Immunohistochemical study of NF-kB activity and IL-8 abundance in oesophageal adenocarcinoma; a useful strategy for monitoring these biomarkers

Abstract

Aims: To determine if immunohistochemistry (IHC) could be used to monitor NF-kB activity in oesophageal adenocarcinoma and pre-malignant (Barrett¡¦s) oesophageal tissues, relative to normal oesophageal mucosa. The pro-inflammatory cytokine IL-8, a transcriptional target of NF-kB, was also studied to better understand NF-kB functionality and 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-kB function, we also assessed changes in gene expression of NF-kB controlled genes (IL-8 and I-kB) in the histological sequence using real-time PCR. More global expression changes were also studied using membrane arrays. Results We found that IHC was effective at monitoring overall NF-kB activity and IL-8 abundance. This method also allowed NF-kB activity and IL-8 abundance to be pin-pointed in specific cell-types. ,We saw significant increases in nuclear NF-kB activity and IL-8 abundance across the histological series by IHC analysis. Gene expression analysis also showed consistent up-regulation of IL-8 confirming the IHC data and demonstrating enhanced transcriptional NF-kB activity. I-kB (another NF-kB target) showed down-regulation in dysplastic and adenocarcinoma tissues. Down-regulation of I-kB gene expression may partly explain increased NF-kB activity. Conclusion We suggest that IHC using antibodies against the NLS of p65 may be useful in monitoring overall NF-kB activity in oesophageal tissues. As IHC is amenable to highthroughput screening (whereas traditional EMSA methods are not), this may lead to the development of a better screening tool for early cancer risk.