Taken together, epoxyazadiradione suppresses cell migration, angiogenesis and breast tumor growth through downregulation of PI3K/Akt-mediated mitochondrial depolarization and induction of caspase-dependent apoptosis and blocking of AP-1 activation and expression of pro-angiogenesis and metastasis genes (Fig. of breast cancer cells in response to epoxyazadiradione. We have also analyzed the effect of SIS3 epoxyazadiradione on breast tumor growth using in vivo mice model. Results In this study, we for the first time investigated that out of 10 major limonoids isolated from as described earlier [12, 19]. Drugs were solubilized in DMSO and DMSO was used as vehicle control. Cell cultures and transfection Human breast cancer cells, MDA-MB-231 and MCF-7 and normal human breast epithelial cells, MCF-10A were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured as per standard conditions. pcDNA6-HA-Akt1 was transiently transfected in MDA-MB-231 cells using Dharmafect-1 (Dharmacon International) as per manufacturers instructions. MTT assay To determine the cytotoxic effect of neem-derived limonoids, MTT assay was performed as described [24]. Briefly, MDA-MB-231 and MCF-7 (1??104 cells/well) cells were plated in 96-well flat-bottom microplate. Further, cells were treated with all ten neem-derived limonoids independently at 100?M and 200?M for 24?h. MTT was added into each well and incubated at 37?C for 4?h. After incubation, formazan crystals were dissolved with isopropanol and optical density of formazan solution, as a measure of cell viability was observed using a microplate reader at 570?nm (Thermo Scientific). In separate experiments, MDA-MB-231, MCF-7 and MCF-10A cells were independently treated with epoxyazadiradione (0C200?M) in time-dependent manner and cytotoxic effect was determined by MTT assay as described above. In other experiments, MDA-MB-231 cells were pre-treated with Caspase 9 inhibitor-I (Calbiochem) or ROS scavenger agents, catalase (CAT) or N-acetyl-cysteine (NAC) (Sigma) independently for 1?h and further incubated with epoxyazadiradione (150?M) for 24?h and MTT assay SIS3 was performed. Annexin V/propidium iodide staining MDA-MB-231 cells were treated with/without epoxyazadiradione (0C150?M) for 24?h and stained with annexin V-FITC followed by propidium iodide (PI) and apoptosis was studied using apoptosis detection kit (BD Pharmingen) according to the manufacturers instructions. Stained cells were analyzed by FACSCalibur cytometer (BD Biosciences). In separate experiments, the effect of epoxyazadiradione on cell-cycle analysis was studied using PI staining as described [24]. Briefly, MCF-7 cells were treated with epoxyazadiradione (0C150?M) for 24?h, stained with PI and analyzed on FACSCalibur cytometer. The cell cycle distribution was analyzed using CellQuest software (BD Immunocytometry System). Immunofluorescence study Cells were grown on cover slips, treated in absence or presence of epoxyazadiradione with increasing concentrations (0C150?M) for 24?h and immunofluorescence analysis was performed as described [31]. MDA-MB-231 or MCF-7 cells were fixed with 2% paraformaldehyde, blocked with 10% FBS and incubated with anti-c-Jun, anti-c-Fos or anti-AIF (Santa Cruz Biotechnology) antibody for overnight followed by fluorescence conjugated Cy2 or Cy3 (Calbiochem) specific antibody. To study the actin cytoskeleton reorganization, epoxyazadiradione treated MDA-MB-231 or MCF-7 cells were stained with FITC conjugated phalloidin (Sigma). Nuclei were stained with DAPI and analyzed under confocal microscope (Zeiss). TUNEL assay To analyze the SIS3 DNA fragmentation in response to epoxyazadiradione, TUNEL assay was conducted using APO-DIRECT? Kit (BD Pharmingen) in MDA-MB-231 cells as per manufacturers instructions. Images were captured using fluorescence microscope (Leica). Determination of ROS production To measure the effect of epoxyazadiradione on intracellular ROS production, MDA-MB-231 or MCF-7 cells were independently treated with increasing concentrations of epoxyazadiradione (0C150?M) for 24?h. These cells were then stained with dihydroethidine (DHE) (Molecular Probes) Tpo for 20?min at 37?C and analyzed on FACSCanto cytometer (BD Biosciences). Measurement of mitochondrial membrane potential (?m) To examine the effect of epoxyazadiradione on mitochondrial membrane potential which is a crucial event.