Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer worldwide. In recent decades, the 5-year mortality rate is approximately 50% around the world. As reliable biomarkers of oral cancer are still lacking, it is necessary to identify new target molecules for early diagnosis, effective therapy, and monitoring of the disease. In the present work, we focused on the expression of the enzyme nicotinamide N-methyltransferase (NNMT). We analyzed enzyme activity in 37 paired tumor and non-tumor tissues and found that activity levels are significantly higher in tumor compared with adjacent normal oral mucosa. Interestingly, oral epithelium surrounding tumor of unfavorable cases (N+) seems to display higher activity levels compared with that of favorable ones (N0). Western blot analyses were performed to evaluate protein levels in saliva samples from patients with OSCC and healthy subjects. Preliminary results indicated an up-regulation of salivary NNMT in tumor. This study shows a marked increase in enzyme activity in oral cancer and suggests that adjacent normal tissue of unfavorable cases seems to change toward cancer. Moreover, it is conceivable to hypothesize that NNMT could represent a potential biomarker for early and non-invasive diagnosis of oral cancer.

The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to assess enzyme activity. Cells were transfected with four shRNA plasmids against NNMT and control cells were treated with transfection reagent only (mock). The efficiency of gene silencing was detected by Real-Time PCR and Western blot analysis. MTT cell proliferation assay and the soft agar colony formation assay were then applied to investigate the functional changes in cancerous cell. NNMT mRNA was detected in cancer cells, showing a very high expression level. In keeping with the results of RT-PCR analysis, the protein level and NNMT enzyme activity were particularly high in KB cells. ShRNA vectors targeted against NNMT efficiently suppressed gene expression, showing inhibition observed at both the mRNA and protein levels. Down-regulation of NNMT significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The present data support the hypothesis that the enzyme plays a role in tumour expansion and its inhibition could represent a possible molecular approach to the treatment of cancer.Tumour arises as a result of gene mutations and abnormal expression that endow the cell with many specific functional capabilities, such as cell proliferation, invasion and metastasis (l). The cellular and biochemical factors that underline cancer dissemination are poorly understood. Treatment of

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