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The role of nitric oxide in oral diseases
Archives of Oral Biology (2003) 48, 93—100
REVIEW
The role of nitric oxide in oral diseases P.A. Brennana,*, G.J. Thomasb, J.D. Langdonc a
Department of Maxillofacial and Head and Neck Surgery, Queen Alexandra Hospital, Portsmouth PO6 3LY, UK b Department of Oral Pathology, Eastman Dental Institute, 256 Grays Inn Road, London WC1X 8LD, UK c Maxillofacial Department, Kings College School of Medicine and Dentistry, London SE5 8RX, UK Accepted 14 October 2002
KEYWORDS Nitric oxide; Review; Oral disease; Cancer
Summary Although previously regarded as a toxic pollutant gas, nitric oxide (NO) is a short-lived molecule that plays a key role in many physiological and pathological processes. It is produced in vivo from the amino acid L-arginine by a complex family of enzymes termed nitric oxide synthase (NOS). Since its discovery as a biological messenger in 1987, NO has been implicated in many disease processes, ranging from septic shock to cancer. It is a highly reactive free radical and causes concentrationdependent conformational changes in proteins, enzymes and DNA, predominantly by its reaction with transition metals and thiol residues. Although high concentrations of NO are cytotoxic, the levels produced in many human cancers possibly facilitate tumour growth and dissemination. The interest in this molecule by scientists and clinicians involved with the oral cavity and head and neck regions is fairly recent, and only a tiny minority of 50,000 papers currently cited on NO relate to diseases in this anatomical area. This review gives an overview of NO, outlining its basic chemistry, formation by NOS and its possible roles in the oral diseases studied to date. The implications for possible therapeutic manipulation of NO are also discussed. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction NO is thought to be one of the oldest molecules on earth, being formed in the primitive atmosphere of the cooling planet.1 Until recently, NO, a toxic oxide of nitrogen produced by internal combustion engines and power stations, had been regarded solely as a cause of acid rain and atmospheric pollution. The Belgian scientist Jan Baptista van Helmont (1577—1644), more widely known for his discovery of carbon dioxide, and for explaining the action of gunpowder by the theory of gaseous expansion, can be credited as the first to synthesise NO in the laboratory in about 1610. In 1772, Joseph
Corresponding author. Present address: 11, Oxlease Close, Romsey, Hants SO51 7HA, UK. Tel.: þ44-1794-519-951. E-mail address: [email protected] (P.A. Brennan).
Priestley (1733—1804) gave it the name ‘nitrous air’ and observed that it did not support plant life, but reduced putrefaction in meat exposed to it. The effects of NO as a bactericidal agent were revisited again in the early 1980s but it was not until 1987 that NO was discovered to be the chemical responsible for the actions of endothelial derived relaxing factor (EDRF).2,3 Following this finding, NO research expanded exponentially, and it is regarded by many in the scientific community as one of the greatest discoveries of the 20th century. In recognition of this, two Nobel prizes have been awarded to researchers in the NO field, and it was named as molecule of the year by the journal Science in 1992. Although initial reports concentrated on its role in the regulation of vascular tone, it was soon realised that NO had a wide and diverging range of both physiological and pathological actions. Many
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(02)00183-8
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Table 1 Diseases and conditions that are associated with altered nitric oxide production. Hypertension and hypotension Thrombo-embolic disease Septic shock Bronchospasm Acute respiratory distress syndrome (ARDS) Pulmonary hypertension Renal failure Immune deficiency HIV induced encephalopathy Impotence Depression Malignancy
of these processes are still poorly understood. Most specialties of both medicine and science are now involved in NO research. It is implicated in various medical conditions ranging from septic shock to impotence (Table 1). The interest by scientists and clinicians in oral and head and neck diseases has been relatively recent, and only a tiny minority of the 50,000 papers currently cited on NO have concerned this anatomical area.
Basic chemistry and actions of NO The arrangement of one atom of nitrogen and one of oxygen leaves an unpaired electron, which makes the molecule a highly reactive free radical. However, it is less reactive than other free radicals (such as superoxide) in that it does not react with itself at physiological temperatures. NO readily reacts with superoxide, oxygen, thiol groups (—SH) to produce a number of other products, such as nitrosothiols (R— SNO), the toxic molecule peroxynitrite (ONOO—) and nitrites (NOx). Peroxynitrite, formed when both NO and superoxide radicals are present, is very toxic, particularly to DNA and to enzymes involved in DNA repair.4 All of these so-called reactive nitrogen oxide species (termed RNOx) react with transition metals (such as zinc and iron in haemcontaining proteins) and thiol groups in various enzymes and proteins. The resulting effects are concentration dependent, but can lead to inhibition of enzymes such as cytochrome c oxidase, resulting in decreased ATP production and cell death. The more familiar poisons cyanide (CN) and carbon monoxide (CO) inhibit cytochrome c oxidase in an analogous fashion to NO.5 Most NO chemistry is due to conformational changes in enzyme structure in a similar fashion to that seen when oxygen binds to haemoglobin. For simplicity, in this article the term NO will encompass all of these molecules.
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A comprehensive review of NO chemistry has been written by Wink et al.4 NO has been preserved throughout evolution as a mediator in many biological systems. For example, in the horseshoe crab, Limulus polyphemus, a species which has not evolved significantly in 500 million years, aggregation of its single type of blood cell, the haemocyte, is modulated by NO in a way similar to that of modern mammalian platelets.6 The physiological activities of NO may be divided into two main groups–—those that occur as a result of its interaction with the haem group in the enzyme guanylate cyclase, with subsequent activation and the generation of the second messenger cGMP, and those that are cGMP independent. Effects following NO-induced guanylate cyclase activation include the regulation of vascular tone,7,8 and a complex role as a neuromodulator in the central nervous system.9 NO has also been implicated as a pain modulator in various conditions, including temporomandibuar joint pain dysfunction syndrome.10 NO actions that are cGMP independent include a pivotal role during the process of angiogenesis,11—14 where it is thought to interact with many key molecules including the potent angiogenic factor vascular endothelial growth factor (VEGF). NO acts both as an upstream activator and downstream effector molecule for VEGF.15,16 It also has immunocytotoxicity against both pathogens and tumours.17 However, the NO concentrations found in human cancers are unlikely to be sufficient to produce tumour cell death or apoptosis, and instead are thought to be responsible for enhancing angiogenesis and tumour dissemination.14,18
Synthesis of NO NO is synthesised by a complex family of enzymes called NO synthases (NOS). These are some of the most complex enzymes known and require many factors and co-factors for activity. The amino acid L-arginine is the substrate for the NOS enzymes, generating NO and the by-product L-citrulline.19 A schematic representation of one of the NOS enzymes (NOS2) is shown in Fig. 1. There are three NOS enzymes, each produced by distinct genes and named in order of discovery.20 NOS1, was the first to be purified and cloned from neural tissue and it is therefore also called nNOS. NOS3 is the isoform first found in endothelial cells, also called eNOS. NOS1 and NOS3 are also termed constitutive since they are expressed continuously in neurones and endothelial cells, respectively. They are also dependent on a rise in tissue calcium
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Figure 1 Schematic representation of NOS2. Cal denotes binding site for calmodulin.
concentration for activity, and therefore produce low, transient concentrations of NO. In contrast, NOS2 is an inducible, calcium-independent isoform, also called iNOS. NOS1 and NOS3 may themselves be induced under certain conditions (such as pregnancy)21 and NOS2 is expressed constantly in certain sites (such as basal skin keratinocyes and salivary duct cells). Therefore the view of the authors is that the numerical nomenclature should be used for the NOS enzymes. Unlike NOS1 and NOS3, induction of NOS2 results in continous production of NO because of tight calmodulin binding, which allow electrons to pass from the haem group to the flavin (FMN, FAD) and nicotinamide (NADPH) electron accepting co-factors (see Fig. 1). From an evolutionary perspective, human NOS2 generates lower NO concentrations than, for example, the equivalent mouse NOS2 enzyme since the latter has much tighter calmodulin binding.22 The promoter/enhancer region of the NOS2 gene is complex and contains at least 22 potential binding sites for transcription factors. In the human gene, this region resides within a 16kilobase segment of the 50 -flanking area. As well as bacterial lipopolysaccharide (LPS) and interferon-g (IFN-g), numerous other factors induce the production of the enzyme. These include the cytokines tumour necrosis factor-a (TNF-a), interleukin (IL)1, IL-10 and IL-12, platelet activating factor (PAF) and nuclear factor-kb (NF-kb). Once induced the enzyme continues to produce much higher NO concentrations than the other two NOS isoforms, for many hours or even days23 until the protein is eventually inactivated, probably as a result of NO-induced protein modification. It is interesting that NOS2 expression is induced by cisplatin and taxols (used in cancer therapy), although NOS2 induction in these instances is likely to be indirect and mediated via TNF-a release. Hypoxia is known to induce mouse macrophage NOS2, acting at a site on the gene termed the ‘hypoxia-responsive element’ (HRE). A synergistic effect occurs when both hypoxia and IFN-g are present.24 Although located in the NOS2 promoter region, the HRE is separate from the other promoter sites and it shows sequence homology with the enhancer site of erythropoietin. Agents that inhibit NOS2 expression include NO itself, transforming growth factor-b (TGF-b), IL-4, IL-6, IL-8 and IL-10 (although paradoxically IL-10
may also induce NOS2), and certain immunosupressive drugs, such as glucocorticoids, cyclosporin and tacrolimus. An important regulator of NOS2 is the tumour suppressor gene p53, which senses raised cellular NO and inhibits NOS2 by a negative feedback loop.25 This relationship has important implications in oral cancer (see below). At a posttranscription level, transforming growth factor-b (TGF-b), and NO may destabilise mRNA.26 The action of corticosteroids in reducing NOS2 expression is not fully understood, but it may involve regulation at both the transcription and translation levels. In chronic inflammatory conditions, NOS2 expression is therefore dependent on the overall cytokine balance. NOS2 is found in various cells including macrophages and natural killer cells in the immune system. It is also constantly expressed by large airway epithelium, in the basal keratinocyte layer of normal skin,27 and in normal salivary ducts.28 In normal skin, the constant NO generation by NOS2 may assist the host defence against pathogens27 although its role in salivary ducts is as yet unknown.
Studies of NO in oral and related diseases There are few studies published on the role of NO in oral diseases. Although there have been few publications concerning this anatomical area, interest in NO is expanding rapidly. Of greatest interest to the authors is the role of NO in the pathogenesis and tumour biology of oral cancer. An overview of oral diseases in which NO has been implicated is discussed below.
Periodontal disease Unsurprisingly, NO synthesis is increased in periodontal disease, as a result of macrophage infiltration in the periodontal tissues.29 It is probable that bacterial plaque is responsible for NOS2 activation, since no NOS2 activity is found in the gingival tissues of sterile animals.30 Recent studies have also demonstrated that gingival fibroblasts in chronic periodontitis have significantly higher NOS2 expression than those in healthy gingival tissue.31 NO might play a role in the pathogenesis of both periodontitis and subsequent bone loss, either directly, or indirectly by modulating the production of other
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pro-inflammatory cytokines. A recent in vitro study found that NOS2 inhibitors reduce gingival fibroblast NO production, and it was postulated that pharmacological inhibition of NO might be therapeutically valuable in the management of this disease.31 It is possible that the induction of NOS2 occurs in response to bacterial LPS, or as a result of cytokines which are formed in periodontal disease. Although NO may be produced in an attempt to kill plaque bacteria, it is likely that (as with oral cancer), the concentrations produced result in host tissue damage instead. Further work is clearly needed in this area.
Odontogenic cysts and periapical infection There have been few published reports in this area and only one paper to date has investigated NOS expression in odontogenic cysts. This study investigated the expression of NOS2 in radicular cysts.32 This type of cyst results from chronic periapical inflammation, and it is not surprising that NOS2 was expressed in this condition. It is not apparent whether NO may modulate bone destruction in such circumstances. We are currently investigating the role of NO in the pathogenesis of odontogenic keratocysts, which can recur following enucleation and often have an aggressive behaviour if they extend into surrounding soft tissue. NO has also been implicated in the pathogenesis of apical infection.33 Again, inflammation-induced NOS2 accounts for NO production, and it has been postulated that NOS inhibitor drugs introduced into the root canals of teeth with periapical infections may lead to resolution of this condition.32
Oral mucosal inflammatory diseases There have been few studies which have investigated the role of NO in oral mucosal disease.34—36 Although oral lichen planus (OLP) is a chronic inflammatory disorder, little or no NOS2 activity or expression was found in this disorder, despite an abundance of CD68 positive macrophages.34 OLP itself is a complex disorder and many cytokines are produced. It was postulated that the balance between NOS2 activation and inhibitory cytokines in OLP favoured NOS2 inhibition over activation, but clearly further research is required in this field. There is still controversy regarding whether erosive and atrophic forms of OLP are associated with the development of oral cancer.37 Chronic inflammatory conditions that induce NOS2 might produce sufficient NO and related products to initiate carcinogenesis.38—40 However, since little or no NOS2 activity was found in OLP, it is unlikely that NO
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would play a role in the transformation of OLP to malignancy, if indeed it occurs at all. In contrast,35 found that salivary nitrite (implying that NO had been produced) was significantly increased in patients with OLP compared to healthy controls. It is clearly not possible to speculate whether salivary NO production was increased as a result of OLP, or whether increased salivary NO has a detrimental effect on the oral mucosa, and might be responsible in part for the development of OLP. Increased salivary nitrite has also been found in patients with recurrent oral apthous ulceration.35 Although the role of salivary NO in normal oral mucosal physiology is as yet unknown, the findings suggest that excessive salivary NO has a potential role in modifying oral mucosal diseases as a patho-physiological regulator.35 At low concentrations, NO may actually protect oral mucosa. A recent study of nonsteroidal anti-inflammatory drug (NSAID)-induced buccal mucosal ulceration found that constitutive NOS activity was reduced when compared with normal controls.41 This situation also occurs in gastric mucosa and provides a plausible explanation for the protective effect of NO on oral mucosa. However, increased NOS activity is also known to occur in chronic ethanol ingestion, and it has been postulated that this interacts with the caspase family of enzymes, thereby promoting apoptosis.42 It is likely that the effects of NO on oral mucosa are concentration dependent.
Salivary gland diseases Few studies have investigated NOS expression in salivary gland disorders. Bentz et al. assessed NOS3 expression in a variety of benign and malignant salivary tumours and found increased NOS3 expression in all 48 tumours studied relative to normal salivary tissue where little NOS3 was found outside blood vessel endothelium.43 It is difficult to comment on the significance of their results as the method of assessing NOS3 expression was by staining intensity only, and NOS3 produces low and intermittent concentrations of NO. We recently found that NOS2 was expressed both in pleomorphic adenoma and normal salivary ducts, and postulated that its expression in this tumour was due to its origin from myoepithelial cells.28 The significance of these findings is as yet unknown as are the effects of NO in saliva. NO produced by NOS1 probably acts as a nonadrenergic, noncholinergic neurotransmitter, whereas that produced by NOS3 exerts a vasodilatory effect.44 NO (or nitrite) formed in saliva although it may serve as an anti-bacterial agent, or be involved in saliva production itself. NO has also been implicated in Sjogrens syndrome (SS).
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Surprisingly, it is the resident salivary gland cells rather than immigrant inflammatory cells that are responsible for raised nitrite concentrations in the saliva of patients with SS. It has been postulated that NO might contribute to inflammatory damage and acinar cell atrophy in SS.44
Role of NO in carcinogenesis Transition from a normal somatic cell to a cancer cell is usually the result of many genetic changes, including the activation of oncogenes or inactivation of tumour suppressor genes. These changes allow the cell to escape normal control mechanisms which control cell proliferation, migration, differentiation and cell death. The frequency of mutations leading to carcinogenesis is largely dictated by chemicals in the cellular microenvironment and to a lesser extent by genetic predisposition. Since high concentrations of NO cause genetic mutations, it could be postulated that long-term NO exposure (from NOS2 activity during chronic inflammation) could have an active role in carcinogenesis.38—40 NO-induced gene mutations can occur via several mechanisms. DNA damage can be due to NOinduced deamination, and nucleic acid transition and/or transversion can also occur following exposure to RNOx species. NO has a great affinity for the thiol group in amino acids, and as a result can inactivate DNA ligase (a DNA repair enzyme), which contains large numbers of cysteine residues.4 Other evidence suggests that NO may promote carcinogenesis by inactivating the tumour suppressor gene, p53. However, there is still controversy as to whether NO can be mutagenic in human cancer, because of the concentrations produced. One in vitro study found that isolated human bronchial cells that had been treated with the NO donor diethyamine/NO (DEA/NO), or transfected with a murine NOS2 gene, showed no appreciable mutations in the p53 gene.45 In contrast, high mutation frequencies were found in this gene (G:C to A:T transition at codon 248) after treatment with the genotoxic agent, ethylnitrosourea (at a concentration of 4 mM). In contrast, a positive correlation was found between total NOS activity and p53 mutations in lung cancer.46 It is difficult to determine whether NO induces p53 mutations or whether the p53 mutation itself resulted in up-regulation of NOS2. The relationship between the tumour suppressor gene p53 and NO is interesting. Forrester et al.25 found that wild-type p53 protein accumulation occurred and NOS2 activity was reduced in a variety of both normal and tumour cells after NO exposure. Only wild-type p53 protein was capable of down regulating NOS2 expression; mutant p53 did not
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have this effect. p53 is a highly sensitive sensor of cellular NO, and will initially down-regulate NOS2 (to reduce NO) before inducing apoptosis if the NO concentrations remain high. Therefore the p53 mutations, a common finding in oral cancer, can lead to unchecked NOS2 activity with potentially detrimental effects. Chen and Lin investigated the immunohistochemical expression of NOS in chemically induced hamster buccal pouch carcinogenesis, finding that NOS expression correlated with the progression from normal mucosa to invasive cancer.47 Although it is not possible to comment on whether NO was directly responsible for this effect, it implies that NOS2 is implicated in this process.
Oral carcinoma and dysplasia Most work published on oral cancer and dysplasia has focused on NOS2. Rosbe et al. were the first to study the immunohistochemical expression of NOS2 in head and neck squamous cell carcinoma.48 The results of 10 cases were published, but no details were given about tumour site. NOS2 was expressed predominantly by tumour cells in eight out of ten cases, with little or no expression being found in oral mucosa. Gallo et al. studied total NOS activity in 27 cases of head and neck squamous cell carcinoma, including six oral cancer cases.14 Again, tumour cells were the predominant source of NOS expression. An increase in NOS activity and microvessel count (assessed using immunochemistry to the endothelial antigen CD31) was seen in tumour samples (taken from the tumour edge) from patients with metastatic neck and advanced disease. Fragments of tumour tissue from neck node positive (n ¼ 5) and node negative (n ¼ 7) patients were implanted into the avascular rabbit cornea. The implanted samples from the neck node positive group induced a more potent angiogenic response, those that had been taken from patients without neck node metastasis. Our own research group has also found that NOS2 correlates with lymph node metastasis in oral cancer.55 Although NO facilitates angiogenesis, it may also have a number of other actions that might be detrimental in oral cancer. NO has been implicated in modulating expression of matrix metalloproteases (MMP), and it is also thought to downregulate the synthesis of their natural inhibitors, tissue inhibitors of matrix metalloproteases (TIMP). MMP are a family of highly homologous proteolytic enzymes involved in the degradation of basement membrane and other extracellular components. They are generally secreted in proenzyme form and are activated by other proteases. Net enzymatic activity
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depends on the balance between levels of activated MMP and TIMP. NO activates several of the MMP family in cartilage, resulting in its degradation.49 The invasion-stimulating effects of NO are mediated, at least in part, by a downregulation of TIMP-2 (and possibly TIMP-3) and up-regulation of MMP-2 and MMP-9.39 We have recently studied the effects of NO on MMP-9 expression (assessed by zymography) in a panel of oral squamous cell carcinoma cell lines. We used a number of NO donor drugs, and specific NOS2 inhibitor agents Our early results suggest that NO donors may increase MMP-9 expression, and that NOS2 antagonist drugs reduce MMP-9 expression in some cell lines. Additionally NOS2 antagonists reduced tumour cell invasion (assessed using Transwell invasion assays) in a number of the cell lines studied. Although these are preliminary studies, it would appear that NO might facilitate tumour cell invasion in oral cancer. As previously discussed, it is unlikely that the NO concentrations found in human cancers are sufficient to induce apoptosis or to cause direct tumour cell death.14,18 However, recent in vitro experiments have found that NO donor drugs induced apoptosis of oral cancer cells at higher concentrations, and eventually caused direct cytotoxicity and cell death.50 It is unlikely that this effect could be utilized clinically, since the concentrations of NO required would have marked systemic side effects, such as hypotension, vasodilation and increased vascular permeability. To date, there has only been one clinical paper published on NOS3 expression in head and neck cancer and dysplasia.43 The study reported the immunohistochemical expression of NOS3 in cases of hyperplasia, dysplasia and invasive cancer and found significantly increased NOS3 staining intensity in cancer cases, compared with normal oral mucosa. NOS3 staining was also found in cases of dysplasia and hyperplasia. It is difficult to comment on the significance of low, intermittent NO concentrations produced by NOS3, but since NOS2 itself requires NO for activation, it might play a part in this process. NOS2 has been studied in oral dysplasia using immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) techniques.56,51 NOS expression and activity is not found in normal oral mucosa57,52 but it correlates with the severity of dysplasia.56 Positive correlations in have also been found between NOS2 and both vascular endothelial growth factor expression (VEGF)58 and p53 expression in oral dysplasia.53 It is possible that NO production facilitates angiogenesis in the complex transformation from dysplasia to malignancy. The role of NOS2 in the pathogenesis of oral cancer and dysplasia warrants further research. The use of
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specific NOS2 antagonist drugs may also play a part in the future management of patients with oral cancer. These drugs are highly specific for NOS2 and in animal studies have reduced invasiveness and metastasis by up to 60%.54 They also have minimal systemic side effects and can be given orally. The latest agent is currently undergoing phase I trials and if successful, it will be evaluated clinically in patients with oral cancer.
Summary and conclusions There has been an exponential rise in interest in NO since its discovery as a biological messenger in 1987. It has been preserved throughout evolution and has a wide repertoire of actions in both physiological and pathological processes. Although relatively little has been published on its role in oral diseases, it almost certainly has important and damaging actions in diseases ranging from periodontal disease to cancer. There is a great opportunity to both study and potentially control NO production in many oral diseases. Despite over 50,000 published papers to date on NO, this tiny molecule still holds many mysteries.
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29. Paquette DW, Williams RC. Modulation of host inflammatory mediators as a treatment strategy for periodontal diseases. Periodontol 2000;24:239—252. 30. Lohinai Z, Stachlewitz R, Virag L, Szekely AD, Hasko G, Szabo C. Evidence for reactive nitrogen species formation in the gingivomucosal tissue. J Dent Res 2001;80:470—475. 31. Daghigh F, Borghaei RC, Thornton RD, Bee JH. Human gingival fibroblasts produce nitric oxide in response to proinflammatory cytokines. J Periodontol 2002;73:392—400. 32. Takeichi O, Hayashi M, Tsurumachi T, Tomita T, Ogihara H, Ogiso B, et al. Inducible nitric oxide synthase activity by interferon-g-producing cells in human radicular cysts. Int Endod J 1999;32:124—130. 33. Shimauchi H, Takayama S, Narikawa-Kiji M, Shimabukuro Y, Okada H. Production of interleukin-8 and nitric oxide in human periapical lesions. J Endod 2001;27:749—752. 34. Brennan PA, Umar T, Palacios-Callender M, Spedding AV, Mellor T, Buckley J, et al. A study to assess inducible nitric oxide synthase expression in oral lichen planus. J Oral Pathol Med 2000b;29:249—254. 35. Ohashi M, Iwase M, Nagumo M. Elevated production of salivary nitric oxide in oral mucosal diseases. J Oral Pathol Med 1999;28:355—359. 36. Bentz BG, Haines GK, Lingen MW, Pelzer HJ, Hanson DG, Radosevich JA. Nitric oxide synthase type 3 is increased in squamous hyperplasia, dysplasia, and squamous cell carcinoma of the head and neck. Ann Otol Rhinol Laryngol 1999; 108:781—787. 37. Mollaoglu M. Oral lichen planus: a review. Br J Oral Maxillofac Surg 2000;38:370—377. 38. Liu RH, Hotchkiss JH. Potential genotoxicity of chronically elevated nitric oxide: a review. Mutation Res 1995;339: 73—89. 39. Lala PK, Orucevic A. Role of nitric oxide in tumor progression: lessons from experimental tumors. Cancer Metastasis Rev 1998;17:91—106. 40. Murata J, Tada M, Iggo RD, Sawamura Y, Shinohe Y, Abe H. Nitric oxide as a carcinogen: analysis by functional yeast assay of inactivating p53 mutations induced by nitric oxide. Mutation Res 1997;379:211—218. 41. Slomiany BL, Slomiany A. Nonsteroidal anti-inflammatory drugs impair oral mucosal repair by eliciting disturbances in endothelin-converting enzyme-1 and constitutive nitric oxide synthase. J Physiol Pharmacol 2001;52:81—92. 42. Slomiany BL, Piotrowski J, Piotrowski E, Slomiany A. Activation of apoptotic caspase-3 and NOS2 in buccal mucosa with chronic alcohol ingestion. J Biochem Mol Biol Int 1998;45:1199—1209. 43. Bentz BG, Haines K, Hanson DG, Radosevich JA. Endothelial constitutive nitric oxide synthase (ecNOS) localisation in normal and neoplastic salivary tissue. Head Neck 1998;20: 304—309. 44. Konttinen YT, Platts LA, Tuominen S, Eklund KK, Santavirta N, Tornwall J, et al. Role of nitric oxide in Sjogren’s syndrome. Arthritis Rheum 1997;40:875—883. 45. Felley-Bosco E, Mirkovitch J, Ambs S, Mace K, Pfeifer A, Keefer LK, et al. Nitric oxide and ethinylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes. Carcinogenesis 1995;16:2069—2074. 46. Ambs S, Bennett WP, Merriam WG, Ogunfusika MO, Oser SM, Khan MA, et al. Vascular endothelial growth factor and nitric oxide synthase expression in human lung cancer and the relation to p53. Br J Cancer 1998;78:233—239. 47. Chen YK, Lin LM. Immunohistochemical expression of inducible nitric oxide synthase in DMBA-induced hamster buccal pouch carcinogenesis. Oral Oncol 2000;36:221—224.
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48. Rosbe KW, Prazma J, Petrusz P, Mims W, Ball SS, Weissler MC. Immunohistochemical characterization of nitric oxide synthase activity in squamous cell carcinoma of the head and neck. Otolaryngol Head Neck Surg 1995;113:541—549. 49. Murrell GAC, Jang D, Williams RJ. Nitric oxide activates matrix metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun 1995;206:15—21. 50. Shang ZJ, Li JR, Li ZB. Effects of exogenous nitric oxide on oral squamous cell carcinoma: An in vitro study. J Oral Maxillofac Surg 2002;60:905—910. 51. Chen YK, Hsue SS, Lin LM. The mRNA expression of inducible nitric oxide synthase in DMBA-induced hamster buccalpouch carcinomas using reverse transcription-polymerase chain reaction. J Oral Pathol Med 2002;31:82—86. 52. Gavilanes J, Moro MA, Lizasoain I, Lorenzo P, Pe ´rez A, Leza JC, et al. Nitric oxide synthase activity in human squamous cell carcinoma of the head and neck. Laryngoscope 1999;109:148—152. 53. Brennan PA, Conroy B, Spedding AV. Expression of NOS2 and p53 in oral epithelial dysplasia. Oral Surg Oral Med Oral Pathol 2000c;90:624—629.
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54. Thomsen LL, Scott JMJ, Topley P, Knowles RG, Keerie AJ, Frend AJ. Selective inhibition of inducible nitric oxide synthase inhibits tumour growth in vivo. Studies with 1400W, a novel inhibitor. Cancer Res 1997;57:3300—3304. 55. Brennan PA, Palacios-Callender M, Spedding AV, Zaki GA, Langdon JD. Type II nitric oxide synthase expression correlates with lymph node status in oral squamous cell carcinoma. J Oral Pathol Med 2001;30:129—134. 56. Brennan PA, Palacios-Callender M, Spedding AV, Zaki GA, Langdon JD. Does type II nitric oxide synthase expression correlate with cellular proliferation in oral squamous cell carcinoma and dysplasia? Head Neck 2001;27:217—222. 57. Brennan PA, Palacios-Callender M, Umar T, Tant S, Langdon JD. Expression of type 2 nitric oxide synthase and p21 in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2002; 31:200—205. 58. Brennan PA, Umar T, Wilson AW, Mellor TK. Expression of type 2 nitric oxide synthase and vascular endothelial growth factor in oral dysplasia. J Oral Maxillofac Surg 2002;60: 1455—1460.
REVIEW
The role of nitric oxide in oral diseases P.A. Brennana,*, G.J. Thomasb, J.D. Langdonc a
Department of Maxillofacial and Head and Neck Surgery, Queen Alexandra Hospital, Portsmouth PO6 3LY, UK b Department of Oral Pathology, Eastman Dental Institute, 256 Grays Inn Road, London WC1X 8LD, UK c Maxillofacial Department, Kings College School of Medicine and Dentistry, London SE5 8RX, UK Accepted 14 October 2002
KEYWORDS Nitric oxide; Review; Oral disease; Cancer
Summary Although previously regarded as a toxic pollutant gas, nitric oxide (NO) is a short-lived molecule that plays a key role in many physiological and pathological processes. It is produced in vivo from the amino acid L-arginine by a complex family of enzymes termed nitric oxide synthase (NOS). Since its discovery as a biological messenger in 1987, NO has been implicated in many disease processes, ranging from septic shock to cancer. It is a highly reactive free radical and causes concentrationdependent conformational changes in proteins, enzymes and DNA, predominantly by its reaction with transition metals and thiol residues. Although high concentrations of NO are cytotoxic, the levels produced in many human cancers possibly facilitate tumour growth and dissemination. The interest in this molecule by scientists and clinicians involved with the oral cavity and head and neck regions is fairly recent, and only a tiny minority of 50,000 papers currently cited on NO relate to diseases in this anatomical area. This review gives an overview of NO, outlining its basic chemistry, formation by NOS and its possible roles in the oral diseases studied to date. The implications for possible therapeutic manipulation of NO are also discussed. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction NO is thought to be one of the oldest molecules on earth, being formed in the primitive atmosphere of the cooling planet.1 Until recently, NO, a toxic oxide of nitrogen produced by internal combustion engines and power stations, had been regarded solely as a cause of acid rain and atmospheric pollution. The Belgian scientist Jan Baptista van Helmont (1577—1644), more widely known for his discovery of carbon dioxide, and for explaining the action of gunpowder by the theory of gaseous expansion, can be credited as the first to synthesise NO in the laboratory in about 1610. In 1772, Joseph
Corresponding author. Present address: 11, Oxlease Close, Romsey, Hants SO51 7HA, UK. Tel.: þ44-1794-519-951. E-mail address: [email protected] (P.A. Brennan).
Priestley (1733—1804) gave it the name ‘nitrous air’ and observed that it did not support plant life, but reduced putrefaction in meat exposed to it. The effects of NO as a bactericidal agent were revisited again in the early 1980s but it was not until 1987 that NO was discovered to be the chemical responsible for the actions of endothelial derived relaxing factor (EDRF).2,3 Following this finding, NO research expanded exponentially, and it is regarded by many in the scientific community as one of the greatest discoveries of the 20th century. In recognition of this, two Nobel prizes have been awarded to researchers in the NO field, and it was named as molecule of the year by the journal Science in 1992. Although initial reports concentrated on its role in the regulation of vascular tone, it was soon realised that NO had a wide and diverging range of both physiological and pathological actions. Many
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(02)00183-8
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Table 1 Diseases and conditions that are associated with altered nitric oxide production. Hypertension and hypotension Thrombo-embolic disease Septic shock Bronchospasm Acute respiratory distress syndrome (ARDS) Pulmonary hypertension Renal failure Immune deficiency HIV induced encephalopathy Impotence Depression Malignancy
of these processes are still poorly understood. Most specialties of both medicine and science are now involved in NO research. It is implicated in various medical conditions ranging from septic shock to impotence (Table 1). The interest by scientists and clinicians in oral and head and neck diseases has been relatively recent, and only a tiny minority of the 50,000 papers currently cited on NO have concerned this anatomical area.
Basic chemistry and actions of NO The arrangement of one atom of nitrogen and one of oxygen leaves an unpaired electron, which makes the molecule a highly reactive free radical. However, it is less reactive than other free radicals (such as superoxide) in that it does not react with itself at physiological temperatures. NO readily reacts with superoxide, oxygen, thiol groups (—SH) to produce a number of other products, such as nitrosothiols (R— SNO), the toxic molecule peroxynitrite (ONOO—) and nitrites (NOx). Peroxynitrite, formed when both NO and superoxide radicals are present, is very toxic, particularly to DNA and to enzymes involved in DNA repair.4 All of these so-called reactive nitrogen oxide species (termed RNOx) react with transition metals (such as zinc and iron in haemcontaining proteins) and thiol groups in various enzymes and proteins. The resulting effects are concentration dependent, but can lead to inhibition of enzymes such as cytochrome c oxidase, resulting in decreased ATP production and cell death. The more familiar poisons cyanide (CN) and carbon monoxide (CO) inhibit cytochrome c oxidase in an analogous fashion to NO.5 Most NO chemistry is due to conformational changes in enzyme structure in a similar fashion to that seen when oxygen binds to haemoglobin. For simplicity, in this article the term NO will encompass all of these molecules.
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A comprehensive review of NO chemistry has been written by Wink et al.4 NO has been preserved throughout evolution as a mediator in many biological systems. For example, in the horseshoe crab, Limulus polyphemus, a species which has not evolved significantly in 500 million years, aggregation of its single type of blood cell, the haemocyte, is modulated by NO in a way similar to that of modern mammalian platelets.6 The physiological activities of NO may be divided into two main groups–—those that occur as a result of its interaction with the haem group in the enzyme guanylate cyclase, with subsequent activation and the generation of the second messenger cGMP, and those that are cGMP independent. Effects following NO-induced guanylate cyclase activation include the regulation of vascular tone,7,8 and a complex role as a neuromodulator in the central nervous system.9 NO has also been implicated as a pain modulator in various conditions, including temporomandibuar joint pain dysfunction syndrome.10 NO actions that are cGMP independent include a pivotal role during the process of angiogenesis,11—14 where it is thought to interact with many key molecules including the potent angiogenic factor vascular endothelial growth factor (VEGF). NO acts both as an upstream activator and downstream effector molecule for VEGF.15,16 It also has immunocytotoxicity against both pathogens and tumours.17 However, the NO concentrations found in human cancers are unlikely to be sufficient to produce tumour cell death or apoptosis, and instead are thought to be responsible for enhancing angiogenesis and tumour dissemination.14,18
Synthesis of NO NO is synthesised by a complex family of enzymes called NO synthases (NOS). These are some of the most complex enzymes known and require many factors and co-factors for activity. The amino acid L-arginine is the substrate for the NOS enzymes, generating NO and the by-product L-citrulline.19 A schematic representation of one of the NOS enzymes (NOS2) is shown in Fig. 1. There are three NOS enzymes, each produced by distinct genes and named in order of discovery.20 NOS1, was the first to be purified and cloned from neural tissue and it is therefore also called nNOS. NOS3 is the isoform first found in endothelial cells, also called eNOS. NOS1 and NOS3 are also termed constitutive since they are expressed continuously in neurones and endothelial cells, respectively. They are also dependent on a rise in tissue calcium
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Figure 1 Schematic representation of NOS2. Cal denotes binding site for calmodulin.
concentration for activity, and therefore produce low, transient concentrations of NO. In contrast, NOS2 is an inducible, calcium-independent isoform, also called iNOS. NOS1 and NOS3 may themselves be induced under certain conditions (such as pregnancy)21 and NOS2 is expressed constantly in certain sites (such as basal skin keratinocyes and salivary duct cells). Therefore the view of the authors is that the numerical nomenclature should be used for the NOS enzymes. Unlike NOS1 and NOS3, induction of NOS2 results in continous production of NO because of tight calmodulin binding, which allow electrons to pass from the haem group to the flavin (FMN, FAD) and nicotinamide (NADPH) electron accepting co-factors (see Fig. 1). From an evolutionary perspective, human NOS2 generates lower NO concentrations than, for example, the equivalent mouse NOS2 enzyme since the latter has much tighter calmodulin binding.22 The promoter/enhancer region of the NOS2 gene is complex and contains at least 22 potential binding sites for transcription factors. In the human gene, this region resides within a 16kilobase segment of the 50 -flanking area. As well as bacterial lipopolysaccharide (LPS) and interferon-g (IFN-g), numerous other factors induce the production of the enzyme. These include the cytokines tumour necrosis factor-a (TNF-a), interleukin (IL)1, IL-10 and IL-12, platelet activating factor (PAF) and nuclear factor-kb (NF-kb). Once induced the enzyme continues to produce much higher NO concentrations than the other two NOS isoforms, for many hours or even days23 until the protein is eventually inactivated, probably as a result of NO-induced protein modification. It is interesting that NOS2 expression is induced by cisplatin and taxols (used in cancer therapy), although NOS2 induction in these instances is likely to be indirect and mediated via TNF-a release. Hypoxia is known to induce mouse macrophage NOS2, acting at a site on the gene termed the ‘hypoxia-responsive element’ (HRE). A synergistic effect occurs when both hypoxia and IFN-g are present.24 Although located in the NOS2 promoter region, the HRE is separate from the other promoter sites and it shows sequence homology with the enhancer site of erythropoietin. Agents that inhibit NOS2 expression include NO itself, transforming growth factor-b (TGF-b), IL-4, IL-6, IL-8 and IL-10 (although paradoxically IL-10
may also induce NOS2), and certain immunosupressive drugs, such as glucocorticoids, cyclosporin and tacrolimus. An important regulator of NOS2 is the tumour suppressor gene p53, which senses raised cellular NO and inhibits NOS2 by a negative feedback loop.25 This relationship has important implications in oral cancer (see below). At a posttranscription level, transforming growth factor-b (TGF-b), and NO may destabilise mRNA.26 The action of corticosteroids in reducing NOS2 expression is not fully understood, but it may involve regulation at both the transcription and translation levels. In chronic inflammatory conditions, NOS2 expression is therefore dependent on the overall cytokine balance. NOS2 is found in various cells including macrophages and natural killer cells in the immune system. It is also constantly expressed by large airway epithelium, in the basal keratinocyte layer of normal skin,27 and in normal salivary ducts.28 In normal skin, the constant NO generation by NOS2 may assist the host defence against pathogens27 although its role in salivary ducts is as yet unknown.
Studies of NO in oral and related diseases There are few studies published on the role of NO in oral diseases. Although there have been few publications concerning this anatomical area, interest in NO is expanding rapidly. Of greatest interest to the authors is the role of NO in the pathogenesis and tumour biology of oral cancer. An overview of oral diseases in which NO has been implicated is discussed below.
Periodontal disease Unsurprisingly, NO synthesis is increased in periodontal disease, as a result of macrophage infiltration in the periodontal tissues.29 It is probable that bacterial plaque is responsible for NOS2 activation, since no NOS2 activity is found in the gingival tissues of sterile animals.30 Recent studies have also demonstrated that gingival fibroblasts in chronic periodontitis have significantly higher NOS2 expression than those in healthy gingival tissue.31 NO might play a role in the pathogenesis of both periodontitis and subsequent bone loss, either directly, or indirectly by modulating the production of other
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pro-inflammatory cytokines. A recent in vitro study found that NOS2 inhibitors reduce gingival fibroblast NO production, and it was postulated that pharmacological inhibition of NO might be therapeutically valuable in the management of this disease.31 It is possible that the induction of NOS2 occurs in response to bacterial LPS, or as a result of cytokines which are formed in periodontal disease. Although NO may be produced in an attempt to kill plaque bacteria, it is likely that (as with oral cancer), the concentrations produced result in host tissue damage instead. Further work is clearly needed in this area.
Odontogenic cysts and periapical infection There have been few published reports in this area and only one paper to date has investigated NOS expression in odontogenic cysts. This study investigated the expression of NOS2 in radicular cysts.32 This type of cyst results from chronic periapical inflammation, and it is not surprising that NOS2 was expressed in this condition. It is not apparent whether NO may modulate bone destruction in such circumstances. We are currently investigating the role of NO in the pathogenesis of odontogenic keratocysts, which can recur following enucleation and often have an aggressive behaviour if they extend into surrounding soft tissue. NO has also been implicated in the pathogenesis of apical infection.33 Again, inflammation-induced NOS2 accounts for NO production, and it has been postulated that NOS inhibitor drugs introduced into the root canals of teeth with periapical infections may lead to resolution of this condition.32
Oral mucosal inflammatory diseases There have been few studies which have investigated the role of NO in oral mucosal disease.34—36 Although oral lichen planus (OLP) is a chronic inflammatory disorder, little or no NOS2 activity or expression was found in this disorder, despite an abundance of CD68 positive macrophages.34 OLP itself is a complex disorder and many cytokines are produced. It was postulated that the balance between NOS2 activation and inhibitory cytokines in OLP favoured NOS2 inhibition over activation, but clearly further research is required in this field. There is still controversy regarding whether erosive and atrophic forms of OLP are associated with the development of oral cancer.37 Chronic inflammatory conditions that induce NOS2 might produce sufficient NO and related products to initiate carcinogenesis.38—40 However, since little or no NOS2 activity was found in OLP, it is unlikely that NO
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would play a role in the transformation of OLP to malignancy, if indeed it occurs at all. In contrast,35 found that salivary nitrite (implying that NO had been produced) was significantly increased in patients with OLP compared to healthy controls. It is clearly not possible to speculate whether salivary NO production was increased as a result of OLP, or whether increased salivary NO has a detrimental effect on the oral mucosa, and might be responsible in part for the development of OLP. Increased salivary nitrite has also been found in patients with recurrent oral apthous ulceration.35 Although the role of salivary NO in normal oral mucosal physiology is as yet unknown, the findings suggest that excessive salivary NO has a potential role in modifying oral mucosal diseases as a patho-physiological regulator.35 At low concentrations, NO may actually protect oral mucosa. A recent study of nonsteroidal anti-inflammatory drug (NSAID)-induced buccal mucosal ulceration found that constitutive NOS activity was reduced when compared with normal controls.41 This situation also occurs in gastric mucosa and provides a plausible explanation for the protective effect of NO on oral mucosa. However, increased NOS activity is also known to occur in chronic ethanol ingestion, and it has been postulated that this interacts with the caspase family of enzymes, thereby promoting apoptosis.42 It is likely that the effects of NO on oral mucosa are concentration dependent.
Salivary gland diseases Few studies have investigated NOS expression in salivary gland disorders. Bentz et al. assessed NOS3 expression in a variety of benign and malignant salivary tumours and found increased NOS3 expression in all 48 tumours studied relative to normal salivary tissue where little NOS3 was found outside blood vessel endothelium.43 It is difficult to comment on the significance of their results as the method of assessing NOS3 expression was by staining intensity only, and NOS3 produces low and intermittent concentrations of NO. We recently found that NOS2 was expressed both in pleomorphic adenoma and normal salivary ducts, and postulated that its expression in this tumour was due to its origin from myoepithelial cells.28 The significance of these findings is as yet unknown as are the effects of NO in saliva. NO produced by NOS1 probably acts as a nonadrenergic, noncholinergic neurotransmitter, whereas that produced by NOS3 exerts a vasodilatory effect.44 NO (or nitrite) formed in saliva although it may serve as an anti-bacterial agent, or be involved in saliva production itself. NO has also been implicated in Sjogrens syndrome (SS).
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Surprisingly, it is the resident salivary gland cells rather than immigrant inflammatory cells that are responsible for raised nitrite concentrations in the saliva of patients with SS. It has been postulated that NO might contribute to inflammatory damage and acinar cell atrophy in SS.44
Role of NO in carcinogenesis Transition from a normal somatic cell to a cancer cell is usually the result of many genetic changes, including the activation of oncogenes or inactivation of tumour suppressor genes. These changes allow the cell to escape normal control mechanisms which control cell proliferation, migration, differentiation and cell death. The frequency of mutations leading to carcinogenesis is largely dictated by chemicals in the cellular microenvironment and to a lesser extent by genetic predisposition. Since high concentrations of NO cause genetic mutations, it could be postulated that long-term NO exposure (from NOS2 activity during chronic inflammation) could have an active role in carcinogenesis.38—40 NO-induced gene mutations can occur via several mechanisms. DNA damage can be due to NOinduced deamination, and nucleic acid transition and/or transversion can also occur following exposure to RNOx species. NO has a great affinity for the thiol group in amino acids, and as a result can inactivate DNA ligase (a DNA repair enzyme), which contains large numbers of cysteine residues.4 Other evidence suggests that NO may promote carcinogenesis by inactivating the tumour suppressor gene, p53. However, there is still controversy as to whether NO can be mutagenic in human cancer, because of the concentrations produced. One in vitro study found that isolated human bronchial cells that had been treated with the NO donor diethyamine/NO (DEA/NO), or transfected with a murine NOS2 gene, showed no appreciable mutations in the p53 gene.45 In contrast, high mutation frequencies were found in this gene (G:C to A:T transition at codon 248) after treatment with the genotoxic agent, ethylnitrosourea (at a concentration of 4 mM). In contrast, a positive correlation was found between total NOS activity and p53 mutations in lung cancer.46 It is difficult to determine whether NO induces p53 mutations or whether the p53 mutation itself resulted in up-regulation of NOS2. The relationship between the tumour suppressor gene p53 and NO is interesting. Forrester et al.25 found that wild-type p53 protein accumulation occurred and NOS2 activity was reduced in a variety of both normal and tumour cells after NO exposure. Only wild-type p53 protein was capable of down regulating NOS2 expression; mutant p53 did not
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have this effect. p53 is a highly sensitive sensor of cellular NO, and will initially down-regulate NOS2 (to reduce NO) before inducing apoptosis if the NO concentrations remain high. Therefore the p53 mutations, a common finding in oral cancer, can lead to unchecked NOS2 activity with potentially detrimental effects. Chen and Lin investigated the immunohistochemical expression of NOS in chemically induced hamster buccal pouch carcinogenesis, finding that NOS expression correlated with the progression from normal mucosa to invasive cancer.47 Although it is not possible to comment on whether NO was directly responsible for this effect, it implies that NOS2 is implicated in this process.
Oral carcinoma and dysplasia Most work published on oral cancer and dysplasia has focused on NOS2. Rosbe et al. were the first to study the immunohistochemical expression of NOS2 in head and neck squamous cell carcinoma.48 The results of 10 cases were published, but no details were given about tumour site. NOS2 was expressed predominantly by tumour cells in eight out of ten cases, with little or no expression being found in oral mucosa. Gallo et al. studied total NOS activity in 27 cases of head and neck squamous cell carcinoma, including six oral cancer cases.14 Again, tumour cells were the predominant source of NOS expression. An increase in NOS activity and microvessel count (assessed using immunochemistry to the endothelial antigen CD31) was seen in tumour samples (taken from the tumour edge) from patients with metastatic neck and advanced disease. Fragments of tumour tissue from neck node positive (n ¼ 5) and node negative (n ¼ 7) patients were implanted into the avascular rabbit cornea. The implanted samples from the neck node positive group induced a more potent angiogenic response, those that had been taken from patients without neck node metastasis. Our own research group has also found that NOS2 correlates with lymph node metastasis in oral cancer.55 Although NO facilitates angiogenesis, it may also have a number of other actions that might be detrimental in oral cancer. NO has been implicated in modulating expression of matrix metalloproteases (MMP), and it is also thought to downregulate the synthesis of their natural inhibitors, tissue inhibitors of matrix metalloproteases (TIMP). MMP are a family of highly homologous proteolytic enzymes involved in the degradation of basement membrane and other extracellular components. They are generally secreted in proenzyme form and are activated by other proteases. Net enzymatic activity
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depends on the balance between levels of activated MMP and TIMP. NO activates several of the MMP family in cartilage, resulting in its degradation.49 The invasion-stimulating effects of NO are mediated, at least in part, by a downregulation of TIMP-2 (and possibly TIMP-3) and up-regulation of MMP-2 and MMP-9.39 We have recently studied the effects of NO on MMP-9 expression (assessed by zymography) in a panel of oral squamous cell carcinoma cell lines. We used a number of NO donor drugs, and specific NOS2 inhibitor agents Our early results suggest that NO donors may increase MMP-9 expression, and that NOS2 antagonist drugs reduce MMP-9 expression in some cell lines. Additionally NOS2 antagonists reduced tumour cell invasion (assessed using Transwell invasion assays) in a number of the cell lines studied. Although these are preliminary studies, it would appear that NO might facilitate tumour cell invasion in oral cancer. As previously discussed, it is unlikely that the NO concentrations found in human cancers are sufficient to induce apoptosis or to cause direct tumour cell death.14,18 However, recent in vitro experiments have found that NO donor drugs induced apoptosis of oral cancer cells at higher concentrations, and eventually caused direct cytotoxicity and cell death.50 It is unlikely that this effect could be utilized clinically, since the concentrations of NO required would have marked systemic side effects, such as hypotension, vasodilation and increased vascular permeability. To date, there has only been one clinical paper published on NOS3 expression in head and neck cancer and dysplasia.43 The study reported the immunohistochemical expression of NOS3 in cases of hyperplasia, dysplasia and invasive cancer and found significantly increased NOS3 staining intensity in cancer cases, compared with normal oral mucosa. NOS3 staining was also found in cases of dysplasia and hyperplasia. It is difficult to comment on the significance of low, intermittent NO concentrations produced by NOS3, but since NOS2 itself requires NO for activation, it might play a part in this process. NOS2 has been studied in oral dysplasia using immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) techniques.56,51 NOS expression and activity is not found in normal oral mucosa57,52 but it correlates with the severity of dysplasia.56 Positive correlations in have also been found between NOS2 and both vascular endothelial growth factor expression (VEGF)58 and p53 expression in oral dysplasia.53 It is possible that NO production facilitates angiogenesis in the complex transformation from dysplasia to malignancy. The role of NOS2 in the pathogenesis of oral cancer and dysplasia warrants further research. The use of
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specific NOS2 antagonist drugs may also play a part in the future management of patients with oral cancer. These drugs are highly specific for NOS2 and in animal studies have reduced invasiveness and metastasis by up to 60%.54 They also have minimal systemic side effects and can be given orally. The latest agent is currently undergoing phase I trials and if successful, it will be evaluated clinically in patients with oral cancer.
Summary and conclusions There has been an exponential rise in interest in NO since its discovery as a biological messenger in 1987. It has been preserved throughout evolution and has a wide repertoire of actions in both physiological and pathological processes. Although relatively little has been published on its role in oral diseases, it almost certainly has important and damaging actions in diseases ranging from periodontal disease to cancer. There is a great opportunity to both study and potentially control NO production in many oral diseases. Despite over 50,000 published papers to date on NO, this tiny molecule still holds many mysteries.
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