Upregulation of cyclooxygenase-2 is accompanied by increased expression of nuclear factor-kappa B and I kappa B kinase-alpha in human colorectal cancer epithelial cells

Abstract

Cyclooxygenase-2 (COX-2) is selectively overexpressed in colorectal tumours. The mechanism of COX-2 induction is not fully understood, but requires de novo messenger RNA and protein synthesis, indicating regulation at the transcriptional level. Sequence analysis of the 5'-flanking region of the COX-2 gene shows two nuclear factor-kappa B (NF-kappa B) sites. Inhibition of this protein in model cell culture systems attenuates COX-2 expression and implies that NF-kappa B plays an important role in COX-2 induction. We measured COX-2, NF-kappa B and I kappa B kinase alpha (IKK alpha) protein expression in matched colonic biopsy samples comprising both nontumour and adjacent tumour tissue from 32 colorectal cancer patients using immunohistochemistry. There was none or very little expression of COX-2, NF-kappa B and IKK alpha in non-neoplastic colon epithelial cells, while the expression of all three of these proteins was significantly increased (P<0.05, Wilcoxon's signed rank test) in adjacent cancerous cells. Moreover, all three proteins were found to be coexpressed in the neoplastic epithelium, with the expression of COX-2 and NF-kappa B highly correlated (Pearson's correlation, P<0.005). There was no apparent correlation between enhanced COX-2, NF-kappa B or IKK alpha expression and tumour Dukes' stages. Our results are compatible with the hypothesis that IKK alpha and NF-kappa B are involved in COX-2 induction in these tumours and the lack of association between COX-2 expression and severity of disease as measured by Dukes' stage is consistent with the proposal that COX-2 expression is an early postinitiation event.

Figure 1

Immunohistochemical localisation of COX-2, NF-κB and IKKα in normal and malignant colonic epithelia from the same patient. The presence of the immunoreactive protein is indicated by brown staining. (A) Normal and (E) tumour tissue treated with preimmune sera as primary antibody (negative control); (B) normal and (F) tumour tissue treated with anti-COX-2 as primary antibody; (C) normal and (G) tumour tissue treated with anti-NF-κB primary antibody; (D) normal and (H) tumour tissue treated with anti-IKKα primary antibody. Insets are higher magnification of the same section.

Figure 2

Expression of COX-2, NF-κB and IKKα in matched normal and malignant colonic epithelia from 24 patients. ^*Significantly different (Wilcoxon's signed rank test, P<0.03) from normal tissue.

Figure 3

Expression of NF-κB and IKKα compared to COX-2 in malignant colonic epithelia. Expression of NF-κB is highly correlated with COX-2 expression (Pearson's correlation, two-tailed P-value <0.005, n=30 patients). Values are mean±s.e.m.