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

¹ Swansea School of Medicine, University of Wales Swansea, Swansea, UK
² Department of Surgery, Morriston Hospital, Swansea, UK
³ Department of Surgery, Singleton Hospital, Swansea, UK
⁴ Department of Pathology, Swansea NHS Trust, Swansea, UK

Correspondence to:
Dr G J S Jenkins, Swansea School of Medicine, University of Wales Swansea, Swansea SA28PP, UK; g.j.jenkins{at}swansea.ac.uk

Aims: To determine if immunohistochemistry (IHC) could be used to monitor nuclear factor-B (NF-B) 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-B, was also studied to better understand NF-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-B function, changes in gene expression of NF-B controlled genes (IL-8 and I-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-B activity and IL-8 abundance. This method also allowed NF-B activity and IL-8 abundance to be pinpointed in specific cell types. There were significant increases in nuclear NF-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-B activity. I-B (another NF-B target) showed down-regulation in dysplastic and adenocarcinoma tissues. Down-regulation of I-B gene expression may partly explain increased NF-B activity.

Conclusion: IHC, using antibodies against the NLS of p65, may be useful in monitoring overall NF-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.

Abbreviations: IHC, immunohistochemistry; IL, interleukin; NF-B, nuclear factor-B; NLS, nuclear localisation sequence

Keywords: immunohistochemistry; NF-kB; IL-8; Barrett’s oesophagus; adenocarcinoma; biomarker