Immunohistochemical study of nuclear factor-kappaB activity and interleukin-8 abundance in oesophageal adenocarcinoma; a useful strategy for monitoring these biomarkers.
OBJECTIVE
To determine if immunohistochemistry (IHC) could be used to monitor nuclear factor-kappaB (NF-kappaB) 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-kappaB, was also studied to better understand NF-kappaB 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-kappaB function, changes in gene expression of NF-kappaB controlled genes (IL-8 and I-kappaB) 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-kappaB activity and IL-8 abundance. This method also allowed NF-kappaB activity and IL-8 abundance to be pinpointed in specific cell types. There were significant increases in nuclear NF-kappaB 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-kappaB activity. I-kappaB (another NF-kappaB target) showed down-regulation in dysplastic and adenocarcinoma tissues. Down-regulation of I-kappaB gene expression may partly explain increased NF-kappaB activity.


CONCLUSIONS
IHC, using antibodies against the NLS of p65, may be useful in monitoring overall NF-kappaB 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.
Jenkins
G J S
GJ
Swansea School of Medicine, University of Wales Swansea, Swansea, UK. g.j.jenkins@swansea.ac.uk
Mikhail
J
J
Alhamdani
A
A
Brown
T H
TH
Caplin
S
S
Manson
J M
JM
Bowden
R
R
Toffazal
N
N
Griffiths
A P
AP
Parry
J M
JM
Baxter
J N
JN
D000230:Adenocarcinoma / Q000378:metabolism*; D001471:Barrett Esophagus / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D018450:Disease Progression; D004938:Esophageal Neoplasms / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011230:Precancerous Conditions / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D015854:Up-Regulation
D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D051996:Transcription Factor RelA