Immunohistochemical study of nuclear factor-κB activity and interleukin-8 abundance in oesophageal adenocarcinoma;: a useful strategy for monitoring these biomarkers

Aims: To determine if immunohistochemistry ( IHC) could be used to monitor nuclear factor- kappa B ( NF- kB) activity in oesophageal adenocarcinoma and pre- malignant ( Barrett's) oesophageal tissues, relative to normal oesophageal mucosa. The pro- inflammatory cytokine interleukin- 8 ( IL- 8), a transcriptional target of NF- kappa B, was also studied to better understand NF- kappa B functionality; its RNA and protein levels were assessed in oesophageal tissues. Methods: IHC was employed using an antibody against the nuclear localisation sequence ( NLS) of the p65 subunit as well as an antibody against IL- 8. To assess NF- kappa B function, changes in gene expression of NF- kappa B controlled genes ( IL- 8 and I- kappa B) were also assessed in the histological sequence using real- time PCR. More global expression changes were also studied using membrane arrays. Results: IHC was effective at monitoring overall NF- kappa B activity and IL- 8 abundance. This method also allowed NF- kappa B activity and IL- 8 abundance to be pinpointed in specific cell types. There were significant increases in nuclear NF- kappa B activity and IL- 8 abundance across the histological series. Gene expression analysis also showed consistent up- regulation of IL- 8, confirming the IHC data and showing enhanced transcriptional NF kappa B activity. I- kappa B ( another NF- kappa B target) showed down- regulation in dysplastic and adenocarcinoma tissues. Down- regulation of I- kappa B gene expression may partly explain increased NF- kappa B activity. Conclusion: IHC, using antibodies against the NLS of p65, may be useful in monitoring overall NF- kappa B activity in oesophageal tissues. As IHC is amenable to high- throughput screening ( whereas traditional electrophoretic mobility shift assay methods are not), this may lead to the development of a better screening tool for early cancer risk.