Increased expression of inducible nitric oxide synthase for human buccal squamous-cell carcinomas: immunohistochemical, reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR studies.

Background: The inducible nitric oxide synthase (iNOS) is involved primarily in inflammatory and carcinogenesis processes. An enhanced expression of iNOS at the protein level has been reported previously for human oral squamous cell carcinoma; however, the expression of iNOS at the mRNA level has not yet been demonstrated. Furthermore, no studies have addressed whether iNOS expression at mRNA level correlates with cervical lymph node metastasis.

Methods: Specimens of the squamous cell carcinoma of the buccal mucosa obtained from tissue samples of surgically resected tumors from 25 male patients were evaluated with immunohistochemical assessment of iNOS protein and IS-RT-PCR, as well as RT-PCR for iNOS mRNA. We also analyzed the iNOS expression status with clinical parameters to determine whether it had any prognostic significance in this homogenous population.

Results: Inducible NOS protein (16 of 25, 64%) and mRNA (13 of 25, 53%) activities were detected for the oral carcinoma specimens examined in this study. Cytoplasmic and/or nuclear stainings were observed in the specimens of both well-differentiated SCC (10 of 15, 67%), and moderately to poorly differentiated SCC (6 of 10, 60%). The cellular location of iNOS mRNA was noted to be consistent with the finding using immunohistochemical technique (cytoplasm and/or nuclei stainings of the tumor islands). Using in situ RT-PCR, iNOS mRNA activity was detected in nine specimens of well-differentiated squamous cell carcinoma (9 of 15, 60%) and four specimens of moderately to poorly differentiated squamous cell carcinoma (4 of 10, 40%). With RT-PCR, an electrophoretic band corresponding to a 907-bp PCR product was observed for nine specimens of well-differentiated squamous cell carcinoma (9 of 15, 60%) and four specimens of moderately to poorly differentiated squamous-cell carcinoma (4 of 10, 40%). Neither iNOS protein nor mRNA was noticed in the samples of normal buccal mucosa or in the negative control samples. There was a significant relationship between iNOS expression (at both protein and mRNA levels) and whether patients had nodal disease.

Conclusions: We have demonstrated an enhanced expression of iNOS protein and mRNA in a number of human buccal carcinomas compared with normal buccal mucosa. Such an observation suggests that the iNOS protein and mRNA expression is chiefly derived from a subset of oral cancerous tissues. Our observation also indicates that iNOS expression correlates with cervical lymph node metastasis in oral squamous cell carcinomas. Therefore, it may also indicate that a subset of oral cancers with greater metastatic potential, as evidenced by increased expression iNOS protein and mRNA, is identified.