INVITED REVIEW
Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis

Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Foundation, and Medical School, Rochester, Minnesota 55905

	ABSTRACT

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

Chronic inflammation of gastrointestinal tissues is a well-recognized risk factor for the development of epithelial cell-derived malignancies. Although the inflammatory mediators linking chronic inflammation to carcinogenesis are numerous, current information suggests that nitric oxide (NO) contributes to carcinogenesis during chronic inflammation. Inducible nitric oxide synthase (iNOS), expressed by both macrophages and epithelial cells during inflammation, generates the bioreactive molecule NO. In addition to causing DNA lesions, NO can directly interact with proteins by nitrosylation and nitosation reactions. The consequences of protein damage by NO appear to be procarcinogenic. For example, NO inhibits DNA repair enzymes such as human 8-oxodeoxyguanosine DNA glycosylase 1 and blocks apoptosis via nitrosylation of caspases. These cellular events permit DNA damage to accumulate, which is required for the numerous mutations necessary for development of invasive cancer. NO also promotes cancer progression by functioning as an angiogenesis factor. Strategies to inhibit NO generation during chronic inflammation or to scavenge reactive nitrogen species may prove useful in decreasing the risk of cancer development in chronic inflammatory gastrointestinal diseases.

apoptosis; cytokines; oxidative DNA damage; DNA repair; p53

	INTRODUCTION

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

GASTROINTESTINALLY DERIVED epithelial cell malignancies are common afflictions in the western world. Although many of these cancers arise de novo without an identifiable predisposing disease process, chronic inflammation of gastrointestinal epithelia unequivocally predisposes to the development of cancers. For example, chronic esophagitis, gastritis, pancreatitis, hepatitis, cholangitis, and colitis are all known risk factors for cancers of the esophagus, stomach, pancreas, liver and biliary tract, and colon, respectively (16, 44, 52, 84). Although considerable information is now available on the genetic and epigenetic changes contributing to these cancers, the mechanisms by which chronic inflammation contributes to these genetic alterations has not been adequately explored. Insight into the mechanisms linking chronic inflammation to gastrointestinal epithelial cell cancers may help in the development of treatment strategies for reducing malignant transformation in these inflammatory diseases. Moreover, information on the inflammatory mediators contributing to cancer development may also help identify new mechanisms of carcinogenesis [e.g., cyclooxygenase-2 (COX-2) and colon cancer] (69, 99).

Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) is a cardinal feature of inflamed tissues including those of the gastrointestinal tract (37, 40). iNOS overexpression with high levels of NO generation provides a plausible link between inflammation and cancer initiation, progression, and promotion (Fig. 1). Indeed, there is substantial evidence implicating NO in cancer as an endogenous mutagen (127), an angiogenesis factor (43), an enhancer of protooncogene expression (3), and an inhibitor of apoptosis (59). Several human gastrointestinal neoplasms express iNOS including gastric cancer (86), colonic adenomas (100), Barrett's esophagus and associated adenocarcinomas (73), hepatocellular carcinoma (84), and cholangiocarcinoma (33, 40) (Fig. 2). These observations suggest that iNOS may play a fundamental role in the initiation and promotion/progression of cancers arising within a background of inflammation. Therefore, it is both timely and topical to review the role of iNOS-generated NO in carcinogenesis of gastrointestinal epithelial cells.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Roles of nitric oxide (NO) in carcinogenesis. NO has been implicated in modulating several events involved in malignant transformation and cancer promotion/progression. NO can directly damage DNA, inhibit DNA repair, block apoptosis, and enhance oncogene expression. NO modulates transcription factors, especially redox-sensitive and zinc finger motifs containing transcription factors. NO has also been found to contribute to angiogenesis.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Inducible nitric oxide synthase (iNOS) expression in response to inflammatory diseases in the gastrointestinal tract. There are emerging clinical data showing that chronic inflammatory conditions along the digestive tract, liver, and pancreas have increased expression of iNOS. Moreover, expression of iNOS is retained by the associated cancers, suggesting that iNOS is important in the development and expression of these malignancies. PSC, primary sclerosing cholangitis.

	NITRIC OXIDE SYNTHASES AND NITRIC OXIDE

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

NO is a 30-kDa gaseous biological mediator synthesized in mammalian cells by a family of three NOS isoforms. The human genes for NOS are characterized as neuronal or nNOS (NOS1), endothelial or eNOS (NOS3), and iNOS (NOS2) (23). NOS catalyzes the conversion of the terminal guanidino nitrogen of the amino acid L-arginine to form NO plus citrulline in a complex reaction involving molecular oxygen and NADPH as cosubstrates with enzyme-bound heme, FAD, flavin mononucleotide (FMN), and tetrahydrobiopterin (BH₄) as cofactors (5, 110). The two constitutive isoforms of NOS, eNOS, and nNOS, are controlled by calcium fluxes and produce only nanomolar NO concentrations. In contrast, the dimeric iNOS protein is permanently active and capable of generating micromolar concentrations of NO on induction in the absence of changes in cellular calcium (68, 72). The iNOS gene is under transcriptional control of inflammatory cytokines and lipopolysaccharides (25, 39, 79). The human iNOS gene was first cloned from synergistic activation of tumor necrosis factor (TNF)-, interleukin (IL)-1, and interferon (IFN-) in hepatocytes (24, 83). iNOS gene expression is regulated by the transcription factors nuclear factor (NF)-B, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, and c-Jun NH₂-terminal kinase (JNK)-activator protein-1 (AP-1) pathways (103). The enzyme is downregulated by steroids, transforming growth factor (TGF)-, the heat shock response element, p53, and NO itself (111). The complexity of iNOS catalysis and its regulation is reflected in the broad range of biological and cell/tissue-specific physiological and pathophysiological functions of NO (6). Studies have shown very little correlation between iNOS mRNA expression and enzyme activity, suggesting a considerable degree of posttranscriptional regulation. iNOS induction by cytokines and NF-B likely accounts for its ubiquitous expression and activity during inflammation.

	INOS EXPRESSION IN GASTROINTESTINAL EPITHELIA

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

Almost every cell in the body has the capacity to express iNOS (78). In inflamed gastrointestinal tissue, iNOS is richly expressed by infiltrating and resident activated macrophages (15, 78). The NO generated by activated macrophages may have significant physiological benefits (82). For example, NO produced by macrophage iNOS has important antimicrobial functions (6, 119) and inhibits the growth of viruses (45), parasites (27), and gram-positive organisms (35) that invade the gastrointestinal tract. Although macrophage-derived NO appears to be important in innate immunity, the high diffusion efficiency of NO makes it potentially reach unintended targets (e.g., neighboring epithelial cells). Inflammatory cytokines will also trigger iNOS expression by epithelial cells (112). Indeed, iNOS has been identified in gastric metaplasia of the esophagus (73), mucosal cells of the stomach in chronic gastritis (66), hepatocytes in viral hepatitis (65, 84), cholangiocytes in primary sclerosing cholangitis (38), and colonocytes in inflammatory bowel disease (100). In contrast to macrophage-generated NO, epithelial cell NO has ready access to epithelial cell targets including those important in carcinogenesis. Thus the salutary effects and potentially detrimental consequences of NO generation in inflammation exist along a continuum related to the magnitude and chronicity of NO exposure. This continuum of iNOS activity and NO generation in immunoregulatory functions can be divided into three stages: 1) under physiological conditions, normal to low quantities of NO produced by constitutive and inducible NOS act as an intracellular activation signal; 2) when NO production is increased for longer periods of time via iNOS, NO functions as an autocrine or paracrine immunoregulatory molecule; and 3) chronic and sustained generation of NO can be associated with direct reactions between NO and cellular constituents and generation of reactive nitrogen species. It is this latter condition that has been postulated to play a pivotal role in carcinogenesis. This interplay is exemplified in Helicobacter pylori infection of the stomach, an infection well documented to be a risk factor for gastric cancer (66, 121). In this process, H. pylori elicits a host inflammatory response with iNOS generation of NO to potentially eradicate the organism. However, because of this bacterium's ability to persist, the inflammatory response becomes chronic and predisposes to cancer with persistence of iNOS expression by the malignancy (86).

	NO AND CELLULAR DNA AND PROTEIN DAMAGE

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

The toxicity of NO during chronic inflammation occurs by two chemical processes, 1) oxidation of NO with superoxide to form peroxynitrite (ONOO) and nitrosating species, namely, NO (nitrates), NO (nitrites), and N₂O₃ (126), referred to as reactive nitrogen oxide species (RNOS), and 2) direct reactions between proteins and NO by nitrosylation events.

NO can damage DNA through a variety of different mechanisms that may be cell specific (107). Nitrosation of purines and pyrimidines results in hydrolytic deamination of cytosine to uracil and guanine to xanthine (81, 109). GC  AT and GC  CG transitions (replacement of purine by pyrimidine base or vice versa) are major mutations observed in naked DNA and human cells exposed to NO (127). Sequence data of gene mutations from patients with hepatocellular carcinoma and gastric carcinogenesis reveal the same GC  AT transitions (12, 121). N₂O₃ is a powerful electrophilic nitrosating agent that causes the formation of carcinogensN-nitroso compounds, DNA strand breaks, and cross-linking of DNA (109). Peroxynitrite can also react directly with DNA and lead to mutations and DNA strand breaks (106). Besides deamination, NO and/or peroxynitrite can cause DNA base oxidation measured as conversion of guanine to 8-nitroguanine and 8- oxoguanine (8-oxodG). H. pylori infections in humans have been associated with an increased release of 8-oxoguanine in urine and the formation of nitrotyrosine in gastric antral biopsies (66, 129). Acute hepatic injury and/or cell necrosis was accompanied by significant induction of iNOS in the liver and other organs with increased nitrosamine formation and release in human urine (92). In fact, increased endogenous formation of nitrate and nitrosamines is recognized as a risk factor for cholangiocarcinoma in humans infested with Opisthorchis viverrini (94, 124) and for hepatocellular carcinoma in cirrhosis patients (61).

NO can also directly nitrosylate susceptible thiol residues on proteins, resulting in the loss of their catalytic activity (87). In many proteins, this is a reversible reaction, but in proteins containing zinc, copper, or iron, the thiol nitrosylation results in irreversible ejection of the metal and denaturation of the protein (58). The reaction of NO with iron-sulfur centers may also perpetuate radical reactions. NO and peroxynitrite react rapidly with iron-sulfur centers, and the resultant free iron may then generate hydroxyl free radical-induced damage via Fenton chemistry (125). Peroxynitrite and NO can oxidize protein and nonprotein thiols, protein sulfides, lipids, and low-density lipoproteins (14, 85). 3-Nitrotyrosine is a stable marker of oxidative protein damage by RNOS (93). The presence of nitrotyrosine has been identified by immunohistochemistry in relation to gut tissue subjected to chronic inflammation with increased iNOS expression such as H. pylori gastritis (66), primary sclerosing cholangitis (38), cholangiocarcinoma (40), pancreatic cancer (122), and esophageal squamous cell cancer (46). Thus there is ample evidence for the presence of reactive oxygen species and NO-mediated DNA and protein damage in gastrointestinal epithelia during chronic inflammation.

	NO AND DNA REPAIR PROTEINS

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

Although NO and reactive oxygen species can directly damage DNA, mammalian cells have rich and complex mechanisms to repair the genome. Several DNA repair processes are now recognized including base excision repair (BER), nucleotide excision repair, transcription-coupled repair, double strand break repair, and mismatch repair (Table 1; Refs. 20, 97, and 130). Accumulation of DNA damage during inflammation, therefore, represents the net result of both direct damage and ineffective repair. Unfortunately, DNA repair enzymes also appear to be protein targets of NO and reactive oxygen species (102, 128). Thus iNOS-generated NO may give the cell a double hit by both damaging the DNA and inhibiting its repair processes. This effect of NO and its by-products may make NO one of the pivotal mediators linking inflammation to carcinogenesis.

View this table: [in this window] [in a new window]   Table 1.   DNA repair pathways

Several key DNA repair enzymes are inhibited by NO-mediated nitrosylation of active site cysteine. The DNA repair enzymes with vulnerable active site cysteine residues include 6-O-alkyl DNA transferase (56), foramidopyrimidine glycosylase (54), xeroderma pigmentosum A protein (76), and DNA ligases with active site lysine residues (60). We recently demonstrated (40) that cytokine-mediated induction of iNOS with NO production is associated with diminished global DNA repair capacity in cholangiocarcinoma cell lines. Although DNA oxidative lesions, the predominant mechanism of DNA damage in inflammation, can be excised from DNA by multiple pathways, BER is quantitatively the most important (41, 55). 8-oxodG, the most abundant oxidative DNA lesion, is excised in humans by 8-oxodeoxyguanosine DNA glycosylase 1 (hOgg1) (51, 95, 131). This glycosylase contains critical thiol moieties in a zinc finger at its active sites (91, 108). Our group has demonstrated that the hOgg1 repair enzyme is inhibited by NO and peroxynitrite generation because of formation of S-nitrosylation at their active site cysteine residues and ejection/loss of zinc (38, 40). This is a good example of how iNOS overexpression due to inflammatory cytokines can be mechanistically linked to accumulation of DNA damage resulting from inhibition of a specific important DNA repair enzyme (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   NO inhibits hOgg1 base excision repair activity. NO causes oxidative DNA damage [e.g., 8-oxoguanine (8-oxodG)] and potentiates the accumulation of the damage by inhibiting the repair activity of its key repair enzyme, human 8-oxydeoxyguanosine DNA glycosylase 1 (hOgg1). Unrepaired 8-oxodG causes GC to TA transitions and is mutagenic.

	NO AND APOPTOSIS

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

In addition to repairing damaged DNA, the organism has another safeguard against cellular transformation, programmed cell death by a process known as apoptosis (34, 104). DNA damage that cannot be repaired is a potent inducer of apoptosis (53). For example, DNA-damaging drugs, -radiation, and ultraviolet light all trigger apoptosis by inducing DNA damage (8). In these circumstances apoptosis is mediated by activation of caspases, a family of cysteine proteases (42). Caspase activation occurs by one of two broad mechanisms, death receptors and/or mitochondrial cytochrome c release (26, 67). The death receptor pathway is initiated by caspase-8, whereas the mitochondrial pathway is dependent on caspase-9. Both death receptor- and mitochondria-initiated apoptotic cascades involve the downstream caspases-3, -6, and -7, which execute the apoptotic program (Fig. 4) (118). Although either or both pathways can be disrupted in established malignancies, dysregulation of the mitochondrial pathway appears to be very important in cancer development and therapy (47). In this pathway, apoptotic signals are initiated by the release of cytochrome c from mitochondria (34, 63). By inhibiting caspase activity through S-nitosylation of the active site cysteine, NO inhibits apoptosis in hepatocytes (48), endothelial cells (10), and several tumor cell lines (9, 63). Indeed, NO has been shown to directly nitrosylate caspases-3, -8, and -9, resulting in their inactivation and inhibition of apoptosis (49, 50). Studies have shown that prevention of NO protein damage induced by intestinal ischemia-reperfusion can protect against hypoxia-induced apoptosis in HT-29 colon carcinoma cells by inhibiting cytochrome c release from the mitochondria (34, 63, 64). In addition to the NOS isoforms described above, mitochondria are also capable of generating NO, although the mechanism responsible remains obscure (26). It is possible that this mitochondria-derived NO is capable of inhibiting apoptosis at the level of this organelle in addition to caspase nitrosylation. Thus NO may inhibit apoptosis at several steps, further allowing the DNA-damaged cell to survive.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Inhibition of caspase activation by NO prevents apoptosis. NO blocks both the death receptor and mitochondrial pathways by nitrosylation of caspase-8 and caspases-9 and -3, respectively. The resultant failure of apoptosis allows the survival of genetically altered cells and is thought to play a pivotal role in carcinogenesis. Bid, BH3-interacting domain death agonist.

	NO, P53, AND TUMORIGENESIS

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

NO and reactive oxygen species generated during chronic inflammation may initiate and enhance carcinogenesis in humans by the biochemical interactions discussed above. Infection by bacteria (H. pylori), parasites (liver fluke helminthes), or viruses (hepatitis B and C) and chronic tissue inflammation are recognized risk factors in the etiology and development of cancers. The process of carcinogenesis is a multistep process involving inactivation of tumor suppressor genes and activation of oncogenes by either DNA mutations or deletions. The DNA repair molecule p53 plays a important role in the cellular response to DNA damage. As mentioned above, the NO-mediated deamination of DNA causes GC  AT transitions. NO-induced DNA damage results in upregulation of p53, an important trans-repressor of iNOS expression in vivo, and attenuates NO production by a regulatory negative feedback mechanism (3, 19). The p53 gene in many human cancers, including liver cancer from hepatitis B (32) and H. pylori-associated gastric cancer (121), has GC  AT transitions. These transitions occur mainly at CpG islands of the p53 gene and occur in a number of human cancers, including colon, gastric, and hepatocellular cancers (1, 28). NO production via iNOS may directly induce these GC  AT mutations in p53, providing another molecular link between chronic inflammation and cancer development. For example, a relationship between p53 mutations and iNOS expression has been established in human colorectal cancer and several human carcinoma cell lines (2, 37). Not only may NO contribute to the development of p53 mutations, but the loss of repressor activity of the mutated p53 would increase iNOS, resulting in enhanced cellular NO generation, further DNA damage, and the development of additional transforming mutations (Fig. 5).

View larger version (9K): [in this window] [in a new window]   Fig. 5.   p53 and NO-mediated proliferation of malignant cells. The tumor suppressor p53 gene is upregulated in response to increased iNOS/NO generation with associated DNA damage. The upregulation of p53 inhibits iNOS expression and arrests the cell cycle, allowing repair to be initiated. However, p53 in cancers (gastric, hepatocellular, hereditary nonpolyposis colorectal cancer, etc.) is mutated. Mutated p53, required in failure of this negative feedback loop, causes enhanced iNOS expression, thereby aggravating the consequences of enhanced chronic iNOS expression. RNOS, reactive nitrogen oxide species.

	NO AND ANGIOGENESIS

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

Because neovasculature is necessary for tumor growth and immunohistochemical studies have indicated that iNOS is overexpressed in solid tumors (22), production of NO may promote tumor growth by stimulating angiogenesis (43, 74), increasing vascular permeability, and suppressing the immune response (57). Angiogenesis is a complex, multistage process, regulation of which requires release of vascular endothelial growth factor (VEGF), angiogenin, adhesion molecules, and basic fibroblast growth factors delivered by the tumor cells themselves, the extracellular matrix, or incorporated macrophages (18). Initial studies suggest that NO plays a central role in the angiogenic cascade by demonstrating that VEGF released as a purified protein or produced by tumor cells requires a functional NO/cGMP pathway within the endothelial compartment to promote neovascular growth (22, 75, 135). Another mechanism by which NO may promote tumor growth is by modulating the production of prostaglandins. NO activates COX-2 (98), which stimulates the production of proangiogenic factors and prostaglandins that facilitate tumor growth by increasing vascular permeability, and by concomitant inhibition of apoptosis from enhanced Bcl-2 protein production (120). The use of iNOS inhibitors as antiangiogenesis agents is now supported by several reports in the literature (reviewed in Ref. 116).

	NO, CHRONIC INFLAMMATION, AND THERAPEUTIC INTERVENTION

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

The use of oxygen by aerobic organisms invariably results in the formation of reactive oxygen species. Cells counterbalance prooxidants with antioxidants. For example singlet oxygen and superoxide are dismuted by Mn superoxide dismutase in the mitochondria and Cu/Zn superoxide dismutase in the cytosol, and H₂O₂ is detoxified by glutathione peroxidase, a selenium enzyme, and by catalases. Besides these defenses, the cell has other proteins like thioredoxin, glutaredoxin, and thioltransferase that participate in maintaining the equilibrium between prooxidant and antioxidant. In addition, many small molecules such as -tocopherol, ascorbate, albumin, uric acid, and glutathione act as scavengers of reactive oxygen species. Genetic stability is also maintained by the activity of efficient DNA repair enzymes. However, during chronic inflammation, the cell is bombarded with the accelerated and sustained production of oxygen and nitrogen free radicals and ions that saturate the antioxidant capacity of the cell, inhibit DNA repair function and programmed cell death, and are potentially carcinogenic. The recognition of the multifaceted role of NO in modulating mutagenesis in chronic inflammatory diseases suggests several therapeutic approaches (36, 77).

iNOS and its associated mediators have been targeted for therapeutic intervention. The current potential strategies for intervention include 1) scavengers of NO and peroxynitrite, 2) repression of iNOS expression by drugs, antisense technology, and p53, and 3) inhibition of iNOS protein and its cofactors. Carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) (132) and PTIO (29) are potent scavengers for NO. Trolox (96), ebselen (4) and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, (17) have been used successfully to scavenge for peroxynitrite. The efficacy of these scavengers in a malignant murine model has been gratifying (30, 123). Curcumin (from Curcuma longa), a naturally occurring polyphenolic phytochemical, is in preclinical trial for cancer chemoprevention. Curcumin inhibits activation of free radical-activated transcription factors such as NF-B and AP-1 and reduces the production of proinflammatory cytokines such as TNF-, IL-1, and IL-8. In addition to inhibiting cytokine-mediated activation of iNOS, curcumin has been shown to inhibit iNOS gene expression in mouse liver (11). Inhibition of gene expression by hybridization of antisense oligonucleotides to the transcripts also has therapeutic potential (13). An antisense oligodeoxynucleotide to iNOS mRNA designed to inhibit translation of iNOS was found to partially inhibit nitrite production in cytokine-stimulated cells (115). Selective inhibitors of iNOS that blocked iNOS dimerization and NO production both in vitro and in vivo were identified in an encoded combinatorial chemical library. These inhibitors were >1000-fold more potent than substrate-based iNOS inhibitors such as 1400W (70). Irreversible inhibition of iNOS was achieved by using flavoprotein iodonium inhibitors that prevented the binding of cofactors NADPH, FMN, and FAD (21). BH₄, another essential cofactor of iNOS, can be inhibited by N-acetylserotonin and dicumarol (101). iNOS isoform-selective substrate-based arginine analog inhibitors can be exploited for therapeutic purposes because arginine binding domains for the isoforms are different. Several general analogs of arginine such as N^G-monomethyl-L-arginine (L-NMMA) acetate and L-N⁶-(1-iminoethyl) lysine (L-NIL) are effective nonspecific inhibitors of iNOS. 1400W, a selective inhibitor of iNOS, inhibits the growth of solid murine tumors expressing iNOS (117). Aminoguanidine and mercaptoethylguanidine are inhibitors of iNOS and have anti-inflammatory properties because they protect against peroxynitrite-induced oxidative damage (105). Chemopreventive approaches may include inhibition of iNOS expression with tetracycline and minocycline (133, 134). Preclinical animal studies involving iNOS inhibitors have been effective in chronic colitis (88, 90). Expression of p53 in cancer cells lacking this tumor suppressor gene can lead to cell apoptosis or cycle arrest and may also inhibit the angiogenesis required for tumor growth by decreasing iNOS expression. Studies have initiated the use of adenoviral vectors for effective locoregional delivery and transient overexpression of p53 and consequently effective p53 gene therapy for cancer (80).

	SUMMARY

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

The current literature suggests that NO is involved in many of the pathophysiological processes linking inflammation to cancer development and progression in the gastrointestinal tract. NO, generated by iNOS in these disease states, is a reactive biomolecule. When the generation of NO exceeds the antioxidant capacity of the cell, NO and its reactive nitrogen oxide species are capable of damaging DNA and proteins. In particular, NO-mediated nitrosylation of DNA repair proteins inhibits their enzymatic activity, potentiating accumulation of DNA damage. NO can also nitrosylate and inactivate the effector proteins of apoptosis, especially caspases. Inhibition of caspases results in dysregulation of apoptosis, further promoting malignant transformation. Because it is associated with VEGF formation, NO can also be considered an angiogenic factor, thereby promoting cancer progression. These concepts provide a model to test the utility of iNOS inhibitors and NO scavengers as chemopreventive agents to minimize the risk of human cancers in these premalignant diseases. We encourage these studies and await their results.

	ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-59427 (G. J. Gores) and DK-24031 (N. F. LaRusso) and by the Mayo Comprehensive Cancer Center, Rochester, MN (CA-15083).

	FOOTNOTES

Address for reprint requests and other correspondence: G. J. Gores, Mayo Medical School, Clinic, and Foundation, Rochester, MN 55905 (E-mail: gores.Gregory{at}mayo.edu).

	REFERENCES

TOP ABSTRACT INTRODUCTION NITRIC OXIDE SYNTHASES AND... INOS EXPRESSION IN... NO AND CELLULAR DNA... NO AND DNA REPAIR... NO AND APOPTOSIS NO, P53, AND TUMORIGENESIS NO AND ANGIOGENESIS NO, CHRONIC INFLAMMATION, AND... SUMMARY REFERENCES

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