title: Recombinant rubistatin (r-Rub), an MVD disintegrin, inhibits cell migration and proliferation, and is a strong apoptotic inducer of the human melanoma cell line SK-Mel-28.
abstract: Disintegrins are low molecular weight peptides isolated from viper venom. These peptides bind to integrin receptors using a conserved binding motif sequence containing an RGD or similar motif. As a consequence, disintegrins can inhibit platelet aggregation and inhibit cell migration, proliferation, and initiate apoptosis in cancer cell lines. Rubistatin is a MVD disintegrin cloned from a Crotalus ruber ruber venom gland. The biological activity of MVD disintegrins is poorly understood. Recombinant rubistatin (r-Rub) was cloned into a pET32b plasmid and expressed in reductase-deficient Escherichia coli. Expression was induced with IPTG and the resulting fusion peptide was affinity purified, followed by thrombin cleavage, and removal of vector coded sequences. r-Rub peptide inhibited ADP-induced platelet aggregation by 54% ± 6.38 in whole blood. We assessed the ability of r-Rub to initiate apoptosis in three human cancer cell lines. Cultures of SK-Mel-28, HeLA, and T24 cells were grown for 24 h with 2.5 μM r-Rub followed by Hoechst staining. Chromatin fragmentation was observed in treated SK-Mel-28, but not in T24 or HeLA cells. A TUNEL assay revealed that 51.55% ± 5.28 of SK-Mel-28 cells were apoptotic after 18 h of treatment with 3.5 μM of r-Rub. Cell migration and proliferation assays were performed in order to further characterize the biological effects of r-Rub on SK-Mel-28 cells. At 3 μM, r-Rub inhibited cell migration by 44.4% ± 0.5, while at 3.5 μM it was able to inhibit cell proliferation by 83% ± 6.0.
authors.affiliation: Biological Sciences Department, San José State University, One Washington Square, DH 254, San José, CA 95192-0100, USA.
authors.forename: Clayton M
authors.initials: CM
authors.lastname: Carey
authors.forename: Raymund
authors.initials: R
authors.lastname: Bueno
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Gutierrez
authors.forename: Christopher
authors.initials: C
authors.lastname: Petro
authors.forename: Sara E
authors.initials: SE
authors.lastname: Lucena
authors.forename: Elda E
authors.initials: EE
authors.lastname: Sanchez
authors.forename: Julio G
authors.initials: JG
authors.lastname: Soto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry / Q000494:pharmacology; D017839:Crotalus; D019483:Disintegrins / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology*; D017422:Sequence Analysis, DNA
chemical_list: D003435:Crotalid Venoms; D019483:Disintegrins; D009842:Oligopeptides; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid