Research Article
Expression of Inducible Nitric Oxide Synthase (iNOS) in Astrocytomas of Various WHO Grades with and without Malignant Progression
Serge Weis1*, Hella Wolf2, Frank Weiner2, Wolf G3 and Johannes Haybaeck2
1Division of Neuropathology, Neuromed Campus, Kepler University Hospital, School of Medicine, Johannes Kepler University, Linz, Austria
2Institute of Pathology, Otto-von-Guericke University, Magdeburg, Germany
3Institute of Medical Neurobiology, Otto-von-Guericke University, Magdeburg, Germany
*Corresponding author: Neuropathology, Neuromed Campus, Kepler University Hospital, School of Medicine, Johannes Kepler University, Wagner-Jauregg-Weg 15, A-4020 Linz, Austria
Published: 16 May, 2017
Cite this article as: Weis S, Wolf H, Weiner F, Wolf G,
Haybaeck J. Expression of Inducible
Nitric Oxide Synthase (iNOS) in
Astrocytomas of Various WHO Grades
with and without Malignant Progression.
Clin Oncol. 2017; 2: 1297.
Abstract
Nitric Oxide (NO), a free radical gas implicated in a wide variety of biological processes, is generated
in many mammalian cells by a family of enzymes, i.e. Nitric Oxide Synthases (NOS). Nitric oxide
produced by the inducible NOS isoform (iNOS or NOS II) seems to play an important role in tumor
biology showing both tumor promoter and antitumor activity.
The aim of the present study was to determine the cellular localization of iNOS in astrocytoma
(WHO grade II), anaplastic astrocytoma (WHO grade III) and glioblastoma (GBM) (WHO grade
IV) by immunohistochemistry using commercially available antibodies, as well as to detect possible
changes in astrocytomas with malignant progression compared to the non-progressive group.
Positive iNOS immunostaining was detected in all samples of astrocytoma specimens, whereas
non-tumorous brain tissue adjacent to the tumor did not show any iNOS positivity. The tumor
tissue revealed a highly inhomogeneous staining pattern. We found uniformly stained tumor
specimens and groups of markedly iNOS-positive tumor cells, besides unstained tumor tissue as
well as randomly scattered individual tumor cells expressing a marked staining. Immunoreactivity
was located within the cytoplasm of neoplastic astrocytes.There were no statistically significant
differences among astrocytomas of WHO grades II, III, and IV, between astrocytomas with and
without malignant progression. Furthermore, there was no clear correlation between the density of
iNOS-immunopositive cells and survival time.
Further studies will be necessary to elucidate the role of iNOS in concert with other signaling
pathways played in astrocytomas in order to see how and to what extent they are functionally
interrelated.
Keywords: Inducible nitric oxide synthase (iNOS); Astrocytomas; Glioblastoma; Malignant progression
Introduction
Nitric Oxide (NO) is a short-lived pleiotropic biomolecule with a multitude of biologic
functions. Since its discovery as a biologically active molecule in the late 1980s, NO is thought
to play a role as a signal molecule in many organs, in immunological and defense mechanisms
[1-3], in multiple signal transduction pathways [1,2], as well as in carcinogenesis [2,4-9]. Under physiological conditions, NO can travel long distances in order to act as an intercellular messenger
in the brain. Its targets include adjacent neurons and astrocytes. Opposite effects of NO, harmful
as well as protective, have been observed [10]. NO is a product of the conversion of L-arginine to
L-citrulline by Nitric Oxide Synthase (NOS), which exists as three enzyme classes: the calciumdependent
endothelial isoform (eNOS or NOS III), the neuronal or brain isoform (nNOS or NOS
I), and a calcium-independent inducible or immunologic isoform (iNOS or NOS II) [11]. At the
present time, the role of NO during tumor development reveals a complex picture. It may be found
in macrophages and microglial cells, in hepatocytes, neutrophils, endothelial cells, and astrocytes.
This form is important in the tumoricidal activity of T lymphocytes and the bacteriostatic response
of reticuloendothelial cells [3].
The observation that levels of calcium-independent NOS (iNOS)
are higher in malignant tissue, and the localization of iNOS to
intratumoral macrophages and microglial cells or endothelial cells
of the tumor vasculature suggests that the intratumoral environment
of these cancers results in the induction of this special isoform
(iNOS) [12,13,15-18]. Studies showing an increase in the growth
rate, vascular density and invasiveness of a human tumor cell line
transfected to constitutively express iNOS support this assumption
[19-21]. Consistently, administration of a highly selective inhibitor
of iNOS limited the invasion and growth rate of iNOS transfected
tumor cell lines as well as of other tumors expressing this isoform
[22]. In addition it was shown that lipoproteins can induce the
formation of reactive astrocytes, inducing iNOS giving experimental
support to a role played by LDL and HDL inducing a reactive
response [8]. Moreover, NO was reported to play a critical role in
neurodegenerative disorders [23,24]. So far, iNOS has been detected
in tumor cells of the human brain, breast, lung, stomach, colon,
prostate, cervix, ovary, kidney, liver, pancreas, and urinary bladder,
among others [13,15-17,25-36].
Astroglial tumors are the most frequently encountered brain
tumors. They are tumors of neuroepithelial tissue. For each tumor
entity, predicting the biological behaviour by means of histological
grading was introduced by the WHO. Astrocytoma (WHO grade II)
is a diffusely infiltrating tumor that typically affects young adults and
is characterized by a high degree of cellular differentiation and slow
growth; the tumor occurs throughout the CNS but is preferentially
located supratentorially and has an intrinsic tendency for malignant
progression to anaplastic astrocytoma and, ultimately, glioblastoma.
Anaplastic astrocytoma (WHO grade III) is a diffusely infiltrating,
malignant astrocytoma that primarily affects adults, preferentially
located in the cerebral hemispheres, and which is histologically
characterized by nuclear atypia, increased cellularity and significant
proliferative activity. The tumor may arise from diffuse astrocytoma
WHO grade II or de novo, i.e. without evidence of a less malignant
precursor lesion, and has an inherent tendency to undergo
progression. Glioblastoma or Glioblastoma Multiforme (GBM)
(WHO grade IV) is a highly malignant neuroectodermal tumor
composed of densely packed, anaplastic, and highly dedifferentiated
tumor cells making the histogenetic typing difficult.
Several pathophysiological properties important for tumor cell
survival and tumor pathology may be mediated by NO. Recent studies
have suggested a role of NO in causing increased tumor blood flow,
edema, and vascular permeability [4-6,37]. These features of tumors
are particularly prominent in pathologically high-grade tumors of
the CNS. Furthermore, cytokines found in brain tumors, such as
interleukin 1, tumor necrosis factor alpha, and gamma-interferon
induce NOS activity in vitro [7,38]. Up to now, little is known about the
expression of iNOS in astrocytomas or in progressive astrocytomas.
An increased expression of the brain and endothelial forms of NOS
(NOS I and NOS III, respectively) in astrocytic tumors was described
by [31]; the highest levels of expression were found in higher grade
tumors. Each of these two isoforms was found in tumor cells and
tumor endothelial cells. The macrophage isoform of NOS was less
frequently detected and expressed at a lower level, predominantly
in tumor endothelial cells. NADPH diaphorase staining for NOS
activity paralleled this pattern of NOS expression.
In the present study, the local evidence of iNOS in tumor tissue
of astrocytomas without and with malignant progression was
investigated immunohistochemically using a commercially available
iNOS antibody and by Western blotting technique. The study was
aimed to compare iNOS expression in astrocytomas of different
grades and behavior in order to discuss its potential roles in brain
tumor biology.
Table 1
Materials and Methods
Tissues from the following brain tumor entities were investigated:
(a) 11 cases of astrocytomas WHO grade II, (b) 10 cases of anaplastic
astrocytomas WHO grade III, (c) 17 cases of Glioblastoma Multiforme
(GBM) WHO grade IV, and (d) 12 cases of astrocytomas showing
malignant progression. Detailed information about the patients with
tumor progression is provided in Table 1. All patients received the
appropriate, state-of the art treatment.
Immunohistochemistry
Resected specimens were fixed in 4% formaldehyde and embedded
in paraffin. Deparaffinized and rehydrated 4μm thick sections were
washed in tap water, then in distilled water and finally in 0.05M Tris
buffer (pH 7.6). Blocking of unspecific binding sites was done with a
commercially available protein blocking agent (Ultra-Tech., Coulter-
Immunotech, Marseille, France) for 10 min. After removing the excess
blocking reagent, sections were incubated with the primary polyclonal
rabbit antibody (Transduction Laboratories, Biomol, Hamburg,
Germany), diluted to 1:1200 in RPMI-medium (Life Technologies,
Eckenstein, Germany). The antibody is directed against the mouse
iNOS C-terminal peptide (1131-1144) plus additional N-terminal
Cys conjugated to KLH (CKKGSALEEPKATRL). According to the
manufacturer, the antibody recognizes the iNOS 130 kDa protein
in humans, rats and mice without cross-reaction to eNOS or nNOS.
For negative control, the sections were incubated with RPMImedium
alone. Colorectal cancer specimens, likewise formalin-fixed
and paraffin embedded, were used as positive control for the iNOS
antigen in immunohistochemistry (Wolff “et al”. 2000).
After an 18h incubation of the primary antibody at 4°C, sections
were rinsed with Tris buffer and incubated with a secondary,
biotinylated goat anti-rabbit antibody (Vectastain, ABC-AP Elite
Kit; Vector Laboratories, Burlingame, USA). After incubation
with Vectastain ABC-AP reagent (avidin-biotin-complex-alkaline
phosphatase, as described by the manufacturer), sections were stained with the Fast Red chromogen system (Coulter-Immunotech.) for 15
min, counterstained with hematoxylin and mounted with glycerol.
In frozen specimens of six GBMs, the NOS activity was colocalized
by the NADPH-diaphorase reaction according to [39].
Western blotting
Protein homogenates from brain tumor specimens were prepared
in 10 volumes of buffer A (25mm Tris-HCl, pH 7.4-100mm NaCl
EDTA-1nm [ethylenebis(oxyethyl-enenitrilo)] tetraacetic acid-1mm
phenylmethylsulfonyl fluoride). Following centrifugation at 15,000
rpm for 20 min, soluble extracts were partially purified using 2',5'-
ADP agarose chromatography as described [40]. Affinity-purified
samples were fractionated on a 7.5% SDS-polyacrylamide gel,
transferred to nitrocellulose, and probed with the iNOS antibody
used for immunohistochemistry (dilution 1:1200). Immunoreactive
species were visualized by enhanced chemoluminescence.
Morphometry
The morphometrical evaluation was made at a 400x magnification
with careful registration of the morphological features. The numerical
density of immunopositive cells, of immunonegative cells, and of all
cells was determined following the 'random systematic sampling'
[41]. Briefly, in 'random systematic sampling', the first measuring
field was positioned randomly within the structure of interest: the
next measuring field was then positioned systematically adjacent
to the previous one; every second measuring field was considered
resulting in a total of ten measuring fields. Further details of the
morphometric methods have been described previously [41]. While
counting the cells, the rules of the unbiased grid were applied. Briefly,
two lines of the measuring field were defined as forbidden lines. All
cell profiles hitting the forbidden lines as well as their extensions were
not counted. All cells falling within the measuring field or touching
the non-forbidden lines and their extensions were counted [42]. The
numerical density was calculated as the number of cells per square
millimeter (n/mm2), and the Labeling Index (LI) expressed in percent,
i.e. the number of immunopositive cells divided by the number of all
cells multiplied by 100, was also determined. The numerical density of
"all cells", i.e. the number of immunonegative cells plus the number of
immunopositive cells, was also calculated.
The evaluated data were processed using the Statistical Package
for the Social Sciences (SPSS). Correlation, one-way analysis of
variance (ANOVA), Chi-square statistics, Student's t-test, and the
non-parametric Mann-Whitney U-test were used.
Figure 1
Figure 1
Immunoreactivities for iNOS in specimens of astrocytoma WHO
grade II (A), anaplastic astrocytoma WHO Grade III (B), and glioblastoma
multiforme WHO grade IV (C, D) (avidin-biotin complex, alkaline phosphatase,
ABC-AP, counterstained with hematoxylin). (E) Band at 130kDa showing the
presence of iNOS in tissues of four cases of GBM WHO grade IV on Western
blot analysis.
Results
The clinicopathological data (i.e. age of the patient, gender WHO
grade, change of grade with progression, interval in years between
surgical interventions) of the examined astrocytoma cases with
malignant progression are given in Table 1.
Positive iNOS immunostaining was detected in all samples of
tumor tissue (Figure 1). The presence of iNOS in the tumor tissue
was also detected by Western blotting technique showing a band of
130 kDa (Figure 1E). In the negative control, immunoreactivity was
completely lacking, while the positive control (colorectal carcinoma)
displayed a clear-cut immunopositivity, which was co-localized with
NADPH-diaphorase activity of the tumor cells.
Immunoreactive tumor cells were both, diffusely or focally,
distributed throughout the tumor tissue. The immunoreaction was
seen homogeneously only in the cytoplasm of the astrocytic tumor
cells. In general, it has to be noted that for all examined groups the
quantitative data showed a high interindividual variability reflected
by high values of the standard deviation and the standard error of
the mean.
Astrocytomas without progression
When analyzing the overall differences among astrocytomas of
grades II, III, and IV, no significant differences were seen on ANOVA
analysis for the numerical density of all cells, immunopositive or
immunonegative cells, as well as for the labeling index (Table 2). A
post-hoc analysis comparing the three grades separately with each
other did also not show significant differences between grades II and
III, grades III and IV, and grades II and IV although the numerical
density of immunopositive cells was increased in the groups with the
higher grades.
Astrocytomas with progression.
When analyzing the overall differences between grades II, III,
and IV in astrocytomas with malignant progression, no significant
differences were seen by ANOVA analysis for the numerical density
of all cells, immunopositive or immunonegative cells, as well as for
the labeling index (Table 3). A post-hoc analysis comparing the three
grades did also not show significant differences between grades II and
III, grades III and IV, and grades II and IV although the numerical
density of immunopositive cells was increased in the groups with the
higher grades.
Differences between progression versus no progression
The comparison of the tumor group with progression with
that without progression for all grades (i.e. II, III, IV) did show a
significant increase in the numerical density of immunopositive
cells (Table 4). When assessing the differences between these two
groups, by analyzing the three WHO grades separately, no significant
differences between the progressive and the non-progressive group
were noted (Table 4).
Number of operations
Within the group of astrocytomas with malignant progression,
no significant differences were noted between the different number
of operations (Table 5). When comparing the first operation with the
subsequent operations grouped together in one group, no significant
differences were obvious.
Correlation with survival
No significant correlation could be drawn between the evaluated
morphometric parameters and survival time.
Table 2
Table 2
Numerical density (n/mm2) of immunopositive (im-pos) cells and
immunonegative (im-neg) cells and labeling index (%) in astrocytomas without
Malignant Progression.
Table 3
Table 3
Numerical density (n/mm2) of immunopositive (im-pos) cells and immunonegative (im-neg) cells and labeling index (%) in astrocytomas with malignant
progression: differences among grades II, III, and IV.
Discussion
The clinicopathological data (i.e. age of the patient, gender WHO
grade, change of grade with progression, interval in years between
surgical interventions) of the examined astrocytoma cases with
malignant progression are given in Table 1.
Positive iNOS immunostaining was detected in all samples of
tumor tissue (Figure 1). The presence of iNOS in the tumor tissue
was also detected by Western blotting technique showing a band of
130 kDa (Figure 1E). In the negative control, immunoreactivity was
completely lacking, while the positive control (colorectal carcinoma)
displayed a clear-cut immunopositivity, which was co-localized with
NADPH-diaphorase activity of the tumor cells.
Immunoreactive tumor cells were both, diffusely or focally,
distributed throughout the tumor tissue. The immunoreaction was
seen homogeneously only in the cytoplasm of the astrocytic tumor
cells. In general, it has to be noted that for all examined groups the
quantitative data showed a high interindividual variability reflected
by high values of the standard deviation and the standard error of
the mean.
Astrocytomas without progression
When analyzing the overall differences among astrocytomas of
grades II, III, and IV, no significant differences were seen on ANOVA
analysis for the numerical density of all cells, immunopositive or
immunonegative cells, as well as for the labeling index (Table 2). A
post-hoc analysis comparing the three grades separately with each
other did also not show significant differences between grades II and
III, grades III and IV, and grades II and IV although the numerical
density of immunopositive cells was increased in the groups with the
higher grades.
Astrocytomas with progression
When analyzing the overall differences between grades II, III,
and IV in astrocytomas with malignant progression, no significant
differences were seen by ANOVA analysis for the numerical density
of all cells, immunopositive or immunonegative cells, as well as for
the labeling index (Table 3). A post-hoc analysis comparing the three
grades did also not show significant differences between grades II and
III, grades III and IV, and grades II and IV although the numerical
density of immunopositive cells was increased in the groups with the
higher grades.
Differences between progression versus no progression
The comparison of the tumor group with progression with
that without progression for all grades (i.e. II, III, IV) did show a
significant increase in the numerical density of immunopositive
cells (Table 4). When assessing the differences between these two
groups, by analyzing the three WHO grades separately, no significant
differences between the progressive and the non-progressive group
were noted (Table 4).
Number of operations
Within the group of astrocytomas with malignant progression,
no significant differences were noted between the different number
of operations (Table 5). When comparing the first operation with the
subsequent operations grouped together in one group, no significant
differences were obvious.
Correlation with survival
No significant correlation could be drawn between the evaluated
morphometric parameters and survival time.
Table 4
Table 4
Numerical density (n/mm2) of immunopositive (im-pos) cells and
immunonegative (im-neg) cells and labeling index (%) in astrocytomas:
progression versus no progression.
Table 5
Table 5
Numerical density (n/mm2) of immunopositive (im-pos) cells and
immunonegative (im-neg) cells and labeling index (%) in astrocytomas with
malignant progression: Differences between time of operations (OP).
Discussion
So far, only few and rather controversial immunohistochemical
studies describing the occurrence of iNOS in various brain tumors
were published. The pattern of expression of the three NOS isoforms in
primary CNS neoplasms including astrocytic tumors, meningiomas,
schwannomas, ependymomas, medulloblastomas, and mixed gliomas
was examined immunohistochemically [31]. Unfortunately, the
authors mixed up the roman numbering of the three isoforms with
their respective letter designations. Thus, endothelial NOS reads as II
instead of III and inducible NOS reads as III instead of II. With regard
to the inducible NOS isoforms, these authors obtained the following
results: (1) iNOS immunoreactivity was less prevalent in the tumors
and was usually confined to the tumor vasculature, (2) distinctively
higher levels of iNOS were expressed in high grade astrocytic
tumors compared to WHO grade II tumors and normal brain tissue,
(3) iNOS immunoreactivity was rarely detectable in tumor cells,
although moderate staining was seen in tumor endothelial cells, and
(4) Western blots revealed no immunoreactivity in lysates of either
normal brain or tumor [31].
Other results were published, reporting upregulation of iNOS in
nearly 50% of gliomas, although only nNOS expression correlated
with tumor grade [36]. The co-expression of NOS I-III was studied
in 220 GBMs showing that all of the specimens revealed some NOS
expression with NOS II expression in macrophages, microglia
and endothelial cells, NOS III and I in GBM cells, and NOS III in endothelial cells [43]. Inducible NOS II in any expression grade was
observed in 47.5% of the specimens. Significant correlations were
observed for the expression of the macrophage marker Ki-MIP with
NOS II, and VEGF-R1 with NOS II and NOS III [43]. Low numbers of
macrophages/microglia expressing NOS II in GBMs were described;
no information was provided as to whether tumor cells were stained
[44].
Tumor cells expressing all three NOS isoforms in brain tumors
except for GBM and metastatic adenocarcinoma were reported [45].
In four tumors, cells (lymphocytes and macrophages) were intensely
labeled with MacNOS in and around the blood vessels. The authors
concluded that nitric oxide is produced in the tumor cells and
endothelium of tumor vasculature while occasionally glial cells may
also produce it [45]. NOS and 5’-nucleotidase were observed in GBM
tumor cells [46]. suggesting that both the cytotoxic effects due to NO
production by tumor cells and the non-catalytic role of membrane 5’
nucleotidase acting as an adhesive molecule favor tumor invasiveness
[46]. The apparent association of increased iNOS immunoreactivity
and NADPH-diaphorase activity with the histological grade of
astrocytic tumors suggested that NO may be an important molecule
in mediating pathological processes characteristic of highly malignant
tumors. High levels of iNOS and NO production may influence brain
tumor growth in vivo [47].
Glioma Stem Cells (GSCs) produce nitric oxide via high NOS2
expression which correlates with decreased survival in human
glioma patients. NOS2 inhibition slows glioma growth in a murine
intracranial model. Thus, NOS2 inhibition may be an efficacious
approach to treating this devastating disease [48]. Reactive glial
cells and the endothelium of small blood vessels displayed strong
NADPH-d and iNOS activities in edematous peritumoral tissue
[49]. In tumor cortex, NADPH-d and iNOS positive neurons were
reduced in number and their dendrites were thin and interrupted,
and infiltrates of NADPH-d and iNOS-positive tumor cells were
frequent. Expression of eNOS in tumor vessels was significantly
correlated with histological grade and proliferative potential [50].
Induction of VEGF gene expression in human A‑172 GBM cells
by NO is mediated through guanylate cyclase activity and requires
on‑going protein synthesis [51]. NO generated by iNOS expression
inhibits hypoxia-inducible factor-1 (HIF-1) activity in hypoxic C6
glioma cells reveals a negative feedback loop in the HIV-1 → iNOS
cascade [52]. As a matter of fact, the authors showed increased NO
synthesis by cytokine exposure or iNOS overexpression neutralized
the cytotoxicity of BCNU and CCNU, but not cisplatin in rat C6
glioma cells. Expression of iNOS inhibited HIF-1 activity under
hypoxia in C6 glioma cells transfected with a VEGF promotordriven
luciferase gene. Pretreatment of C6 cells with the antioxidant
N-acetyl-1-cysteine, mollified the inhibitory effect of iNOS on HIF-1
binding. HIF-1 is known to be present at high levels in human tumors
and that it is crucial in tumor promotion by up-regulating several
target genes. HIF-1 stimulates the production of NO through the
induction of inducible NO synthase (iNOS). HIF-1alpha and iNOS
expressions did not correlate with patient survival [53].
By investigating immunohistochemical expression of COX-2,
iNOS, and VEGF in 51 high-grade astrocytomas the relationship with
microvessel density and prognostic significance were determined.
Stepwise increase of immunoreactive scores for COX-2, iNOS, and
VEGF was found from astrogliosis, through low-grade to high-grade
astrocytoma. The COX-2 expression strongly correlated with iNOS,
VEGF, and high MVD, both overall and in all tumors, whereas
iNOS expression was weakly associated with VEGF and high MVD.
Univariate analysis revealed a significant association between COX-2
overexpression and a poor outcome [54].
To verify the hypothesis that NO is involved in p53‑dependent
response to many kinds of stress, such as heat shock and changes
in cellular metabolism, the effect of NO produced endogenously by
heat‑shocked cells on nonstressed cells using a human glioblastoma
cell line, A‑172, and its mutant p53 (mp53) transfectant (A‑172/
mp53) was examined [55]. The accumulation of iNOS was caused
by heat treatment of the mtp53 cells but not of the wild‑type p53
(wtp53) cells. The accumulation of heat shock protein 72 (hsp72) and
p53 was observed in nontreated mtp53 cells cocultivated with heated
mp53 cells, and the accumulation of these proteins was suppressed
by the addition of a specific iNOS inhibitor, aminoguanidine, to
the medium. Furthermore, the accumulation of these proteins was
observed in the wtp53 cells after exposure to the conditioned medium
by preculture of the heated mp53 cells, and the accumulation was
completely blocked by the addition of a specific NO scavenger, 2‑
(4‑carboxyphenyl)‑ 4,4,5,5‑tetramethyl‑imidazoline‑1‑ oxyl‑3‑oxide,
to the medium. The accumulation of hsp72 and p53 in NO‑recipient
cells cocultivated with heated NO‑donor cells provides the first
evidence for an intercellular signal transduction pathway via NO as
intermediate without cell‑to‑cell interactions such as gap junctions.
Furthermore, modifications of p53 in gliomas in vivo could be
mediated by peroxynitrite, a highly reactive metabolite of NO [47].
Accumulation of iNOS was caused by X irradiation of mutant TP53
but not of the wild-type TP53 glioblastoma cells [55].
Several pathways might be involved in the patheogenic repertoire
of NO. There is evidence for a regulatory axis of high grade glioma cell movement from NO through MMP-1, with NOS inhibitor
results showing promise for future pharmacologic investigation
[56]. Intracellular NO affects IRE1-alpha-dependent CREB
phosphorylation in human glioma cells. Therefore, an IRE1-alphadependent
phospho-CREB signaling pathway responsive to NO/
Ca(2+) may play an important role in regulating ER-related cell death
in glioma [57]. Results have been reported indicating that the NO/
Ca2þ/CaM/ERK signalling pathway is a mechanism mediating the
mitogenic effect of IL-1b in human astrocytes. A nitric oxide/Ca2þ/
calmodulin/ERK1/2 mitogenactivated protein kinase pathway is
involved in the mitogenic effect of IL-1ß in human astrocytoma cells
[77]. It was observed that the stimulatory effect of 5-AzadC on iNOS
expression was independent of DNA demethylation. α9β1 integrinmediated
cell migration utilizes the iNOS pathway, and inhibition of
the migratory potential of glioma cells by simultaneous knockdown
of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin
and iNOS levels [59].
iNOS levels in GBM were positively correlated with IL-1ß mRNA,
but not with other cytokines like TNF-alpha, interferon-gamma, TGF
ß1 and TGF ß2 [60]. The survival duration was enhanced when levels
of IL-1ß mRNA were elevated or when levels of TGFß2 were low,
but was independent of the level of iNOS mRNA within the tumor
[60]. Incubation of C6 astrocytoma cells with bacterial endotoxin
(lipopolysaccharide; LPS) plus interferon‑gamma (IFN‑γ), or with a
combination of cytokines (TNF‑α, IL-1ß, and IFN‑γ) leads to high
levels of iNOS expression [61]. It was concluded that astrocytoma
cells possess a cytokine‑inducible Ca(++)‑calmodulin‑independent
NO‑synthase, whose activation seems to occur with a mechanism
different from that described for LPS [62]. Lipopolysaccharide and
interferon‑γ, which are able to strongly induce iNOS in astrocytoma
cells, can rapidly inhibit the NO production generated by the
constitutive NOS isoform [63]. Thus, the results suggest a possible
role of a constitutive NOS isoform in astrocytes as a control function
on iNOS induction [63]. Data show that IFN-γ increases the synthesis
and release of NO by cultured astrocytoma cells and this could
co‑participate in the MHC II antigen expression by this cell type [64].
Several papers were published, in which the production of NO
by brain tumor cells and the influence of possible regulatory factors
was studied in cell culture systems. RhoA was identified as a negative
regulator of iNOS expression via the inactivation of NF-kappaB
in transformed brain cell lines C(6) glioma, human astrocytoma
(T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes.
It was reported that downregulation of RhoA by lovastatin resulted
in increased iNOS expression via the activation of NF-kappaBCBP/
p300 pathway in transformed brain cells [9,65-67]. It has been
observed that conditioned medium from activated microglia resulted
in the induction of iNOS in C6 cells, and IL-1ß was shown to be a key
regulator of iNOS induction [68-70].
Some therapeutically used agents were described to potentiate
the iNOS activity. As astrocytes could modulate CNS autoimmunity
iNOS-mediated production of immunoregulatory free radical NO,
the effect of taxol, a microtubule-stabilizing agent, on NO synthesis
has been investigated in rat astrocytes. Taxol, either alone or in
combination with interferon-γ, induced NO generation in primary
astrocytes and astrocytoma C6 cells in a dose- and time-dependent
manner NO release by taxol-stimulated astrocytes was blocked with
the microtubule-depolymerizing agent colchicine [71]. Although a
heterogeneous nitric oxide production by xenografted glioma cells
may impact VEGF-A and Cyclin D1 expression levels, a reduction
of nitric oxide levels by nitric oxide scavenging could be an efficient
approach to treat glioma [72]. The reciprocal activation of glioma cells
and microglia via ROS-dependent iNOS/NO elevation at least partially
mediated by TNF-α and MCP-1 was shown by [73]. L‑NMMA and
aminoguanidine, competitive inhibitors of iNOS, suppressed NO
production as measured by the NO by-product, nitrite, as did IFN‑ß.
Dexamethasone enhanced the NO production, and IFN‑ß decreased
the amount of the enhancement. Neither IL‑10 nor TGF‑ß inhibited
nitrite production. High levels of NO suppress IL‑8 production by
T98G GBM cells, and murine IL‑1 alpha plays a major role in the
induction of IL‑8 production by these T98G cells [74]. It is, therefore,
possible that excessive production of NO during the interaction
of glioma cells with macrophages may play a regulatory role in
chemokine production, thus mitigating inflammatory responses.
IL-17 caused a dose-dependent enhancement of IFN-γ-triggered
NO synthesis in both mouse and rat primary astrocytes [75]. IFN-γ-
triggered expression of mRNA for iNOS, but not for its transcription
factor interferon regulatory factor-1, which was markedly elevated in
IL-17-treated astrocytes.
Massive production of NO by iNOS has been shown to exert
tumoricidal effects. However, NO may enhance vasodilation and
promote neovascularization, thereby facilitating tumor growth.
Compared to the effects of NO on tumor cell death and survival,
correlation between NO and cytotoxicity of chemotherapeutic
reagents in glioma have been less well characterized. HIF-1
contributes to iNOS induction under hypoxia. It was reported that that
increased NO synthesis by cytokine exposure or iNOS overexpression
neutralized the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea
(BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU),
but not cisplatin, in rat C6 glioma cells. That NO generated by
iNOS expression inhibits HIF-1 activity in hypoxic C6 cells reveals
a negative feedback loop in the HIF-1 → iNOS cascade [52]. Oral
administration of L-arginine or hydroxyurea selectively increased
tumor permeability, which is likely mediated by alteration in cGMP
levels. The findings of the authors suggest that use of oral NO donors
may be a strategy to enhance the delivery of chemotherapeutics to
malignant brain tumors [76].
Tumors prone to edema formation, such as high grade
astrocytomas, were more likely to have higher levels of NOS II and
NOS III reactivity than tumors which are not characterized by edema
formation, such as juvenile pilocytic astrocytomas or schwannomas
[31]. Thus, NO produced by these tumor capillaries is likely to
contribute to edema formation, and one strategy for selectively
reducing tumor blood flow and edema might involve inhibition of
NO production. Interestingly, dexamethasone, a corticosteroid used
to treat increased intracranial pressure due to brain tumor edema, is a well-known inhibitor of NOS III but not of the constitutive
NOS I and NOS II isoforms [77]. Also, the presence of NOS II and
NOS I in tumor endothelial cells, often at high levels, suggests that
other inhibitors of NOS which block NO production by these NOS
isoforms may be effective in further reducing tumor edema and blood
supply to the tumor by selectively blocking NOS activity in the tumor
cells and tumor endothelial cells.
NO plays an important role in recent conceptions of tumor
pathogenesis, although the biologic role NO plays in malignancies is
still unclear. The supraphysiologic production of NO in malignant
tissue by iNOS may cause cytotoxicity, and supports as inhibits immune defense mechanisms as described [78]. Furthermore, NO
may increase tumor blood flow and promote angiogenesis with
NO having both pro- and antitumor-actions depending on its
concentration As seen in the present study, iNOS expression in tumor
cells is highly inhomogeneous and, hence, the role of NO must be
quite different with regard to the situation within the NO-producing
cell and transcellular effects on surrounding tissue.
NO may be responsible for increasing blood flow to tumors
expressing high levels of NOS [4]. Neuronal NO regulates cerebral
blood flow in the normal brain, and endothelial-derived NO results
in vasodilatation of normal blood vessels and inhibits platelet
aggregation [79]. NO has been identified as an important regulator
of tumor blood flow in experimental tumors in mice, and inhibition
of NO production in these tumors by systemically administered NOS
inhibitors decreased tumor blood flow and reduced tumor growth
[80]. Rapidly growing tumors such as GBMs are highly vascular and
have altered blood flow dynamics [81]. NO production by tumor
cells and tumor endothelial cells may play, therefore, a critical role
in ensuring maximum blood flow to the tumor cells. NO production
by capillary endothelial cells influences the degree of vascular
permeability in blood vessels, and NOS inhibitors can decrease local
edema formation by experimental tumors in mice [5].
In summary, nitric oxide has both physiological roles (e.g.,
neurotransmitter-like activity, stimulation of cyclic GMP), and
pathophysiological roles (e.g., neoplastic transformation, tumor
neovascularization, induction of apoptosis, free radical damage).
Moreover, whether nitric oxide is neuroprotective or neurotoxic
in a given disease state, or whether it enhances or diminishes
chemotherapeutic efficacy in malignant neoplasia, is unresolved [82].
Emerging knowledge of the dual and diverging role of nitric oxide
in glioma biology has focused on possibilities to achieve anti-glioma
effects by modulation of Nitric Oxide (NO) release and function in
these tumors. NO has been shown to influence proliferation of glioma
cells, vascular function in gliomas, invasive capacity of gliomas,
effects of chemo and radiotherapy and also immune reactivity against
these tumors. The mechanisms behind the reported diverse and dual
effects of NO in glioma biology are multiple. Some of the diversity can
be explained by different experimental setups as in vitro versus in vivo
models but the cellular sources, timing, absolute levels and gradients
play a decisive role for the effects of NO on glioma biology. Current
research in this field is hampered by the lack of inhibitors and donors
approved for clinical use [83].
In the present study, the expression of the inducible
isoform of the NO producing enzyme, iNOS, could be distinctly
demonstrated in astrocytomas of various grades of malignancy.
The immunohistochemical iNOS reactions did not show a clear
correlation with tumor grade or survival. Further investigations of
the pathophysiologic and protective roles of NO and the influence
of NO inhibitors on tumor growth in brain tumors will be necessary
to elucidate the role of iNOS in astrocytomas and to see how and to
what extent they are functionally interrelated. NO plays an intriguing,
controversial and complicated role in tumor growth, survival and
invasion.
Acknowledgement
The help of Dr. KH Smalla in preparing the Western blots and of Ida C. Llenos, MD, in correcting the manuscript is highly appreciated.
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