Fig. 6.

Epoxyazadiradione attenuates PI3K/Akt pathway-dependent AP-1 DNA binding, VEGF expression and breast cancer cell migration and induces apoptosis: a MDA-MB-231 cells were treated with epoxyazadiradione. The level of phosphorylation of p85 (Tyr458) and Akt (Ser473) and c-Jun and c-Fos were analyzed by immunoblot. Actin was independently reprobed with respective original blots and served as loading control. b Ratio of densitometric analysis of p-Akt and Akt. Values are represented in mean ± SEM of three independent experiments; **, p < 0.004; ***, p < 0.0004 with untreated control cells. c The expressions of c-Fos and c-Jun were detected by confocal microscopy at indicated conditions. d Nuclear fractions isolated from MDA-MB-231 cells treated with epoxyazadiradione for 24 h and used for EMSA. Nuclear extracts were incubated with γ-³²P-labeled oligonucleotides containing the AP-1 consensus binding site. Arrow indicates the AP-1-DNA bound complex. e The expression of p-Akt, c-Jun and VEGF were analyzed by immunoblot in MDA-MB-231 cells treated with either epoxyazadiradione (50 μM) or perifosine (15 μM) for 24 h (left panel). MDA-MB-231 cells were transiently transfected with pcDNA6-HA-Akt1 and then treated with epoxyazadiradione (50 μM) for 24 h. The expression of c-Jun and VEGF was analyzed by western blot (right panel). f Flow cytometry analysis of JC-1 staining in MDA-MB-231 cells either treated with epoxyazadiradione (150 μM) or perifosine (30 μM) or overexpressed with Akt1 and then treated with epoxyazadiradione (150 μM) for 24 h. g MDA-MB-231 cells were either treated with epoxyazadiradione (150 μM) or perifosine (30 μM) or overexpressed with Akt1 and then treated with epoxyazadiradione (150 μM) for 24 h and cell viability was determined using MTT assay. Data were analyzed statistically and represented graphically. h Wound migration assay at indicated conditions. i Transwell migration assay as indicated conditions and cells. Values are represented in mean ± SEM of three independent experiments in each case or representative of a typical experiment. * P < 0.0005