Functional analysis of a recombinant PIII-SVMP, GST-acocostatin; an apoptotic inducer of HUVEC and HeLa, but not SK-Mel-28 cells

Abstract

Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin-V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68% +/− 3.09 of treated HUVEC, and 35.86% +/− 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors.

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

“…However, we have shown that SK-Mel-28 cells lack α1β1 (Seoane et al, 2010). Both HeLa and SK-Mel-28 cell lines express α2β1 (Taklemariam et al, 2011), but r-Rub failed to induce apoptosis of HeLa cells. We also hypothesize that it is unlikely that r-Rub binds to αvβ3, an integrin known to bind to apoptotic-inducing disintegrins (Yeh et al, 1998; Wierzbicka-Patynowski et al, 1999), as this receptor is found in both SK-Mel-28 and T24 cell lines (Seoane et al, 2010; Taklemeriam et al, 2011).…”

Cytotoxic and apoptotic activities of black widow spiderling extract against HeLa cells

“…To further characterize the cytotoxic and apoptotic activities of the extract, it was divided into high-and low-molecular-mass fractions and it was revealed that such activities were primarily attributed to the proteins with higher molecular masses. This seems to differ from other animal venoms, including snake venom, of which a number of proteins and low molecular mass peptides are able to efficiently inhibit the proliferation of HeLa cells (6,31,38). After the high molecular mass fraction was further separated into four subfractions, all were found to significantly decrease cell viability (P<0.01), indicating that the fraction was rich in the relevant active proteins.…”

Purification and Immunoprotection Evaluation of AaHIV from Complex Venom Metalloproteinases ofDeinagkistrodon acutus

“…Studies had shown that SVMPs can hydrolyze fibrinogen, the extracellular matrix, and the basement membrane in vitro, as well as induce apoptosis of vascular endothelial cells, antitumor effects, and the rolling of leukocytes. It is the component of D. acutus VC with the greatest toxicity [18][19][20]. Liu et al [21] constructed a cDNA library of gkistrodon acutus venom.…”