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 E. 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 2.5µM 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 3.5µM 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 3µM, r-Rub inhibited cell migration by 44.4% +/− 0.5, while at 3.5µM it was able to inhibit cell proliferation by 83% +/− 6.0.

Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): A second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells.

“…An approach for the development of innovative chemotherapies is the screening, selection, modification, and synthesis of compounds that can inhibit multiple landmarks of cancer development (for example, angiogenesis, cell migration, metastasis, and cell proliferation) while inducing apoptosis. Our previous research with two recombinant disintegrins (r-Rub and r-Moj-DM) provided us with good models to investigate this approach (Carey et al 2012; Seoane et al 2010). …”

“…The effects of r-Moj-D_ peptides on cell migration were determined using the method described by Carey et al (2012).…”

Functional analysis of four single (RGDWL, RGDWM, RGDWP, RGDMN) and two double (RGDNM, RGDMP) mutants: The importance of methionine (M) in the functional potency of recombinant mojastin (r-Moj)

“…The effects of r-Moj-WL, r-Moj-WM, r-Moj-WP, r-Moj-WN, r-Moj-MN, r-Moj-MP, or r-Moj-NM peptides on SK-Mel-28 cell migration were determined using the method described by Carey et al (2012).…”

“…A Cell BioLabWound Healing Assay was used to measure cell migration inhibition as described by Carey et al (2012). Cells were incubated for 24 h with their respective r-Moj treatments.…”

“…Targeted mechanisms include activation or repression of genes involved in cell death and survival, activation of apoptosis, angiogenesis inhibition, cell division inhibition, and metastasis inhibition. Several investigators have shown that disintegrins have the ability to induce cellular responses that target the mechanisms listed above (McBride et al, 2016; Ramos et al, 2016; Lucena et al 2014, 2015; Ribeiro et al, 2014; Carey et al, 2012; Selistre-de-Araujo et al, 2010; Minea et al, 2010; Seoane et al, 2010; Ren et al, 2006; Alimenti et al, 2004; Brassard et al, 1999; Wu et al, 2003; Hong et al, 2003; Zhou et al, 1999; Yeh et al, 1998). …”