Expression of type 2 nitric oxide synthase and p21 in oral squamous cell carcinoma.

Nitric oxide (NO) has a complex role in tumour biology. Most cancer research has focused on the enzyme nitric oxide synthase-2 (NOS2), an inducible isoform responsible for prolonged NO production. In normal cells exposed to high NO concentrations, the tumour-suppressor gene, p53, promotes apoptosis via the p21 pathway, in an attempt to safeguard against potential NO-mediated DNA damage. In cancer cells with mutant p53, this pathway is unlikely to occur directly, although, p53-independent p21 expression and subsequent apoptosis can occur at higher NO concentrations. In this study, the possible direct association between NOS2 and p21 was assessed in oral squamous cell carcinoma. Immunohistochemistry was performed for NOS2 and p21 on 56 cases, and NOS2 activity was determined with citrulline assays in selected cases. A significant relationship was demonstrated between the immunohistochemical expression of NOS2 and its activity (P<0.001), but not between NOS2 and p21 expression (P=0.76). It is unlikely that the NO concentrations found in oral cancer (up to 10.3 pmol NO min(-1) mg protein(-1)) are sufficient to cause direct (p53-independent) p21 accumulation and subsequent apoptosis. As with many other tumours, since NO production has a detrimental role, its pharmacological inhibition in oral cancer represents an exciting area for possible future therapeutic manipulation.

Does type II nitric oxide synthase expression correlate with cellular proliferation in oral squamous cell carcinoma and dysplasia?

BACKGROUND: Nitric oxide (NO) has been implicated both in tumor progression and inhibition. This study investigated whether type II nitric oxide synthase (NOS2) expression correlated with cell proliferation in oral squamous cell carcinoma (OSCC) and dysplasia. METHODS: Paraffin-embedded tissue samples of normal oral mucosa, OSCC, and dysplasia were assessed immunohistochemically using monoclonal antibodies to NOS2 and Ki-67 antigen. We used Western blotting to confirm NOS2 antibody specificity and protein expression in select cases. RESULTS: NOS2 staining was increased in OSCC relative to normal oral mucosa, in which no expression was found. Both NOS2 expression and Ki-67 indices independently correlated with grade of dysplasia (p < .001) but not with the degree of tumor differentiation. A positive correlation was found between NOS2 expression and Ki-67 in cases of mild and moderate dysplasia (p < .001), but not in severe dysplasia and OSCC. CONCLUSIONS: No correlation exists between Ki-67 and NOS2 expression in severe dysplasia and OSCC. The findings suggest that the level of NO produced by NOS2 is insufficient to affect cellular proliferation in these conditions. The mechanism of NOS2 activation and the consequences of its expression remain to be fully explained.

Type II nitric oxide synthase (NOS2) expression correlates with lymph node status in oral squamous cell carcinoma.

In tumour biology, nitric oxide (NO) has a complex array of concentration-dependent actions, including both inhibitory and promoting effects. It is thought that the levels of NO found in many human cancers lead to enhanced angiogenesis and tumour dissemination. In the current study, we assessed the immunohistochemical expression of the enzyme type II nitric oxide synthase (NOS2) in 41 cases of oral squamous cell carcinoma and correlated the findings with lymph node status. A significant relationship was found between NOS2 expression and lymph node metastasis (P<0.0002). Furthermore, lymph node metastasis correlated with the degree and intensity of staining seen (P<0.001). No correlation was found between the size of the primary tumour, degree of tumour differentiation or smoking status and NOS2 staining. Western blotting confirmed NOS2 protein expression in select cases. As with many other human tumours, NOS2 is not a ubiquitous finding in oral cancer. Its expression may be of value in assessing lymph node status prior to surgery, and it represents a target for possible therapeutic manipulation.

Biphasic regulation of NF-kappa B activity underlies the pro- and anti-inflammatory actions of nitric oxide.

Expression of inducible NO synthase (iNOS) by macrophages is a prerequisite for the production of high output NO, which mediates many bactericidal and tumoricidal actions of these immune cells. The expression of iNOS in mammalian cells is governed predominantly by the transcription factor, NF-kappa B, which regulates the expression of many host defense proteins. In the present study, we characterize a novel, biphasic effect of NO on NF-kappa B activity in murine macrophages. This mechanism depends on the local concentration of NO and enables it both to up- and down-regulate the expression of host defense proteins including iNOS, cyclooxygenase-2, and IL-6. This biphasic activity of NO appears to play a pivotal role in the time course of activation of these immune cells and, by inference, in facilitating the initiation of a defense response against pathogenic stimuli and in its termination to limit tissue damage. This mechanism may explain at least in part the reported ability of NO to act in both a pro- and anti-inflammatory manner.

Enhanced peroxynitrite formation is associated with vascular aging.

Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (.O(2)(-)) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented.O(2)(-) release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Correlation between type II nitric oxide synthase and p53 expression in oral squamous cell carcinoma.

Prolonged nitric oxide (NO) production by the enzyme type II nitric oxide synthase (NOS2) has been implicated in angiogenesis and metastasis of human cancers. In animal models, wild-type p53 (but not mutant) protein results in down-regulation of NOS2 expression, which reduces both tumour growth and dissemination. In the current study, we aimed to find out whether a correlation was present in oral squamous cell carcinoma. Fifty-six cases of squamous cell carcinoma were assessed immunohistochemically using antibodies to NOS2 and p53 (clone DO-7). We also confirmed NOS2 protein expression in selected cases using immunoblotting. The results were correlated with clinicopathological findings. Statistical analysis showed a significant relationship between p53 and NOS2 expression (P= 0.001). No relationship was found between size of tumour or histological degree of differentiation, and NOS2 expression in the primary tumour.

A study to assess inducible nitric oxide synthase expression in oral lichen planus.

Nitric oxide (NO) has been implicated in a variety of diseases but has not been previously studied in oral lichen planus (OLP). Since OLP has a complex immunogenesis with abundant macrophage infiltration, this study determined by immunohistochemistry whether or not the expression of the inducible form of nitric oxide synthase (iNOS) was increased in this condition relative to normal mucosa. Thirty cases of OLP and 10 normal buccal mucosa biopsies were studied utilising primary antibodies to iNOS and CD68, a myelomonocytic marker. iNOS activity was additionally assessed using a [(14-)C]-labelled arginine to citrulline assay. CD68 expression was significantly increased in the cellular infiltrate of all 30 cases of OLP compared with normal mucosa (P<0.009). Although iNOS staining was seen in a minority of cells in nine cases, this was not statistically significant when compared with the absent staining in normal oral mucosa (P=0.26). Furthermore, the minimal iNOS activity found in OLP was similar to that in normal mucosa. We conclude that expression of iNOS by macrophages is downregulated in OLP and discuss the possible reasons for this finding.

Does expression of inducible nitric oxide synthase correlate with severity of oral epithelial dysplasia?

The small molecule nitric oxide (NO) has generated an exponential amount of research since its discovery as a biological messenger in 1987. It has a vast number of actions, many of which are poorly understood. It has been studied in a variety of human cancers and has been implicated both in tumour promotion and inhibition. Although NO is produced by three distinct isoforms of the enzyme nitric oxide synthase (NOS), most cancer research is directed towards the calcium-independent form, iNOS which following induction, produces much higher quantities of NO than the other two. In this study the expression of iNOS is assessed by immunohistochemistry in 26 cases of oral epithelial dysplasia ranging in severity from mild to severe. iNOS staining was found in all 26 cases of dysplasia with the degree of staining correlating to the severity of dysplasia (p < 0.001). There was no iNOS staining seen in adjacent normal epithelium. The possible role of iNOS in the complex transformation from dysplasia to invasive oral cancer and the clinical applications are discussed.