LAWSONIA ALBA LEAVES INDUCE APOPTOSIS AND CELL CYCLE ARREST IN B16F10 MELANOMA CELLS

Objective: The present study was designed to investigate the anti-melanoma activity of the ethyl acetate fraction of Lawsonia alba lam leaves (ELA) against B16F10 cells.Methods: Cytotoxicity of ELA on B16F10 cells was determined by MTT assay and supported with the morphology of apoptotic and necrotic cells under phase-contrast microscope, fluorescence microscopy with AO/EtBr, confocal microscopy with PI, Agarose gel electrophoresis and Annexin V-FITC/PI staining, mitochondrial membrane potential and cell cycle arrest by FACS was also performed on B16F10 cells.Results: Cytotoxic effect of ELA on B16F10 melanoma cell was confirmed by MTT assay with an IC50 value of 14.10μg/ml. Morphological study showed arrays of both the nuclear changes including chromatin condensation and apoptotic body formation indicating that the treatment with ELA and 5-Fluorouracil (standard) causes apoptotic changes in the melanoma cells compared to the untreated control. Agarose gel electrophoresis study showed fragmented DNA in the form of ladder. The depolarization of mitochondrial membrane potential was confirmed. Flow cytometric analysis showed appreciable number of cells in early apoptotic stage. The cells were arrested mostly in G0/G1 phase of cell cycle.Conclusion: Ethyl acetate fraction of Lawsonia alba L. leaves possesses potent apoptotic activity against B16F10 cells.