible nitric oxide synthase inhibitor
in colon carcinogenesis, administered alone or in combination
with celecoxib, a selective cyclooxygenase-2 inhibitor. Cancer
Res 2002; 62: 165–70.
22. Schleiffer R, Duranton B, Gosse F, et al. Nitric oxide
synthase inhibition promotes carcinogen-induced preneoplas-
tic changes in the colon of rats. Nitric Oxide 2000; 4: 583–9.
23. Massela R, Benedetto RD, Vari R, et al. Novel
mechanisms of natural antioxidant compounds in biological
systems: involvement of glutathione and glutathione-related
enzymes. J Nutr Biochem 2005; 16: 577–86.
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