Nuclear factor-kappaB p65 (RelA) transcription factor is constitutively activated in human colorectal carcinoma tissue

Abstract

Aim: Activation of transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to play a role in cell proliferation, apoptosis, cytokine production, and oncogenesis. The purpose of this study was to determine whether NF-kappaB was constitutively activated in human colorectal tumor tissues and, if so, to determine the role of NF-kappaB in colorectal tumorigenesis, and furthermore, to determine the association of RelA expression with tumor cell apoptosis and the expression of Bcl-2 and Bcl-x(L).

Methods: Paraffin sections of normal epithelial, adenomatous and adenocarcinoma tissues were analysed immunohistochemically for expression of RelA, Bcl-2 and Bcl-x(L) proteins. Electrophoretic mobility shift assay (EMSA) was used to confirm the increased nuclear translocation of RelA in colorectal tumor tissues. The mRNA expressions of Bcl-2 and Bcl-x(L) were determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method.

Results: The activity of NF-kappaB was significantly higher in adenocarcinoma tissue in comparison with that in adenomatous and normal epithelial tissues. The apoptotic index (AI) significantly decreased in the transition from adenoma to adenocarcinoma. Meanwhile, the expressions of Bcl-2 and Bcl-x(L) protein and their mRNAs were significantly higher in adenocarcinoma tissues than that in adenomatous and normal epithelial tissues.

Conclusion: NF-kappaB may inhibit apoptosis via enhancing the expression of the apoptosis genes Bcl-2 and Bcl-x(L). And the increased expression of RelA/nuclear factor-kappaB plays an important role in the pathogenesis of colorectal carcinoma.

Figure 1

Immunohistochemical staining of RelA in tissue sections of colorectal adenoma (A) and adenocarcinoma (B, C). RelA protein is mainly expressed in the cytoplasm of tumor cells and nuclear accumulation of RelA is also detected. × 200.

Figure 2

Electrophoretic mobility shift assay demonstrating increased nuclear translocation and DNA binding of NF-kB. A: lane 1, positive control (using Hela nuclear extract); lane 2, normal; lanes 3-6, adenocarcinoma; lanes 7-10, adenoma. B: lane 1, positive control (using Hela nuclear extract); lanes 2-3, specific competitor (using excess of unlabeled oligonucleotide); lanes 4-5, adenoma; lanes 6-7, adenocarcinoma; lane 4 and 6, supershift (addition of p65 antibodies to the nuclear extracts).

Figure 3

Immunohistochemical staining of Bcl-2 in tissue sections of colorectal adenoma (A) and adenocarcinoma (B). Bcl-2 expression is restricted to the cytoplasm of cancer cells. × 200.

Figure 4

Immunohistochemical staining of Bcl-xL in tissue sections of colorectal adenoma (A) and adenocarcinoma (B). Bcl-x_L expression is restricted to the cytoplasm of cancer cells. × 200.

Figure 5

The mRNA expressions of Bcl-2 and Bcl-x_L were assessed using RT-PCR standardized by coamplifying the housekeeping gene b-actin. A: the mRNA expression of Bcl-2. lanes 1-3, adenocarcinoma; lanes 4-6, adenoma; lane 7, normal; lane 8, marker. B: the mRNA expression of Bcl-2. lane 1, marker; lane 2, normal; lanes 3-5, adenoma; lanes 6-8, adenocarcinoma.

Figure 6

TUNEL staining in tissue sections of colorectal adenoma (A) and adenocarcinoma (B). TUNEL staining is restricted to the nucleus of apoptotic cells. × 200.