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Expression of inducible and neuronal nitric oxide synthase in 20-methyl cholanthrene (20-MCA) induced fibrosarcoma


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Objective: Studies have shown that expression and localisation of nitric oxide synthase (NOS) depends on the type and differentiation status of different types of tumours. The present study was conducted to evaluate the expression of NOS in carcinogen induced tumour. Method: Swiss albino mice were injected with 20-methyl cholanthrene. Seventy six percent of the animals were positive for fibrosarcoma at the end of 100 days following exposure. The tumours were excised when they reached a predetermined size and immunostained for inducible and neuronal NOS. The staining was graded on an increasing intensity scale. Results: The tumour sections stained very strongly for iNOS as compared to controls (identical sections from tumour negative mice). Weak staining was detected for nNOS in the tumour sections unlike controls. Conclusions: NO may be involved in carcinogen induced fibrosarcoma as seen from the expression of its synthesising enzymes.
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Indian Journal of Pharmacology 1999; 31: 315-318 SHORT COMMUNICATION
Correspondence: S.K. Gupta
Departments of 1Pharmacology, 2Biochemistry, 3Institute Rotary Cancer Hospital,
All India Institute of Medical Sciences, New Delhi 110029.
Manuscript Received: 19.3.99 Revised: 18.4.99 Accepted: 24.5.99
Studies have shown that expression and localisation of nitric oxide synthase (NOS) depends
on the type and differentiation status of different types of tumours. The present study was conducted
to evaluate the expression of NOS in carcinogen induced tumour.
Swiss albino mice were injected with 20-methyl cholanthrene. Seventy six percent of the
animals were positive for fibrosarcoma at the end of 100 days following exposure. The tumours were
excised when they reached a predetermined size and immunostained for inducible and neuronal NOS.
The staining was graded on an increasing intensity scale.
The tumour sections stained very strongly for iNOS as compared to controls (identical
sections from tumour negative mice). Weak staining was detected for nNOS in the tumour sections
unlike controls.
NO may be involved in carcinogen induced fibrosarcoma as seen from the expression of
its synthesising enzymes.
Nitric oxide nNOS iNOS 20MCA carcinogenesis fibrosarcoma mice
Since the discovery of nitric oxide (NO), an endo-
genously generated molecule 1, has been implicated
in diverse physiological roles including intracellular
signalling, as a transcellular messenger or as a cyto-
toxic agent 2,3. It is synthesised by the enzyme nitric
oxide synthase (NOS) in the arginine to citrulline
pathway. Of the three isoforms of NOS, endothelial
NOS (eNOS) and neuronal NOS(nNOS) are consti-
tutive calcium dependent enzymes while inducible
NOS (iNOS) is expressed in response to immuno-
logic stimuli and is capable of sustained NO release
at concentrations which could be cytostatic or cyto-
Studies have demonstrated the presence of NOS in
human tumours4. Cobb’s
et al
suggested that NO pro-
duction could be associated with pathophysiological
processes important to tumourogenesis5. To the best
of our knowledge, no studies have however been con-
ducted to implicate NO in carcinogen induced
cancer. The present study was thus conducted to
evaluate the involvement of NO in carcinogenesis.
Swiss albino mice (body wt 15-16g, either sex) were
procured from the Central Experimental Animal Facil-
ity of AIIMS and housed 5 per cage with food and
water available
ad libitum
. They were acclimatised in
standard controlled laboratory conditions for 1 week
prior to inclusion in the experiment. On the day of the
experiment all the mice were injected subcutaneously
in the left flank with 20MCA (Sigma, USA) at a dose
of 2mg/kg, in DMSO (Sigma, USA) as vehicle. They
were then housed in sterile conditions and observed
daily until the tumours were palpable. The animals
were sacrificed under chloroform anaesthesia when
the tumours reached a size of 5 x1000 cu mm and
the tumours were immediately excised. Half of the
tumour was processed for H&E staining after fixing
in 10% formalin. The other half was fixed in 4%
et al.
Figure 1. (a) Cross section of the tumour (H&E) at an early stage. The transformed cells can be seen infiltrating the underlying
muscles. (b) Fully developed tumour (5000 cu mm) with H&E staining showing classical Herring bone pattern characteristic
of fibrosarcoma (4X).(c) Negative control tissue stained for iNOS seen at higher magnification and (d) tumour cross section
stained for iNOS with intense staining in the cancer tissues (open arrows). Note the dark stained infiltrated macrophages
stained positively for iNOS (closed arrows). (e) Negative control tissue stained for nNOS at higher power vs (f) positive
nNOS staining in the tumour sections (open arrows) (4X).
The sections were treated with 70% methanol con-
taining 0.1% H2O2 for a period of 20-30 min to block
endogenous peroxidase activity. Sections were
washed thoroughly and then treated with 20% nor-
mal horse serum (for those to be incubated with
mouse monoclonal antibody) and 20% normal goat
serum (for sections to be titrated with rabbit polyclonal
antibodies) for 3h at 4oC.
The sections were incubated for 3 days at 4oC with
primary antibodies against iNOS (1:50 mouse
monoclonal, Transduction laboratories, Kentucky, USA)
and nNOS(1:250, rabbit polyclonal, Transduction labo-
ratories, Kentucky, USA). The antibodies were diluted
in 0.01M phosphate buffer saline containing 0.5%
Triton X and 5% normal sera. Following this the sec-
tions were washed in buffer and put in secondary
paraformaldehyde in 0.1M phosphate buffer for 2-4 h
at 4o C. After several washes in buffer they were
cryoprotected in 30% sucrose overnight. Sections were
cut at 5-10 µµ
µm thickness in a Reichart jung cryostat
and stored in vials containing phosphate buffer at
4o C till use for immunohisto-cytochemistry. However,
6 mice did not develop any tumour till day 150 when
they were sacrificed and the tissue was excised from
the left flank and subjected to the same treatment as
above. Of the 25 mice injected with 20-MCA, 6 mice
were put in control group since they did not develop
tumour and rest 19 were put into the other group con-
sidered as tumour group.
H&E staining
Tissue samples were stained with haematoxylin and
eosin according to standard protocol following par-
affin embedding.
antibodies (1:200) for 5-6h at 4oC. For iNOS the sec-
ondary antibody was horse anti-mouse IgG (Vector
labs, USA) whereas for nNOS it was goat anti-rabbit
IgG (Vector labs , USA). After washing , the sections
were treated with avidin-biotin peroxidase complex
(Vector Labs, USA) for 2 h at room temperature. For
visualisation of the antigen antibody reactions the sec-
tions were treated with 0.05M acetate-imidazole buffer
(pH 7.4) containing 0.3% nickel sulphate as intensi-
fier, for 15-20 seconds. The sections were finally
rinsed in distilled water, mounted on gelatin coated
slides and air dried. They were then dehydrated in
alcohol and mounted in DPX. The slides were exam-
ined by an independent observer and the intensity of
staining was graded as - when there was no detect-
able staining and grades +, +, ++, +++ were used for
weak, moderate, strong and intense staining respec-
tively. The observer was exposed to control slides
from untreated sections to establish a baseline zero
prior to the sections from negative and positive groups.
Of the 25 animals injected with 20MCA, 19 devel-
oped solid tumours characteristic of fibrosarcoma
with Herring bone pattern (Figures 1a and b). The tu-
mours reached 200 cu mm between day 90-100 and
5000cu mm within 18+2 days after reaching 200 cu
mm, characteristically following a Gompertzian curve.
The remaining 6 mice did not develop tumours even
after 150 days of administration of 20MCA and served
as negative controls. The tumour sections stained
very strongly for iNOS (Figure 1d) and was infiltrated
with macrophages, which was not detected in con-
trol sections (Figure 1c). Neuronal NOS expression
was also significantly elevated (Figure 1f) in com-
parison to controls (Figure 1e). Table 1 gives the gra-
dation of intensity of staining by an independent ob-
Nitric oxide (NO) has been implicated in a wide range
of biological functions. It is generated enzymatically
from terminal guanidinonitrogen of L-Arginine by
NOS. Despite intensive investigations, the role of NO,
either as a primary product of LArginine/NOS path-
way or provided by donors, in carcinogenesis and
tumour cell growth remains unclear. NO and its de-
rivatives can cause DNA damage6 and cell death7 at
Table 1. Staining intensity grades for immunostaining in tumour
sections vs control
Tissue iNOS nNOS
Control (n=6) - -
Tumour (n=19) +++ ++
- no detectable staining , + weak, + moderate, ++ strong,
+++ intense staining
higher concentrations and may exert opposite effects
at lower concentrations8. Growth of solid tumours is
regulated by interactions of endothelial cells of tu-
mour vasculature, the tumour cells and the infiltrat-
ing immune cells as macrophages9. Most of these
cellular components have been shown to generate
in vitro
10,11. Indeed in the present study we found
a significant expression of iNOS in macrophages that
had infiltrated into the tumour tissue. However as
reported in other types of tumours12,13, in the present
study fibrosarcoma induced by carcinogen 20MCA
also had a high expression of iNOS unlike the con-
trol group which did not develop any tumour. This in-
dicates that NO synthesised by iNOS does play a
role in the process of tumourogenesis. Studies have
shown that iNOS transfected cells proliferate at a
slower rate indicating that NO exerts a cytostatic ef-
fect on tumour cells14 but it has been found to pro-
mote solid tumours by promoting angiogenesis12. In
the present study too a high degree of vascularisa-
tion was noted in the tumours; however further ex-
periments are warranted to implicate iNOS synthe-
sised NO in angiogenesis in this tumour type.
The surprising observation was the expression of
nNOS in the tumour section. Luck
et al
15 have shown
that nNOS system is of relevance to myogenic dif-
ferentiation and it is present at an elevated level in
inflammatory myopathies16. The expression of nNOS
in the present system could thus indicate the differ-
entiation, muscle cell death or apoptosis occurring
in the tumour. This suggests that both the isoforms
of NOS have a significant role in the process of
carcino/tumourigenesis. The present study thus opens
the door for further pharmacological experiments with
selective inhibitors of NOS with the possibility of in-
hibiting/altering the progress of the pathology. Indeed
early results have shown that cancer prevention by
et al.
curcumin could be related to inhibition of NOS gene17
or that administration of NG-nitro-L-arginine-methyl-
ester (L-NAME) a NOS inhibitor could reduce the
incidence and number of metastasis18.
SS would like to thank AIIMS for the award of Geeta
Mittal gold medal for basic research in oncology as
the present paper formed part of the material on which
the award was made.
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... Staining results were interpreted according to the scheme reported by previous authors. 21,22 ...
... Staining results were interpreted according to the scheme reported by previous authors. 21,22 ...
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This review has identified several general themes in tumor biology that indicate directions requiring research emphasis in the future: 1. A more thorough understanding of the cellular and metabolic characteristics of tumors in humans in relation to laboratory models. Development of appropriate experimental models for studying questions of relevance to human tumors is important. 2. The significance of differences of intrinsic cellular sensitivity for determining tumor control and response to radiation therapy. The sensitivity of the clonogenic stem cells and the relative importance of their growth status at the time of treatment and subsequently, their repair capacity, and effects of local extrinsic pathophysiological microenvironments such as hypoxia need to be determined for human tumors. 3. Improvements of noninvasive methods to measure various pathophysiological and metabolic indicators of tumor response to therapy. An emphasis on studies that relate measurements obtained with invasive techniques to those obtained noninvasively is necessary to interpret and appropriately use the information to improve therapy. Many of these research areas require long-term studies and commitments both by investigators and funding support agencies to understand basic mechanisms as well as to develop and characterize appropriate models and new technologies as applied to studies of human tumors. For many of the scientific questions posed there is an opportunity to apply new molecular biology approaches so that significant differences in tumor genetic and phenotypic properties and changes associated with progression of malignancy can be identified at a level of sensitivity not previously possible. Such information may then be applied to develop ways to intervene therapeutically and improve cancer control rates in humans.