Fig 1.
AEE788 inhibits cell proliferation, induces apoptosis and alters cell cycle in colorectal cancer cells.
A) Cell proliferation was evaluated after 72h of treatment with different doses of AEE788. B) Inhibition of cell proliferation by AEE788 was tested in cells growing in the presence of EGF (100 ng/ml). C) The fraction of apoptotic cells was estimated after 48 h of treatment with different doses of AEE788 of cells growing in the presence of EGF (100 ng/mL). D) Analysis of cell cycle was performed by flow cytometry after 48 h of treatment with different doses of AEE788 of cells growing in the presence of EGF (100 ng/mL). Data are means ± SEM of three independent experiments (*p <0.05, compared with the control).
Fig 2.
AEE788 inhibits EGFR signaling in colorectal cancer cells.
The phosphorylated and non-phosphorylated forms of EGFR, ERK 1/2 and Akt were detected by Western-blot using specific antibodies. Cells were grown in the absence or presence of EGF (100 ng/mL) and treated with AEE788 (2.5 µM) for 5, 10 or 15 min. The expression level of -actin was included as loading control.
Fig 3.
Inhibition of COX-2 enhances the antitumor efficacy of AEE788 in colorectal cancer cells.
A) EGF-driven cell proliferation was assayed in cells growing in the presence of EGF (100 ng/ml) and in the presence or absence of AEE788 (2.5 μM), celecoxib (10 μM) and NS-398 (10 μM). B) Analysis of cell cycle was performed by flow cytometry after 48 h of treatment with AEE788 (2.5 μM) and/or celecoxib (10 μM) of cells growing in the presence of EGF (100 ng/mL). C) Expression levels of cyclooxygenase-2 (COX-2) were analyzed by western-blot in whole cell extracts form Caco-2 and HCT-116 cells. Expression of β-actin is included as loading control. D) The phosphorylated and non-phosphorylated forms of EGFR, ERK 1/2 and Akt were detected by Western-blot using specific antibodies. Cells were grown in the presence of EGF (100 ng/mL) and treated with AEE788 (2.5 µM) and/or celecoxib (10 μM) for 6h. The expression level of -actin was included as loading control. The corresponding densitometric analysis is also shown. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).
Fig 4.
Celecoxib intensifies the anti-angiogenic activity of AEE788.
A) VEGF-A mRNA expression levels were assayed by real time RT-PCR in colorectal cancer cells growing in the presence of EGF (100 ng/ml) and exposed to the indicated treatments for 48 h. B) VEGFA165 levels were quantified by ELISA in the conditioned media collected from cells growing in the presence of EGF (100 ng/ml) and treated with AEE788 (2.5 µM) and/or celecoxib (10 μM) for 48h. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control). C) The angiogenic activity of media conditioned by Caco-2 cells exposed for 24 h to the indicated treatments was evaluated using the endothelial tube assay as described under Material and Methods. Data are the total length of formed tubes in pixels (px), showing means ± SEM of three independent experiments (*p <0.05, compared with the control). D) Representative images of the formed interconnected networks after the treatment of endothelial cells with the indicated Caco-2 cells conditioned media. The extent of tube formation was quantified as indicated in the Material and Methods section. (Final magnification: X40, scale bar corresponds to 100 microns).
Fig 5.
Combined AE788/Celecoxib treatment reduces the migratory capacity of colorectal cancer cells.
A) Scratch wound healing assay was used to analyze the inhibition of cell migration in colorectal cancer cells treated for 24 h with AEE788 (2.5 µM) and/or celecoxib (10 µM). Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells). Final magnification: X100, scale bar corresponds to 100 microns. B) Representative images of scratched areas in confluent Caco-2 and HCT-116 cell layers. The yellow lines indicate the invasive front in the wound healing assay. Wound closure was photographed at 0h and 24 h after wounding. The scratched area at control (0 h) was arbitrarily assigned as 100%.
Fig 6.
Combined AEE788/Celecoxib treatment alters subcellular distribution of β-catenin in Caco-2 cells.
To determine subcellular localization of β-catenin, both Caco-2 (A) and HCT-116 (B) cells were treated with AEE788 (2.5 µM) and/or celecoxib (10 μM) for 6 h, stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue). Merged images of β-catenin and DAPI staining are also shown. Final magnification: X400. Nuclear β-catenin levels were quantified as the integrated density of β-catenin signal in confocal microscopy images using the Image-J software. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).
Fig 7.
Combined AEE788/Celecoxib treatment downregulates FOXM1 protein levels in colorectal cancer cells.
FOXM1 expression was analized by western-blot in cells were grown in the presence of EGF (100 ng/mL) and treated with AEE788 (2.5 µM) and/or celecoxib (10 μM) for 6h. The expression -actin is included as loading control. The corresponding densitometric analysis is also shown. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).
Fig 8.
Combined AEE788/Celecoxib impairs FOXM1- β-catenin interaction.
To determine subcellular localization of β-catenin and FOXM1, both Caco-2 (A) and HCT-116 (B) cells were exposed to AEE788 (2.5 βM) and/or celecoxib (10 μM) for 6 h, stained for β-catenin (green) and FoxM (red) immunofluorescence, and counterstained with DAPI (blue). Merged images of β-catenin, FOXM1 and DAPI staining are also shown. Final magnification: X400. C) Pearson´s coefficient analysis was performed for the co-localization in cell nuclei of β-catenin and FOXM1. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control). D) Cell extracts of Caco-2 cells after 6 h of the indicated treatments were subjected to IP using β-catenin antibody or control IgG, followed by IB with FOXM1 antibody.
Fig 9.
Combined AEE788/ Celecoxib treatment in colon cancer cells impairs colonosphere formation capability.
Colon cancer cells were pre-treated with 2.5 µM AEE788 as a single agent or in combination with 10 µM celecoxib in the presence of 100 ng/mL EGF for 48h, and then cells were seeded at clonal density with serum free medium in low-adherence plates. After seven days, the number (A), size (B) and appearance (C) of formed colonospheres were evaluated by light microscopy. Spheres size was quantified in micrograph with the imaging software (Image J software). (Final magnification: X100, scale bar corresponds to 100 microns. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).
Fig 10.
Combined AEE788/celecoxib treatment downregulates stemness-related pathways in colorectal cancer cells.
The expression of stem cell markers Oct 3/4, Nanog and Sox-2 was analyzed by western blot in total cell extracts of colon cancer cells after 6h of indicated treatments. The expression of -actin is included as loading control. The corresponding densitometric analysis is also shown. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).