The combination of antitumor drugs, exemestane and erlotinib, induced resistance mechanism in H358 and A549 non-small cell lung cancer (NSCLC) cell lines

Figures & data

Figure 1. Dose-response of (a) exemestane and (b) erlotinib of H23, H358 and A549 cells. Different doses of exemestane or erlotinib were applied on cell lines; and 48 h later, cell number was estimated by colorimetric MTT assay. Combined targeting of aromatase and EGFR with exemestane and erlotinib on (c) H23, (d) H358 and (e) A549, respectively, 48 h after drug application. The results were expressed as cell number ± SEM. Asterisks denote a statistically significant difference compared to untreated cells. *p < 0.05, **p < 0.01 and ***p < 0.001. C: control, Ex: exemestane, Erl: erlotinib and Ex + Erl: exemestane and erlotinib.

Figure 2. The effect of exemestane, erlotinib and their combination on Bcl-2 anti-apoptotic protein levels. (a) Representative blots of three independent experiments for H23, H358 and A549 cells. Quantification of western blot images (b) in H23, (c) in H358 and (d) in A549. Results are expressed as mean ± SEM of the % change compared to the untreated cells. Asterisks denote a statistically significant difference compared to untreated cells. *p < 0.05 and ***p < 0.0001. C: control, Ex: exemestane, Erl: erlotinib and E + E: exemestane and erlotinib.

Table 1. The effect of exemestane and erlotinib on apoptosis of H23, H358 and A549 cells.

	H23		H358		A549
Apoptosis 48 h (%)	%	p	%	p	%	p
Ex.	58.0 ± 26.0	0.025^a	186.3 ± 13.7	0.00017^b	34.7 ± 13.4	0.018^a
Erl.	16.7 ± 5.0	0.023^a	65.3 ± 14.4	0.0105^a	66.3 ± 13.6	0.00816^c
Ex + Erl	44.0 ± 8.5	0.0064^c	406.3 ± 16.5	0.000017^b	37.7 ± 26.8	0.031^a

Results are expressed as the % percentage of annexin⁺ cells ± SEM compared to untreated cells based on at least three independent experiments. Asterisks denote a statistically significant difference compared to untreated cells (C).

^ap < 0.05.

^bp < 0.001.

^cp < 0.01.

Ex: exemestane, Erl: erlotinib, Ex + Erl: exemestane + erlotinib.

Figure 3. The effect of exemestane and erlotinib on beclin-1 protein levels. (a) Representative blots of three independent experiments for H23, H358 and A549 cells. Quantification of western blot images (b) in H23, (c) in H358 and (d) in A549. Results are expressed as mean ± SEM of the % change compared to the untreated cells. Asterisks denote a statistically significant difference compared to untreated cells. *p < 0.05. C: control, Ex: exemestane, Erl: erlotinib, E + E: exemestane and erlotinib.

Figure 4. The effect of exemestane and erlotinib on H23 (a) and H358 (b) migration ability. Results are expressed as mean ± SEM of the % change compared to the untreated cells. Asterisks denote a statistically significant difference compared to untreated cells. *p < 0.05 and **p < 0.01. C: control, Ex: exemestane, Erl: erlotinib, E + E: exemestane and erlotinib.

Figure 5. Effect of exemestane and erlotinib on MMP-9 and MMP-2 secretion by H23, H358 and A549 cells. This picture is a representative gel of three independent experiments.

Figure 6. The effect of exemestane and erlotinib on EGFR localization in H23 cells at the time point of 24 h after drug application. Untreated cells (a) and cells treated with exemestane (b), erlotinib (c) and their combination (d) were stained with anti-EGFR (I), mitochondria dye (II) and nucleus dye (III) and visualized in confocal microscope. The colocalization for three fluorescent dyes is demonstrated in lane (IV). The figure is a representative of at least three independent experiments using a magnification of 63×.