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

St-Germain ME, Gagnon V, Parent S, Asselin E - Mol. Cancer (2004)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biology, Research Group in Molecular and Cellular Biopathology, Medical Biology Section, University of Quebec at Trois-Rivieres, C,P, 500, Trois-Rivieres, Quebec, Canada G9A 5H7. Marie-Eve_St-Germain@UQTR.CA

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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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.
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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.

Mentions: As we showed previously, mutated PTEN endometrial cancer cell lines (RL 95-2 and Ishikawa) expressed high levels of Akt phosphorylation which was concomitant with the presence of high levels of COX-2 mRNA and protein [42]. In the latter study, there was no Akt phosphorylation found and nearly undetectable COX-2 protein in the wild-type cell line (HEC 1-A). PI 3-K inhibition in RL 95-2 and Ishikawa cells directly blocked Akt phosphorylation and caused a reduction of COX-2 mRNA and protein [42]. We wanted to further investigate the involvement NF-κB/IκB pathway in the regulation of COX-2 by Akt. As hypothesized, the results demonstrate that PI 3-K inhibition results in the reduction IκB phosphorylation in mutated PTEN RL 95-2 and Ishikawa cells (Fig. 2). There was no effect of PI 3-K inhibitors in IκB phosphorylation in HEC 1-A wild-type cells. To further confirm that inhibition of IκB phosphorylation leads to the activation and translocation of NF-κB to the nucleus, a NF-κB Chemiluminescent Assay was used to measure NF-κB activity in the nucleus (Fig. 3). The activity of NF-κB was high in mutated-PTEN human endometrial cancer cells compared to wild-type PTEN HEC 1-A cancer cell line. PI 3-K/Akt inhibition with Wortmannin significantly decreased NF-κB activity in both RL 95-2 and Ishikawa and inhibition had no effect in NEC 1-A cells.


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

St-Germain ME, Gagnon V, Parent S, Asselin E - Mol. Cancer (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC394342&req=5

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.
Mentions: As we showed previously, mutated PTEN endometrial cancer cell lines (RL 95-2 and Ishikawa) expressed high levels of Akt phosphorylation which was concomitant with the presence of high levels of COX-2 mRNA and protein [42]. In the latter study, there was no Akt phosphorylation found and nearly undetectable COX-2 protein in the wild-type cell line (HEC 1-A). PI 3-K inhibition in RL 95-2 and Ishikawa cells directly blocked Akt phosphorylation and caused a reduction of COX-2 mRNA and protein [42]. We wanted to further investigate the involvement NF-κB/IκB pathway in the regulation of COX-2 by Akt. As hypothesized, the results demonstrate that PI 3-K inhibition results in the reduction IκB phosphorylation in mutated PTEN RL 95-2 and Ishikawa cells (Fig. 2). There was no effect of PI 3-K inhibitors in IκB phosphorylation in HEC 1-A wild-type cells. To further confirm that inhibition of IκB phosphorylation leads to the activation and translocation of NF-κB to the nucleus, a NF-κB Chemiluminescent Assay was used to measure NF-κB activity in the nucleus (Fig. 3). The activity of NF-κB was high in mutated-PTEN human endometrial cancer cells compared to wild-type PTEN HEC 1-A cancer cell line. PI 3-K/Akt inhibition with Wortmannin significantly decreased NF-κB activity in both RL 95-2 and Ishikawa and inhibition had no effect in NEC 1-A cells.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biology, Research Group in Molecular and Cellular Biopathology, Medical Biology Section, University of Quebec at Trois-Rivieres, C,P, 500, Trois-Rivieres, Quebec, Canada G9A 5H7. Marie-Eve_St-Germain@UQTR.CA

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.

Show MeSH
Related in: MedlinePlus