NF-κB dependent cytokine production in HNSCC cell lines increases with inflammatory state.

A56

Carcinogens found in tobacco products induce nuclear factor-kappaB (NF-κB) activation. This leads to increased production of proinflammatory and proangiogenic cytokines (IL-6, IL-8 and VEGF) that have been implicated in the pathogenesis of head and neck cancers. Relative to controls, research has demonstrated elevated IL-6, IL-8 and VEGF levels in tissue culture of head and neck squamous cell carcinoma (HNSCC) cell lines and in saliva (and serum) of patients with preneoplastic leukoplakia. A correlation between VEGF levels in serum and saliva has also been demonstrated. There is evidence that in the early stages of disease, tumor necrosis factor-alpha (TNF-α) can act as an endogenous tumor promoter in tumor microenvironment. However, the correlation of cytokine production to inflammatory state of cancer cells has not been definitively studied. The hypothesis of the present study is that cytokine levels in HNSCC cell lines also correlate with inflammatory state. This was studied in vitro using HNSCC cell lines UMSCC-9, -38 and human oral keratinocyte (Hok 16B) cell lines. We predicted Hok 16B cells will have the lowest cytokine production and would be least sensitive to changes in inflammation. To stimulate increasing inflammatory state, 24hour treatments of 0ng/ml, 0.2ng/ml, 2ng/ml and 20ng/ml TNF-α in serum free media were added to 60-80% confluent 25cm2 cell culture flasks. The cell number in each individual flask was obtained to determine quantity of cytokine secreted per 1 million cells. IL-6, IL-8 and VEGF concentrations in clarified supernatants were determined by quantitative sandwich enzyme linked immunosorbent assay (ELISA). All experiments were independently repeated 2-3 times. In all three cell lines, IL-6, IL-8 and VEGF levels were 1-5 fold higher with TNF-α treatment. UMSCC-9 exhibited the greatest induction of cytokine release in response to TNF-α. Hok 16B cells expressed the lowest IL-8 levels at each TNF-α concentration. For IL-6, Hok 16B cells released higher levels than UMSCC-9 at 0ng/ml and 0.2ng/ml TNF-α but levels lower than UMSCC-38 at all TNF-α concentrations. In the VEGF ELISAs, Hok 16B cells demonstrated higher or similar VEGF production in comparison to UMSCC-9 and UMSCC-38. However, Hok 16B did not show a higher response to TNF-α stimulation in the VEGF ELISAs. This could be attributable to the relative importance of VEGF as a HIF (hypoxia inducible factor) dependent gene rather than an NF-κB dependent gene. In this study, we show that production of IL-6, IL-8 and VEGF is increased by TNF-α stimulation in UMSCC-9, UMSCC-38 and HOK-16B cells. UMSCC-9 exhibited the greatest response to TNF-α treatment while Hok 16B produced less IL-6 and IL-8. These results suggest that tissue cancer cells may have a higher response to inflammation in tumor microenvironment. More importantly, these cancer cells tend to produce more proinflammatory cytokines with increased inflammation.