Antiproliferative effect of exemestane in lung cancer cells

Abstract

Background: Recent evidence suggests that estrogen signaling may be involved in the pathogenesis of non-small cell lung cancer (NSCLC). Aromatase is an enzyme complex that catalyses the final step in estrogen synthesis and is present in several tissues, including the lung. In the current study we investigated the activity of the aromatase inhibitor exemestane in human NSCLC cell lines H23 and A549.

Results: Aromatase expression was detected in both cell lines. H23 cells showed lower protein and mRNA levels of aromatase, compared to A549 cells. Exemestane decreased cell proliferation and increased apoptosis in both cell lines, 48 h after its application, with A549 exhibiting higher sensitivity than H23 cells. Aromatase protein and mRNA levels were not affected by exemestane in A549 cells, whereas an increase in both protein and mRNA levels was observed in H23 cells, 48 h after exemestane application. Moreover, an increase in cAMP levels was found in both cell lines, 15 min after the administration of exemestane. In addition, we studied the effect of exemestane on epidermal growth factor receptor (EGFR) localization and activation. Exemestane increased EGFR activation 15 min after its application in H23 cells. Furthermore, we demonstrated a translocation of EGFR from cell membrane, 24 h after the addition of exemestane in H23 cells. No changes in EGFR activation or localization were observed in A549 cells.

Conclusion: Our findings suggest an antiproliferative effect of exemestane on NSCLC cell lines. Exemestane may be more effective in cells with higher aromatase levels. Further studies are needed to assess the activity of exemestane in NSCLC.

Figure 1

The effect of exemestane on protein levels of aromatase in H23 and A549 cells. A: H23 and A549 cells were treated with 50 μM and 20 μM exemestane respectively and 48 h later, cells were lysed and analyzed in SDS-PAGE. Actin was used as an internal control. The figure is a representative from at least three independent experiments. B: Quantification of western blot images. The results are expressed as % change of control. Asterisks denote a statistically significant difference (unpaired t-test) compared to control. * P < 0.05.

Figure 2

The effect of exemestane on mRNA levels of aromatase in H23 and A549 cells. H23 and A549 cells were treated with 50 μM and 20 μM exemestane respectively and 48 h later, aromatase mRNA levels were studied with real time RT-PCR. Results are expressed as relative expression and normalized to untreated cells. Asterisks denote a statistically significant difference (unpaired t-test) compared to untreated cells. * P < 0.05.

Figure 3

Dose response of a) exemestane and b) testosterone in H23 and A549 cells. Different doses of exemestane or testosterone were applied on H23 and A549 cells and 48 h later, the number of cells was estimated with the colorimetric MTT assay. Results are expressed as mean ± SEM of the number of cells from at least three independent experiments performed in triplicates. Asterisks denote a statistically significant difference (unpaired t-test) compared to untreated cells. **P < 0.01 and ***P < 0.001.

Figure 4

The effect of exemestane on apoptosis of H23 and A549 cells. A: Flow cytometry analysis of H23 and A549 following exposure to exemestane. A representative image from three independent experiments is shown. H23 cells. C: Control and E 50 μM: Exemestane 50 μM. A549 cells. C: Control and E 20 μM: Exemestane 20 μM. B: Results are expressed as the % percentage of Annexin⁺ cells ± SEM compared to untreated cells from at least three independent experiments. Asterisks denote a statistically significant difference (unpaired t-test) compared to untreated cells (C). ** P < 0.01 and *** P < 0.001.

Figure 5

The effect of a) exemestane and b) testosterone on aromatase activity of H23 and A549 cells. H23 and A549 cells were treated with 50 μM and 20 μM exemestane, respectively and both cell lines were treated with 1 μM testosterone. At the time points of 15 min, 6, 8, 12, 24 and 48 h, samples were analysed as described in Methods. *P < 0.05, ** P < 0.01 and *** P < 0.001.

Figure 6

The effect of exemestane on cAMP levels in H23 and A549 cells. H23 and A549 cells were treated with 50 μM and 20 μM exemestane, respectively. At the time points of 15 and 30 min, samples were analysed as described in Methods. ** P < 0.01.

Figure 7

The effect of exemestane on EGFR localization in H23 and A549 cells. Both cell lines were treated with 50 μM and 20 μM exemestane, respectively. At the time point of 24 h, samples were analyzed as described in Methods. The figure is a representative from at least three independent experiments (magnification at 60×).

Figure 8

The effect of exemestane on EGFR phosphorylation a) in H23 cells using an ELISA kit assay and b) in H23 and A549 cells using immunoblotting. H23 and A549 cells were treated with 50 μM and 20 μM exemestane, respectively and 15 min later they were analyzed as described in Methods. C: untreated cells (H23 or A549 cells) and Exm: H23 or A549 cells treated with 50 μM or 20 μM exemestane, respectively. *P < 0.05.