AND PARASITOLOGY, 4/7, 2001
Bulgarian Academy of Sciences
Nitric oxide and cancer
R. ALEXANDROVA1, M. MILEVA,2 E. ZVETKOVA3
Institute of Experimental Pathology and Parasitology,
BAS, Acad. G. Bonchev Str., Bl.25, 1113 Sofia, firstname.lastname@example.org
Neonatal Research Laboratory, Department of Pediatrics,
New York Medical College, New York 10595, USA
Instutute of Experimental Morphology and Anthropology,
BAS, Acad. G. Bonchev Str., Bl.25, 1113 Sofia
Nitric oxide (NO), a potent biological mediator, plays a key role in physiological as well as pathological pro-
cesses. An area of great interest is the role of nitric oxide in the growth and metastasis of solid tumours, where it
seems to have a complex action including both inhibitory and tumour-promoting activities. Modification of ni-
tric oxide synthetase (NOS) activity in tumours, and hence NO biosynthesis, may be regarded as a promising
means for selective tumour blood flow modification providing a novel approach for reducing tumour oxygen-
ation aimed at enhancing the efficiency of hypoxia-mediated, bioreductively activated anti-cancer drugs.
Key words: nitric oxide (NO), nitric oxide synthetases (NOS), cancer – inhibitory and tumour promoting
Nitric oxide can cause cell death throughout oxidative
Nitric oxide has generated an exponential stress, disrupted energy metabolism, DNA
amount of research since its identification damage, activation of poly(ADP-ribose)
in 1987. During the past decade this small polymerase, or dysregulation of cytosolic
molecule has emerged as an important me- calcium (T h o m s e n, M i l e s, 1998).
diator in physiological and pathological Most of the cellular components as the
processes (D e B e l d e r, R a d o m s k i, tumour mass (tumor cells themselves, and
1994; D a w s o n, D a w s o n, 1995; the immune cells infiltrate) have been
G r o s s, V o l i n, 1995; B r e n n a n et shown to generate NO in vitro. The expres-
al., 1999). NO is the product of the conver- sion of NOS in neoplasms provokes the
sion of L-arginine to L-cytrulline by nitric question about a physiological role for tu-
oxide synthetase (NOS). The wide variety mour associated NO production (M u r t a
of roles described for NO can be placed into et al., 1999).
one of three categories: i) an intracellular Nitric oxide synthetases
signal, ii) a transcellular messenger, or iii) NOS are unique among eukariotic enzymes
a cytotoxic species (Knowles, Moncada, in being dimeric, calmodulin-dependent or
1994). NO has several essential roles in calmodulin-containing cytochrome P-450-
mammals, but unregulated NO production like haemoproteins that combine reductase
and oxygenase catalitic domains in one cell lines studies (T h o m s e n et al.,
monomer. They catalise the formation of 1995a; T h o m s e n, M i l e s, 1998).
NO from L-arginine in the presence of Studies of NOS at the level of mRNA have
NADPH and molecular oxygen shown that some colon epithelial cell lines
(G r i f f i t h, S t u e h r, 1995; C h i n j e, (WiDr, DLD-1, SW-480, SW-620) express
S t r a t f o r d, 1997). NOS exists as three eNOS, with mRNA for nNOS present only
isoforms; the calcium-dependent endothe- in the SW-480 cell line. mRNA for iNOS is
lial (eNOS), neuronal (nNOS) and the cal- expressed by cytokine stimulated and
cium-independent inducible (iNOS) unstimulated SW-480, SW-620, and DLD-
(K n o w l e s, M o n c a d a, 1994). Of 1 cells but none has noted in WiDr cells.
these, the endothelial cell-specific form There is little correlation between mRNA
(eNOS) can play an important role in vascu- expression and enzyme activity suggesting
lar development, maintenance of vascular a considerable degree of post-transcrip-
tone and tumour growth. A third, inducible, tional regulation (T h o m s e n, M i l e s,
calcium-independent form (iNOS) may be 1998).
found in macrophages, hepatocytes, neutro- NO as an inhibitor of tumour growth
phils, endothelial cells and astrocytes. This In human tumours the role of NO has not
form is important in the immunogenic and been established. NO produces multiple ef-
cytotoxic response of T-lymphocytes and fects that can influence the outcome of tu-
macrophages as well as in the bacteriostatic mour growth and metastasis. This molecule
activity of reticuloendothelial cells regulates vasodilatation and platelet aggre-
(B r e n n a n et al., 1999). The expression gation (R a d o m s k i et al., 1990;
of iNOS in human infectious, autoimmune, D a w s o n, D a w s o n, 1995; G r o s s,
and chronically inflammatory diseases as V o l i n, 1995) which affect tumour cell
well as several other human disorders is arrest in capillaries (L i et al., 1991). NO
well established. It is becoming clear that is also a major cytotoxic mediator secreted
NO, including iNOS derived reactive nitro- by activated macrophages (N a t h a n,
gen species, comprises both regulatory and X i e, 1994) and endothelial cells (L i et
effector functions (K r o n k e et al., al., 1991). It is shown to be responsible for
1998). the destruction of tumour cells passing
NOS expression in tumour cell lines through capillary beds. The production of
The first report of NOS expression in hu- endogenous NO is associated with
man tumour cell lines belongs to Radomski apoptosis of tumorigenic cells. (C u i et al.,
et al. (1991) who studied human colorectal 1994). Cytotoxicity as a result of a substan-
adenocarcinoma cell lines from a primary tial NO-formation is established to initiate
tumour (SW-480) and a lymph node me- apoptosis, characterized by upregulation of
tastasis (SW-620) derived from the same the tumour suppressor p53, changes in the
patient. Both cell lines were shown to ex- expression of pro- and anti-apoptotic Bcl-2
press constitutively a calcium-independent family members, cytochrome c relocation,
NOS activity. Constitutive expression of activation of caspases, chromatin condensa-
calcium dependent NOS has also been re- tion, and DNK fragmentation (B r u n e et
ported in the cervical epithelial cell line, al., 1999). Taken together, these results
ME-180. Many reports show induction of suggest the possibility that production of
iNOS in a range of other human cell lines in endogenous NO may be detrimental to tu-
response to cytokine stimulation, including mour cell survival and production of me-
the adenocarcinoma line DLD-1, a glioblas- tastasis (X i e et al., 1995b, 1996). Nu-
toma line A-172, and an ovarian line merous in vivo and in vitro studies support
NIH:OVCAR-3. Constitutive expression this hypothesis (C u i et al., 1994; X i e et
and induction of iNOS, however, has not al., 1995a, b; D o n g et al., 1994). For
been demonstrated in some human breast example, it has been found an inverse corre-
lation between production of endogenous isoforms has been found in tumour cells and
NO and the ability of circulating K-1735 tu- tumour endothelial cells (C o b b s et al.,
mour cells to survive and produce me- 1995).
tastases (D o n g et al., 1994). The data The role of nitric oxide (NO) in tumori-
demonstrate that the introduction of an en- genesis is multifactorial. NO could partici-
zymatically active iNOS gene into highly pate in the complicated process of carcino-
metastatic murine melanoma K-1735 C4 genesis by mediating DNA damage in early
cells (which express low levels of iNOS) in- phases of tumorigenesis, support tumour
duces apoptosis, suppresses growth, and ab- progression through the induction of angio-
rogates metastasis (X i e et al., 1995b, genesis and the suppression of the immune
1996). Some other reports have also sug- response (L a l a, O r u c e v i c, 1998). It
gested that the expression of iNOS can in- has been demonstrated that oxygen radicals
fluence tumour growth and metastasis by and nitrogen oxide derivatives such as
regulating vasodilation and platelet aggre- peroxynitrite and NO2 can effectively dam-
gation (P a l m e r et al., 1988; age DNA despite the presence of multiple
R a d o m s k i et al., 1990), inhibiting an- antioxidant defense and repair systems.
giogenesis (S a k k o u l a et al., 1994) and Such damage is thought to make a signifi-
inducing programmed cell death (A l b i n a cant contribution to the age-related devel-
et al., 1993; B r u n e et al., 1999). opment of cancer (M a e d a, A k a i d e,
NO as a promotor of tumour growth 1998). It is also proposed that increased
While a few reports indicate that the pres- NO production may select mutant p53 cells
ence of NO in tumour cells or their mi- and contribute to human carcinogenesis and
croenvironment is detrimental to tumour tumour progression (A m b s et al., 1997).
cell survival and consequently their meta- There is increasing evidence for a role of
static ability, a large body of clinical and NO in angiogenesis. It has recently been re-
experimental data suggest a promoting role ported that generation of angiogenic activ-
of NO in tumour progression and metasta- ity by human monocytes requires a NOS ef-
sis. Expression of NOS has been found to be fector mechanism (L e i b o v i c h et al.,
correlated with the grade of some human 1994) and that NO plays an important role
tumours, such as breast, ovarian, cervix, in the angiogenesis associated with wound
gastric, head and neck cancer (T h o m s e n healing (K o n t u r e k et al., 1993). NO
et al., 1994, 1995b; R o s b e et al., also functions as a vascular permeability
1995; T h o m s e n, M i l e s, 1998) and factor (D a w s o n, D a w s o n, 1995;
associated with enhanced metastasis G r o s s, Vo l i n, 1995). Other important
(D u e ñ a s - G o n z a l e s et al., 1997). factors that affect the vascular permeability
In these cases high levels of NOS activity of solid tumour are bradykinin and a vascu-
have been detected, while the enzyme activ- lar permeability factor (VPF, also called
ity is below detectable levels in normal tis- vascular endothelial growth factor –
sues from non-cancer patients (T h o m - VEGF). It has been reported that VEGF in-
s e n, M i l e s, 1998; M u r t a et al., duces angiogenesis via formation of NO.
1999). In addition, malignant central ner- Consequently, it is plausible that the angio-
vous system neoplasms express high levels genic potential as well as the vascular per-
of NOS activity which may be associated meability enhancing effect of NO may
with pathophysiological processes deter- faciliate rapid growth of solid tumours,
mining their growth. It has been demon- which have great demands for various nutri-
strated an increased expression of the brain ent factors (M a e d a, A k a i d e, 1998).
and endothelial forms of NOS (nNOS and Furthermore, bradykinin is known to acti-
eNOS) in astrocytic tumours, and the high- vate an eNOS. Because bradykinin is gener-
est levels of expression has been found in ated effectivelly in tumours, bradykinin -
highest grade tumours. Each of these two NO interplay may become an important is-
sue in tumour growth (M a e d a, A k a i d e, man. This has most frequently been mea-
1998). The invasion-stimulating effects of sured by analysis of nitrate concentrations
NO are also due to an upregulation of ma- in plasma, as a breakdown product of NO.
trix metalloproteases and a downregulation Several of these studies associate increased
of their natural inhibitors. Recent reports NO production with the toxic effects of
implicate NOS involvement in degradation cytokine therapy, such as circulatory
of articular cartilage and show that NO acti- changes and hypotension. But there are
vates metalloproteinase enzymes in studies indicating that patients with the
chondrocytes and cartilage tissue from hu- highest plasma nitrate levels show the high-
man, bovine and rabbit sources. The subse- est response to the therapy (M i l e s et al.,
quent loss of integrity of extracellular ma- 1994; T h o m s e n et al., 1992, 1998).
trix and basement membranes would pro- On the other hand, it has been demonstrated
mote angiogenesis, invasion and the meta- that the administration of NOS inhibitors in
static process (T h o m s e n, M i l e s, 1998; mice could decrease IL-2/NO-induced cap-
L a l a, O r u c e v i c, 1998; H a s s a n et illary leakage and improve the IL-2-induced
al., 1998). antitumour effector cell activation, stimu-
Possible mechanism(s) of the dual role lating by this way tumour regression
of NO in tumour biology (O r u c e v i c, L a l a, 1998).
It is proposed that low concentrations of NO It has been proposed that selected NO-
can be pro-angiogenic and pro-tumour blocking drugs may be useful in the treat-
growth inducers, whereas hihger NO con- ment of certain human cancers – either as
centrations can have the opposite effects single agents or as a part of combined thera-
(C h i n j e, S t r a t f o r d, 1997). The effect pies. In accordance with that, treatment of
of NO production in tumour biology may tumour-bearing mice with NO-blocking
change during tumour progression (A m b s agents reduce the growth and vascularity of
et al., 1997). This hypothesis is supported primary tumours and their spontaneous me-
by data investigating the role of NO in can- tastases (L a l a, O r u c e v i c, 1998). The
cer metastasis. After in vitro incubation NO-inhibitory ϖ3 polyunsaturated fatty ac-
with cytokines or lypopolysaccharide (LPS), ids are known to reduce the risk of colon
non-metastatic cells exhibited a high level cancer in both man and rat. Arginine ana-
of inducible NOS activity and NO produc- logues block the promotional phase of neo-
tion, whereas metastatic cells did not (X i e plastic transformation of mouse fibroblasts.
et al., 1996). Cancer growth can be stimu- On the other hand, they could potentiate
lated as well as inhibited by the immune pulmonary metastasis of Lewis lung carci-
system. The intratumoural macrophageal noma and B16 melanoma cells (Z i d e k,
arginine metabolism is one molecular ex- M a s e k, 1998).
planation for the dual ability of the immune Interestingly, dexamethasone, a corticos-
system to inhibit or stimulate tumour teroid used to treat increased intracranial
growth. It has been suggested that arginine pressure due to brain tumour edema, is a
metabolism in the tumour bed, yielding cit- well known inhibitor of iNOS but not of the
rulline and NO, favours tumour rejection, constitutive eNOS and nNOS isoforms. The
whereas production of ornithine and urea inhibition of iNOS may contribute to the
could promote tumour growth (M u r t a et therapeutic effects seen in brain tumour pa-
al., 1999). tients treated with dexamethasone
(R a d o m s k i et al., 1990).
NO, NOS and cancer therapy
Tumour cytokine therapy, using Interleulin-
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T h o m s e n, L. L., D. M i l e s. Role of nitric oxide in Received, 14.11.2001