Determining if αvβ3 integrin is activated in the apoptotic induction of Sk-Mel-28 cells by r-Moj-DM disintegrin

Integrins are heterodimeric transmembrane cell surface receptors that consist of non-covalently bound alpha and beta subunits. Since integrins are involved in cell signaling pathways, it seems plausible that their deregulation or dysfunction play a role in tumor behavior and metastasis. Current research is focused on understanding integrins that activate signal transduction pathways that trigger apoptosis. Disintegrins are small cysteine rich, non-enzymatic peptides with an integrin binding-loop that bind and alter integrin signaling.  A recombinant mojastin disintegrin peptide (r-Moj-DM) was shown to induce apoptosis of a human melanoma cell line (SK-Mel-28). Flow cytometry analysis showed that SK-Mel-28 cells express αvβ3, αv, α6, and β1 integrin subunits. Integrin αvβ3 has been shown to be involved in apoptosis-induction by disintegrins. Given these findings, we hypothesized that r-Moj-DM binds to the αvβ3 integrin receptor activating apoptosis of SK-Mel-28 cells. We investigated this hypothesis using small hairpin RNA lentiviral particles to generate a stable SK-Mel-28 cell line with αv integrin subunit knockdown. The r-Moj-DM peptide (5µM) was added to experimental and control cells for 24 hours, and apoptosis was visualized using a tunnel assay (APO-BRDU). The r-Moj-DM peptide induced apoptosis at similar levels in αv knockdown cells (26.1% ± 3.85), untreated cells (25.6% ± 1.21) and in shRNA scrambled control cells (23.6% ± 0.27), suggesting that αvβ3 integrin is not the receptor r-Moj-DM peptide binds to. These results raise the possibility that the αvβ3 integrin receptor may be involved in up regulating expression or activating survival factors. Research supported by NIH/SCORE grant# 2506-GM008122.