Abstract
BackgroundIn recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line.MethodsBased on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-Tricine-SDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively.ResultsDisintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 μg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 μg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control.ConclusionThus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.
Highlights
In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy
In order to isolate the disintegrin, Fraction 2 was refractionated by reversed phase chromatography on a Fast Protein Liquid Chromatography (FPLC) system with a segmented acetonitrile gradient represented by the dashed line in blue (Fig. 1b)
After the second chromatographic step, it was possible to observe in Fraction 6 a single band of nearly 7 kDa corresponding to disintegrin
Summary
Snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. In 1987 Huang et al observed that a small protein isolated from Trimeresurus gramineus venom, called “trigramin”, presenting the arginine-glycine-aspartic acid (RGD) domain, was able to inhibit platelet aggregation, by preventing the connection between fibrinogen and platelets stimulated by ADP [3]. Disintegrins can be classified according to their structure, considering the numbers of disulfide bonds and amino acid residues: (i) short disintegrins contain 41 to 51 amino acid residues, stabilized by 4 disulfide bonds; (ii) medium-sized disintegrins contain approximately 70 amino acid residues and 6 disulfide bonds; (iii) long disintegrins, with approximately 84 residues and 7 disulfide bonds; (iv) and the last group that comprises homo- and heterodimeric disintegrins, with approximately 67 residues per subunit, 4 intrachain disulfide bonds, in addition to 2 interchain cystine bonds, stabilizing the molecules [7,8,9,10]
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