Figure 1.
Effects of exemestane on MCF-7aro cells morphology.
Phase contrast microscopy (A, D and G), Giemsa staining (B, E and H) and Hoechst staining (C, F and I). MCF-7aro cells were examined in the absence (A, B and C) or in the presence of 10 µM (D, E and F) or 15 µM (G, H and I) of exemestane during 6 days. Treated cells presented, cytoplasm vacuolization (black arrows) in phase contrast microscopy and Giemsa staining, chromatin condensation (yellow filled arrows) and chromatin fragmentation (yellow open arrows) in Hoechst staining.
Figure 2.
Effects of exemestane on MCF-7aro cells viability, membrane integrity, rate of DNA synthesis and on cell cycle distribution.
To study cell viability (A, B) and cell proliferation (C), cells were treated with different concentrations of exemestane for different times. Cells cultured with T represent the maximum of cell viability and cell proliferation and were considered as control. To study cell cycle distribution cells were treated with exemestane during 3 (D) and 6 (E) days and subjected to flow cytometric analysis after PI staining. Data presented in histograms were analysed with FlowJo Software (Tree Star, Inc) by the application of the Watson mathematical model and are representative of one independent assay. Results are the mean ± SEM of three independent experiments, performed in triplicate. Significant differences between the control and cells with exemestane are denoted by * (p<0.05) and *** (p<0.001).
Figure 3.
Annexin V-PE labeling and mitochondrial transmembrane potential following exemestane treatment.
(A) Annexin V-PE labelling of MCF-7aro cells treated with exemestane (15 µM) for 6 and 9 days of treatment. Cells cultured with T were considered as control and for 6 days present 3.54% and for 9 days 5.58% of Annexin V binding. Data presented in histograms were analysed with FlowJo Software (Tree Star, Inc), correspond to cells gated for negative 7-AAD staining and are representative of one independent assay. The grey filled line corresponds to control and the black line to exemestane at 15 µM. (B) Mitochondrial transmembrane potential (ΔΨm) of MCF-7aro cells treated with exemestane (15 µM) with or without 3-MA (1 mM), for 3 and 6 days. Viable cells and cells with ΔΨm loss were identified. Data presented in histograms were analysed with FlowJo Software (Tree Star, Inc) and are representative of one independent assay.
Table 1.
Effects of exemestane on Annexin V-PE labelling in MCF-7aro cells.
Table 2.
Effects of exemestane on mitochondrial transmembrane potential (ΔΨm) in MCF-7aro cells.
Figure 4.
Activation of caspases and ROS production.
Caspase-9 (A), caspase-8 (B) and caspase-7 (C) activities of MCF-7aro cells treated with exemestane after 3 days. The effects of 3-MA on caspase activities were also studied. Cells cultured with T were considered as control, cells treated with STS were considered as positive control and with Z-VAD-FMK was negative control. The results are presented as relative luminescence units (RLU). (D) ROS in MCF-7aro cells treated with exemestane after 3 days. It was also studied the effects of 3-MA. Cells cultured with T were considered as control and cells treated with PMA were as positive control for ROS production. The results are presented as mean fluorescence intensity (MFI). Results are the mean ± SEM of three independent experiments performed in triplicate. Significant differences between the control T versus treated cells, or between the control plus 3-MA versus cells with exemestane plus 3-MA are indicated by * (p<0.05), ** (p<0.01) and *** (p<0.001); between the cells treated with exemestane versus cells with exemestane plus 3-MA are indicated by # (p<0.05); between the cells treated with exemestane versus cells with exemestane plus Z-VAD-FMK are indicated by §§§ (p<0.001).
Figure 5.
(A) Effects of exemestane in the formation of AVOs analysed by fluorescence microscopy. MCF-7aro cells were treated with exemestane with or without 3-MA, during 6 days and stained with AO. The presence of AVOs was indicated by the yellow/orange/red fluorescence. (B) Ultrastructural features of cell death in MCF-7aro cells treated with exemestane for 6 days. The presence of autophagosomes (black arrows) was observed in the cytoplasm of treated cells. (C) Western Blot analysis of LC3-I/LC3-II. Cells were cultured with exemestane and with exemestane plus 3-MA for 3 days. β-tubulin was used as a loading control. Cells cultured with T were considered as control. Results are shown from a single representative of three independent experiments.
Table 3.
Quantification of acid vesicular organelles (AVOs) in MCF-7aro cells treated with exemestane.