Chronic inflammation, with its associated increased generation of reactive nitrogen species (RNS) and reactive oxygen species, is implicated in the pathogenesis of many cancers, including head/neck squamous cell carcinoma (HNSCC). Furthermore, previous studies have demonstrated the contribution of oxidative stress in aberrant activation of the pleiotrophic transcriptional activating factor NFκB in the progression of numerous diseases including cancer. Nitrosative stress is also implicated in the development and progression of HNSCC. To date, however, no studies have investigated the effects of RNS on NFκB activation and subsequent gene expression. This study employed transient transfection of an NFκB reporter gene to assess NFκB activation, while effects on gene expression were monitored by cellular production of two HNSCC relevant cytokines VEGF and IL 8. Studies were conducted on characterized HNSCC cell lines obtained from ATCC, and included a 24 h treatment with these reactive species (RS) generators: TNF (100U/ml-inititates endogenous RS production), H2O2 (freely permeable, 0.2mM), NOC 18 (long lived RN production, 25 μM) and SIN 1 (simultaneous production of O2.- and NO, 100 μM). NFκB activation was assessed by fluorimetric detection of release of the reporter gene protein (data normalized to amount of PCR amplified plasmid DNA) and VEGF and IL 8 production were determined by ELISAs (pg/mg protein). Data were analyzed with a Yates corrected Chi Square. Results from ELISA assays demonstrate cell line and RS generator dependent variability. TNF significantly increased IL 8 levels in SCC 25 cells (p<0.05) and demonstrated a trend toward increased levels in both SCC 9 and 2095 cells. While TNF did not increase VEGF in any of the tested cell lines, these data were not unexpected as NFκB only partially regulates VEGF expression. In contrast, the primary transcriptional regulator of IL 8 is NFκB. Although H2O2 strongly induced VEGF and IL 8 in SCC 9 cells (p<0.001, 0.05), H2O2 had no effect on VEGF protein levels in SCC 2095 or 25 and either reduced IL 8 release (SCC 25, p<0.05) or caused no change (SCC 2095). Most cell lines were refractory to NOC 18 with the exception of reduction of VEGF levels in SCC 2095 (p<0.05). Following SIN 1 challenge, IL 8 and VEGF production increased in SCC 9 cells (p<0.005). SCC 25 SIN 1 results reveled reduced IL 8 (p<0.005) with no change in VEGF, while SIN 1 treatment of SCC 2095 cells reduced levels of both IL 8 and VEGF. Transfection results demonstrate that all RS generators induce NFκB activation in the single cell line yet evaluated (SCC 2095, n=3, p<0.05). Our data show, for the first time, that RNS can directly activate NFκB and elicit downstream effects. While these results are promising, they need to be confirmed in additional cell lines. Ongoing studies entail additional transfection and ELISA analyses, as well as investigations to assess the effects of inclusion of RNS quenching and antioxidant compounds on the HNSCC tumorigenic phenotype.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]