Author : Helen Shiphrah Vethakanraj, Mohamed Aslan S, Jayashree A, Vishnu Priya P and Ashok Kumar Sekar

The present study investigates the apoptotic effect of ceramidase inhibitor, Ceranib 2 and Sphingosine kinase 1 inhibitor, Dimethyl sphingosine in human colon cancer cell line SW480. Cells were treated with different doses of Ceranib 2 and Dimethyl sphingosine for 24, 48 and 72 hours to determine the cell viability using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay). Morphological changes were examined in phase contrast microscope, nuclear changes were observed using DAPI staining. DNA gel electrophoresis was performed to visualize DNA changes, Acridine orange/Ethidium bromide staining was done to discriminate live, early apoptotic, late apoptotic, necrotic cells. Mitochondrial membrane potential was determined by JC-1 staining. MTT assay revealed the cytotoxic effect of Ceranib 2 and Dimethyl sphingosine in SW480 cell line in dose and time dependant manner. Morphological changes and chromatin condensation observed in DAPI staining are morphological hallmarks of apoptosis. DNA ladder pattern observed in drug treated groups with respect to control group is biochemical hallmark of apoptosis. Further, presence of early and late apoptotic cells in Acridine orange/Ethidium bromide staining, loss of mitochondrial membrane potential in drug treated groups confirmed the apoptotic effect of Ceranib 2 and Dimethyl sphingosine in SW480 cells.

Keywords: Colon cancer, Apoptosis, Ceranib 2, Dimethyl sphingosine, Ceramidase, Sphingosine kinase 1