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Introduction
Colon cancer, the third leading cause of cancer in the United States and one of the most common human malignancies in
the Western world, is highly preventable. It is estimated that there will be over 150 000 new cases and
52 000 deaths from colon cancer in 2007 in the United
States[1]. These dreadful statistics underscore the pressing need for the development of new and
effective methods for its prevention. Of the several strategies being currently pursued by various groups around the world,
chemoprevention, defined as the application of natural or synthetic agents to prevent the development or recurrence of
cancer, holds credible promise as an effective modality that will decrease the incidence of cancer in at-risk populations.
The last two decades have witnessed an almost explosive interest in screening both natural and synthetic compounds in
the hopes of identifying suitable candidates for further development. An alternative approach has been to exploit knowledge
from epidemiological studies and then proceed with the rational development of new agents and/or approaches.
Nonsteroidal anti-inflammatory drugs and their prototype aspirin (ASA) represent a case in point. The ample documentation
(epidemiological and recently interventional) of the chemopreventive effect of ASA has led to the exploration of the role of
various NSAIDs in colon cancer chemoprevention. An almost natural extension of this work has been the recent effort to
enhance the pharmacological properties of NSAIDs by combining them with a moiety that releases nitric oxide. This new
class of compounds, the NO-donating NSAIDs (NO-NSAIDs), is the subject of the present review. After
summarizing the salient points of the role of NO and also of
conventional NSAIDs in cancer, we present our current
understanding of the potential chemopreventive role of NO-NSAIDs
against colon cancer.
Nitric oxide and its role in cancer
Nitric oxide (NO) is one of the simplest biological
molecules in nature[2]. The discovery of its physiological role in
the 1980s has had a major influence on many aspects of
current biomedical research. Produced through the
oxidation of L-arginine by the various nitric oxide synthases (NOS),
NO is a free radical, a property that renders it very reactive
and unstable. The 1998 Nobel Prize in Physiology or
Medicine was awarded to R FURCHGOTT, L IGNARRO and F
MURAD for their discoveries concerning NO as a signaling
molecule in the cardiovascular system.
In addition to its role as an endogenous regulator of blood
flow and thrombosis, NO is now accepted as a fundamental
signaling molecule regulating virtually every critical cellular
function, as well as a potent mediator of cellular damage in a
wide range of conditions[3]. NO reacts readily with another
radical, superoxide anion, forming peroxynitrite (this pair is
considered to regulate the concentration of each other in a
cell), which interacts with lipids, DNA, and proteins via
direct oxidative reactions or via indirect, radical-mediated
mechanisms. Chronic inflammatory diseases and cancer are
among the many pathogenetic mechanisms mediated by
peroxynitrite.
Although inflammation and DNA damage engendered
by NO provide general links to cancer, many lines of
evidence suggest the involvement of NO in discrete and
sometimes critical steps of the complicated process that leads
from normalcy to malignancy[4]. For example, NO affects
tumor angiogenesis, metastasis, blood flow and immune
surveillance[5]. Recent published reports indicate that
even endothelial NO synthase (eNOS) can modulate cancer-related
events, such as angiogenesis, apoptosis, cell cycle, invasion,
and metastasis[6]. Furthermore, NO has the potential to
enhance both radiotherapy and chemotherapy, but such
strategies depend on achieving appropriate levels of
NO[5].
NSAIDs and their role in colon cancer
Hippocrates, the Greek physician considered "the father
of medicine," wrote in the 5th century BCE that the willow
bark improves aches, pains and fever. The modern era of
NSAIDs began in 1829 with the isolation of salicin from the
white willow, which was followed by the synthesis of aspirin
in 1899 by Hoffman[7]. Currently, NSAIDs, comprising a
chemically heterogeneous group of drugs, are being used as
antipyretics, analgesics, and anti-inflammatory medications
and in the prevention of myocardial infarction and stroke.
NSAIDs are also used to close patent ductus arteriosus in
neonates and in the treatment of dysmenorrhea. Lawrence
Levine, demonstrating in 1973 that indomethacin reduced
tumor size in fibrosarcoma-bearing
mice[8], was the first to show the anticancer effect of NSAIDs. Soon after, several
epidemiological studies showed that NSAIDs reduce both
the risk of and mortality from colorectal cancer by about
half[9]. Interventional studies conducted by Baron and his
colleagues provided formal proof that aspirin does prevent
colon cancer, albeit at a lower than expected
rate[10,11].
Like all cancers, colon cancer is a congregation of
abnormal cells and represents an imbalance between cell renewal
and cell death. Following our original demonstration that
NSAIDs reduce cell proliferation, induce apoptosis and
change the distribution of cells in the cell
cycle[12], many groups have attempted to decipher their molecular
mechanism of action[13]. A surprising finding was that the effect of
NSAIDs on cancer was independent of their inhibition of
cyclooxygenase (COX), their best known pharmacological
target[14]. This counterintuitive observation, initially received
with skepticism, has been amply confirmed. Over 15
potential mechanisms have been described. Salient amongst them
are inhibition of signaling via NF-κB, COX, NOS, PPAR
(peroxisome-proliferator activated receptor), a family of
nuclear hormone receptor and/or transcription factors, as
well as inhibition of angiogenesis. All of these mechanisms
converge directly or indirectly into a cell kinetic effect that
eliminates preferentially neoplastic cells, thereby
diminishing tumor cell mass. NSAIDs have their main effect in cancer
prevention, being essentially ineffective in cancer treatment.
This observation suggests that the fully developed cancer
cells either have acquired resistance to NSAIDs or that the
lower complexity of early neoplastic cells makes them
vulnerable to NSAIDs.
There are two main pharmacological features of NSAIDs
that essentially preclude their clinical application to colon
cancer chemoprevention[15]. First, their efficacy is below 50%
and, second, their safety profile is not favorable. It is
important to recall that chemoprevention differs substantially from
chemotherapy. In the latter, a medication, even one with
significant side effects, is administered to treat patients with
fully developed cancers that threaten their lives.
Chemopreventive agents, in contrast, are given to healthy subjects
for a cancer they may never develop. Thus the requirements
for efficacy and safety are more stringent for
chemopreventive agents. It is precisely these considerations that
propel the search for alternatives to conventional NSAIDs
as agents for cancer prevention. NO-NSAIDs are a
promising development in this direction.
NO-donating NSAIDs as agents against colon cancer
NO-NSAIDs consist of a conventional NSAID to which
the NO-releasing moiety _ONO2 has been attached via a
chemical linker[16]. Representative members of this class of
drugs are shown in Figure 1. The spacer can vary in its
chemical structure, providing a great number of derivatives.
NO-aspirin (NO-ASA) is at present the best-studied NO-NSAID.
There are three positional isomers of the NO-ASA molecule,
ortho, meta and para, generated by varying the position of
the _CH2ONO2 group with respect to the ester bond linking
the two benzenes[17].
Williams et al were the first to publish data that showed
that NO-ASA, NO-sulindac, and NO-ibuprofen have chemopreventive properties; all three compounds reduced
the growth of cultured HT-29 human colon adenocarcinoma
cells more potently than their corresponding
NSAIDs[18]. This was achieved by inhibition of proliferation, induction of
apoptosis and also inhibition of cell cycle phase transitions.
Beyond classical apoptosis there was another form of cell
death induced by NO-ASA that was named atypical cell
death[17]. Atypical cell death, initially described
in vitro, may actually occur in vivo. Ouyang
et al recently described a sequence of morphological changes in intestinal tumors of
Min mice treated with NO-ASA that strongly suggests its
existence[19]. Gao et al also recorded the induction of
apoptosis by NO-ASA in the SW480 colon cancer cell line
and the induction of oxidative stress evidenced by enhanced
levels of reactive oxygen species[20]. It was also observed
that NO-ASA induced apoptosis in Min mice but did not do
so in their wild type congenic controls.
Recent animal studies indicated that NO-ASA was more
effective than its parental ASA in preventing colon
cancer[21]. Studies on Min mice (they develop spontaneous intestinal
tumors because of a truncating Apc mutation), rats treated
with the carcinogen azoxymethane, and tumor xenografts, all
generated similar results. In Min mice, 3 weeks of treatment
with NO-ASA decreased the number of tumors by
55%[22]. Wallace and co-workers studied carcinogen-treated rats and
used as endpoint aberrant crypt foci, which are the earliest
premalignant lesions in the colon. NO-ASA reduced the
number of aberrant crypt foci by 85% while ASA resulted in a
64% reduction[21]. Recently, Rao et
al assessed the chemopreventive properties of NO-indomethacin (NCX 530) and
meta NO-aspirin (NCX 4016) against azoxymethane-induced
colon cancer in F344 rats[23]. NO-indomethacin and NO-ASA
significantly suppressed both tumor incidence and
multi-plicity. The degree of inhibition was more pronounced with
NO-indomethacin than with NO-aspirin. Finally, the
antitumor activity of para NO-ASA (NCX 4040) in
combination with 5-fluorouracil (5-FU) or oxaliplatin was evaluated
in vitro and in vivo in colon cancer models by Leonetti
et al[24]. NO-ASA and 5-FU, combined
in vitro, were always additive, regardless of the scheme used. Sequential NO-ASA
and oxaliplatin treatment produced strong synergism in cell
lines. In vivo this sequence caused higher reduction in
tumor growth than single-drug treatments. The authors
concluded that NO-ASA sensitizes colon cancer cell lines to the
effect of antitumor drugs and that their combination could
be useful for the clinical management of colon cancer.
A remarkably consistent finding in numerous animal
studies, including those mentioned above, is the safety of
NO-NSAIDs, which appears superior to that of their parent
NSAIDs. Some clinical studies underscore the same, but they
are very limited in scope[25,26].
The molecular mechanism underlying the effect of
NO-NSAIDs remains unknown despite extensive work by us and
others[27]. Significant effects have been noted on a variety of
important signaling cascades including NF-κB, Wnt,
mitogen activated protein kinase (MAPK), and NOS. On all of
them the effect of NO-ASA, the agent studied in greatest
detail, was inhibitory. Regarding the COX pathway, it was
unexpectedly observed that NO-ASA induced the
expression of COX-2[28]. When a comparison was made between
the IC50s for cell growth inhibition and signaling inhibition in
response to NO-ASA, the most important event was the
inhibition of Wnt (β-catenin) signaling that occurred at
concentrations way below those required for the inhibition of
cell growth. In the study of Rao et al mentioned
above[23], NO-indomethacin and NO-ASA inhibited the colon tumors'
COX activity and the levels of various prostaglandins, as
well as NOS2 activity and β-catenin expression. In colonic
crypts and tumors of animals fed these two NO-NSAIDs,
cell proliferation was decreased.
Additional mechanistic studies have revealed
modulation of drug metabolizing
enzymes[29]. Such effects, leading to facilitated elimination of carcinogens, have been
considered to represent a successful strategy for cancer
chemo-prevention. NO-ASA induced the activity and expression
of NAD(P)H:quinone oxireductase (NQO) and glutathione
S-transferase (GST) both in vitro and in the liver of
Min mice, Interestingly, NO-ASA induced the translocation of
Nrf2 into the nucleus, an effect that paralleled the
induction of NQO1 and GST P1-1. The induction of phase II
enzymes by NO-ASA and the modulation of the Keap1-Nrf2
pathway were speculated to be part of NO-ASA's
mechanism of action against colon and other cancers.
What appears to be the critical proximal event in the
action of NO-ASA in cancer, however, is the induction of a
state of oxidative stress in the target
cell[20]. It is conceivable, that many of the changes in the signaling pathways
mentioned above are derivative events, generated by redox
changes that may either lead to reversible redox signaling or
irreversible oxidative stress that culminates in cell death and
thus elimination of the neoplastic cell. Figure 2 recapitulates
the proposed mechanism of action of NO-ASA, highlighting
changes concerning the (converging) apoptosis and Wnt
signaling pathways.
Perspectives
NO-NSAIDs represent a promising recent development
in cancer chemoprevention. The work accomplished to date
by several laboratories exemplifies a rational and
mechanism-based approach to cancer prevention. The NO-NSAID-based
approach is the end result of a logical sequence starting with
epidemiological observations on the effect of NSAIDs
against colon cancer, mechanistic and formal interventional
studies focusing mainly on conventional ASA and then the
rather ingenious combination of NO and NSAIDs aiming to
generate a superior agent.
The NO-based compounds have several promising
features that generate reasonable expectations that they will
prove useful chemopreventive agents. They include a sound
pharmacological basis (NSAIDs plus NO); congruent
preclinical evidence suggesting both efficacy and safety; and
their presumed mechanism of action, which is based on ROS
generation and includes a multi-pronged effect on several
signaling pathways.
Only clinical testing will ascertain whether these
compounds have any clinical value. Although the road to
clinical application is long and the task arduous, several
significant milestones have already been achieved, generating a
level of excitement about these compounds.
Acknowledgments
We thank Stancy JOSEPH for critical comments and
general help with the manuscript.
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