**3.1.2 Leukoplakia, oral lichen planus and oral cancer**

More than 300,000 new cases are being diagnosed with oral squamous cell carcinoma annually in the world (Sudbo & Reith, 2005). Oral leukoplakia is a precancerous lesion characterized by white plaque and hyperkeratosis (Neville & Day, 2002; Reibel, 2003). Five to 15% of leukoplakia is histologically classified as dysplasia (Suarez et al., 1998; Sudbo & Reith, 2005). The presence of epithelial dysplasia may be important in predicting malignant development. A substantial part of dysplasia is reported to develop into oral carcinoma (Lumerman et al., 1995; Sudbo & Reith, 2005). Oral lichen planus (OLP) is a chronic inflammatory mucosal disease (Scully et al., 1998). Several pathological features indicate that OLP is an immunologically mediated inflammatory response, including an intense, band-like infiltrate of predominantly T-lymphocytes subjacent to epithelium. Basal epithelial cells are the target for immune destruction by cytotoxic T-lymphocytes (Tyldesley & Appleton, 1973; Dekker et al., 1997). The most important complication of OLP is development of oral squamous cell carcinoma (OSCC) (Rajentheran et al., 1999; Mignogna et al., 2004).

Antioxidants have induced regression of precancerous oral lesions including leukoplakia (Enwonwu & Meeks, 1995), suggesting that ROS are involved in the pathogenesis of precancerous lesions. ROS are considered to participate in carcinogenesis by forming oxidative DNA lesions, such as 8-oxodG (Kawanishi et al., 2001; Kawanishi et al., 2002).

A chronic inflammatory infiltration was generally present in oral tissues of leukoplakia patients (Rodriguez-Perez & Banoczy, 1982). Expression of COX-2, an inflammatory mediator, was increased in oral mucosa with various lesions of leukoplakia, including hyperplasia and dysplasia (Renkonen et al., 2002; Altorki et al., 2004). These reports implicate that inflammation may mediate the development of oral cancer from leukoplakia. Therefore, we examined whether nitrative stress contributes to the development of oral carcinogenesis from leukoplakia through DNA damage.

We demonstrated that accumulation of 8-nitroguanine and 8-oxodG was observed in oral epithelium of biopsy specimens from patients with OLP and OSCC, whereas no immunoreactivity was observed in normal oral mucosa (Chaiyarit et al., 2005). 8- Nitroguanine and 8-oxodG were also observed in oral epithelium of patients with leukoplakia (Ma et al., 2006). Co-localization of 8-nitroguanine and iNOS was found in oral epithelium of patients with OLP, OSCC and leukoplakia. Immunoreactivity of 3 nitrotyrosine, which is formed by protein tyrosine nitration and considered to be a

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Fig. 6. Localization of 8-nitroguanine, PCNA, p53, iNOS and 3-nitrotyrosine in oral tissues of leukoplakia patients. Paraffin sections were immunostained with anti-8-nitroguanine

biochemical marker for inflammation, was also observed in oral epithelial cells. Accumulation of p53 was observed in oral epithelium in OLP and leukoplakia, and more prominent expression of this protein was observed in OSCC patients. Our findings demonstrate that iNOS-dependent DNA damage may lead to p53 accumulation not only in OLP and leukoplakia, but also in OSCC. It is concluded that the formation of 8-nitroguanine and 8-oxodG may contribute to the development of oral cancer from OLP and leukoplakia.

Fig. 5. Localization of 8-nitroguanine and 8-oxodG and histopathological changes in oral tissues of leukoplakia patients. Paraffin sections were immunostained with anti-8 nitroguanine antibody and anti-8-oxodG antibody using double immunofluorescence technique as described in immunofluorescent staining procedure procedures. Strong 8 nitroguanine and 8-oxodG immunoreactivities are observed in the nucleus of the same epithelial cells in supra-basal and basal layers and epithelial-connective tissue interface area of leukoplakia patients. The distribution of 8-nitroguanine- and 8-oxodG-positive cells is similar to that of dysplastic epithelial cells. Little or no immunoreactivity of 8-nitroguanine and 8-oxodG was observed in normal mucosa. Scale bar represents 50 m.

biochemical marker for inflammation, was also observed in oral epithelial cells. Accumulation of p53 was observed in oral epithelium in OLP and leukoplakia, and more prominent expression of this protein was observed in OSCC patients. Our findings demonstrate that iNOS-dependent DNA damage may lead to p53 accumulation not only in OLP and leukoplakia, but also in OSCC. It is concluded that the formation of 8-nitroguanine and 8-oxodG may contribute to the development of oral cancer from OLP and leukoplakia.

Fig. 5. Localization of 8-nitroguanine and 8-oxodG and histopathological changes in oral tissues of leukoplakia patients. Paraffin sections were immunostained with anti-8 nitroguanine antibody and anti-8-oxodG antibody using double immunofluorescence technique as described in immunofluorescent staining procedure procedures. Strong 8 nitroguanine and 8-oxodG immunoreactivities are observed in the nucleus of the same epithelial cells in supra-basal and basal layers and epithelial-connective tissue interface area of leukoplakia patients. The distribution of 8-nitroguanine- and 8-oxodG-positive cells is similar to that of dysplastic epithelial cells. Little or no immunoreactivity of 8-nitroguanine

and 8-oxodG was observed in normal mucosa. Scale bar represents 50 m.

Fig. 6. Localization of 8-nitroguanine, PCNA, p53, iNOS and 3-nitrotyrosine in oral tissues of leukoplakia patients. Paraffin sections were immunostained with anti-8-nitroguanine

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Fig. 7. Formation of 8-nitroguanine and 8-oxodG and expression of LMP, iNOS, STAT3 and

EGFR in the nasopharyngeal tissues of NPC and chronic nasopharyngitis patients.

antibody and anti-PCNA or anti-3-nitrotyrosine or anti-iNOS, p53 antibody using double immunofluorescence technique. 8-Nitroguanine formation is observed mainly in the nucleus of oral epithelial cells. Strong expression of PCNA and p53 was observed in the nucleus of 8 nitroguanine-positive cells in the basal layer. iNOS expression is strongly observed in inflammatory cells and weakly in the cytoplasm of epithelial cells. Moreover, strong 8 nitroguanine and 3-nitrotyrosine immunoreactivities are observed in the nucleus of the same epithelial cells. Scale bar represents 50 m.
