In-vitro Morphological Assessment of Apoptosis Induced by Nimbolide; A Limonoid from Azadirachta Indica (Neem Tree)

The present study was designed to investigate the in-vitro morphological assessment of apoptotic effect caused by nimbolide on the selected cancer cell lines (DU-145, PC-3, A-549) and normal cell lines (NIH3T3, CCD-18Co). The cells were grown in 6 well tissue culture plates after treatment with different concentrations of nimbolide and untreated control cells. Apoptotic and necrotic cells were measured using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and also by staining with annexin V and propidium iodide through flow cytometric analysis. The activity of caspase 3, 8, and 9 was measured by caspases colorimetric assay kits. The number of apoptotic and necrotic cells were significantly higher in all selected cancer cell lines treated with nimbolide as compared with untreated control cells, whereas in normal cell lines no significant difference was observed between nimbolide treated and untreated cells. The activity of caspase 3, 8, and 9 was also significantly higher in all cancer cell lines treated with nimbolide as compared with untreated control cells while it did not change significantly in normal cell lines as compared with untreated control. The results of the present study suggested that nimbolide induced apoptosis only in cancer cells without affecting the normal cells and one of the apoptosis inducing mechanism is through the activation of caspases signaling pathways. Therefore, nimbolide may be a novel promising candidate as an anticancer drug in future.