Three Recombinant Disintegrins Induce Apoptosis in SK-Mel-28 Cells

Disintegrins are cysteine-rich peptides found in viper venom. The majority of soluble disintegrins possess a peptide motif, arranged in a loop at the C-terminus, which is thought to be responsible for the protein’s adhesive properties and consists of three amino acids: arginine/glycine/aspartic acid (RGD). Previous work conducted by our lab has shown that by altering the two amino acids C-terminal to this RGD binding motif from the wild-type tryptophan/arginine sequence found in Crotalus scutulatus to an aspartic acid/methionine sequence (r-Moj-DM), the recombinant peptide was capable of inducing apoptosis in SK-Mel-28 (melanoma) cells from less than 2% to over 13%, respectively. We hypothesize that recombinant disintegrins, r-Moj-MN, r-Moj-WP, r-Moj-WM, will induce apoptosis of SK-Mel-28 cells. A qualitative method of apoptotic induction known as chromatin fragmentation was used.  Eight wells of an eight-chambered slide were seeded with 50,000 SK-Mel-28 cells per well in EMEM media. Cells were cultured for 24 hrs, and then treated with each recombinant disintegrin or PBS, in duplicate, at 5 µM for 18 hrs. Cells were subsequently fixed with 3% formaldehyde, stained with 500 µL of 10 µg/mL Hoechst, fixed with 30% glycerol, and visualized on a Zeiss fluorescent microscope. Our preliminary results support our hypothesis. Each recombinant disintegrin induced chromatin fragmentation of SK-Mel-28 cells. Tunnel assays will be performed to quantitate the number of cells that undergo apoptosis after disintegrin treatment. ANOVA statistical analysis will also be performed to determine the contribution of each amino acid to apoptosis induction.