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. 2004 Mar 11:3:7.
doi: 10.1186/1476-4598-3-7.

Regulation of COX-2 protein expression by Akt in endometrial cancer cells is mediated through NF-kappaB/IkappaB pathway

Marie-Eve St-Germain  1 , Veronique Gagnon, Sophie Parent, Eric Asselin
Affiliations

Regulation of COX-2 protein expression by Akt in endometrial cancer cells is mediated through NF-kappaB/IkappaB pathway

Marie-Eve St-Germain et al. Mol Cancer. .

Abstract

Background: Cyclooxygenase-2 (COX-2) has been shown to be highly expressed in a broad series of primary endometrial tumors and its expression may be closely associated with parameters of tumor aggressiveness. In human endometrial cancer, tumor suppressor phosphatase tensin homologue (PTEN) is frequently mutated. In the presence of a mutated PTEN protein, Akt phosphorylation levels increase leading to the activation of this survival pathway. The nuclear transcription factor kappaB (NF-kappaB) is a well establish regulator of genes encoding cytokines, cytokine receptors, and cell adhesion molecules that drive immune and inflammatory responses. More recently, NF-kappaB activation has been connected with multiple aspects of oncogenesis, including the control of apoptosis, cell cycle, differentiation, and cell migration. It is known that Akt may act through NF-kappaB pathway and that COX-2 gene has been shown to be regulated at the promoter level by NF-kappaB. Recently, we showed that Akt regulates COX-2 gene and protein expressions in phospho-Akt expressing endometrial cancer cells. The present study was undertaken to determine the involvement of NF-kappaB pathway and IkappaB (an inhibitor of NF-kappaB) in the regulation of COX-2 expression and to determine more precisely the downstream targets of Akt involved in this process.

Results: Three different human endometrial cancer cell lines known to have wild type PTEN (HEC 1-A) or a mutated inactive PTEN protein (RL 95-2 and Ishikawa) were used for these studies. Expression IkappaB and Phospho-IkappaB were evaluated by Western analysis. The presence of IkappaB phosphorylation was found in all cell lines studied. There was no difference between cell lines in term of NF-kappaB abundance. Inhibition of PI 3-K with Wortmannin and LY294002 blocked IkappaB phosphorylation, reduced NF-kappaB nuclear activity, reduced COX-2 expression and induced apoptosis. Transfection studies with a dominant negative Akt vector blocked IkappaB phosphorylation and reduced COX-2 expression. On the opposite, constitutively active Akt transfections resulted in the induction of IkappaB phosphorylation and up-regulation of COX-2.

Conclusion: These results demonstrate that Akt signals through NF-kappaB/IkappaB pathway to induce COX-2 expression in mutated PTEN endometrial cancer cells.

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Figures

Figure 1
Figure 1
Effect of PI 3-K inhibitors on apoptosis in HEC 1-A, RL 95-2 and Ishikawa cells. Control (■), LY294002 (○) and Wortmannin (▲). 2 × 106 cells were plated for 0, 6, 12, 24 h and cultured in the presence of medium and LY294402 or Wortmannin. Cells were trypsinized, pooled with floating cells and collected for Hoechst nuclear staining (right panel) or TUNEL analysis (left panel) to count apoptotic cells. Data represent the mean ± SEM of 4 independent experiments.
Figure 2
Figure 2
Effect of PI 3-K inhibitors on IκB expression and phosphorylation in HEC-1-A, RL-95-2 and Ishikawa cells. Western analysis was performed on cell protein lysates from pooled attached and floating cells. β-actin was used as control to correct for loading. Densitometric analyses were performed using BIO RAD gel doc system and are presented as a ratio (value/β-actin). 2 × 106 cells were plated for 24 h and cultured in medium in the presence or absence of LY294402 or Wortmannin. Data represent the mean ± SEM of 4 independent experiments. * p < 0.05 compared to control.
Figure 3
Figure 3
NF-κB activity in response to Wortmannin. HEC 1-A, RL 95-2 and Ishikawa cells were treated with Wortmannin for 24 hours and cells were recovered and lysed. Nuclear cell lysates were recovered and NF-κB activity was measured using the Chemiluminescent NF-κB Assay. Data represent the mean ± SEM of 4 independent experiments. * p < 0.05 compared to control.
Figure 4
Figure 4
Constitutively active Akt action on IκB activity and COX-2 protein expression. RL 95-2 cells were transfected with constitutively active (CA) Akt expression vector or control vector and (A) Akt protein, (B) phospho-Akt, (C) COX-2, (D) Phospho-IκB et (E) IκB protein levels were measured by Western analysis. β-actin was used as control to correct for loading. Densitometric analyses were performed using BIO-RAD gel doc system and are presented as a ratio (value/β-actin). Data represent the mean ± SEM of 4 independent experiments. * p < 0.05 compared to control.
Figure 5
Figure 5
Dominant negative Akt action on IκB activity. RL 95-2 expressing phospho-Akt cells were transfected with Akt dominant negative (DN) vector or control vector and (A) phospho-IκB and (B) IκB protein levels were measured by Western analysis. β-actin was used as control to correct for loading. Densitometric analyses were performed using BIO-RAD gel doc system and are presented as a ratio (value/β-actin). (C) Cells were trypsinized, pooled with floating cells and collected for Hoechst nuclear staining to count apoptotic cells. Data represent the mean ± SEM of 4 independent experiments. * p < 0.05 compared to control
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