Isolation of an Anti-Tumour Disintegrin: Dabmaurin-1, a Peptide Lebein-1-Like, from Daboia mauritanica Venom

Abstract

In the soft treatment of cancer tumours, consequent downregulation of the malignant tissue angiogenesis constitutes an efficient way to stifle tumour development and metastasis spreading. As angiogenesis requires integrin-promoting endothelial cell adhesion, migration, and vessel tube formation, integrins represent potential targets of new therapeutic anti-angiogenic agents. Our work is a contribution to the research of such therapeutic disintegrins in animal venoms. We report isolation of one peptide, named Dabmaurin-1, from the hemotoxic venom of snake Daboia mauritanica, and we evaluate its potential anti-tumour activity through in vitro inhibition of the human vascular endothelial cell HMECs functions involved in tumour angiogenesis. Dabmaurin-1 altered, in a dose-dependent manner, without any significant cytotoxicity, HMEC proliferation, adhesion, and their mesenchymal migration onto various extracellular matrix proteins, as well as formation of capillary-tube mimics on Matrigel^TM. Via experiments involving HMEC or specific cancers cells integrins, we demonstrated that the above Dabmaurin-1 effects are possibly due to some anti-integrin properties. Dabmaurin-1 was demonstrated to recognize a broad panel of prooncogenic integrins (αvβ6, αvβ3 or αvβ5) and/or particularly involved in control of angiogenesis α5β1, α6β4, αvβ3 or αvβ5). Furthermore, mass spectrometry and partial N-terminal sequencing of this peptide revealed, it is close to Lebein-1, a known anti-β1 disintegrin from Macrovipera lebetina venom. Therefore, our results show that if Dabmaurin-1 exhibits in vitro apparent anti-angiogenic effects at concentrations lower than 30 nM, it is likely because it acts as an anti-tumour disintegrin.

Keywords: angiogenesis; anti–angiogenic peptide; anti–tumour; cell adhesion molecules; disintegrin; endothelial cells; integrin; snake venom.

Figure 1

Isolation of active peptide: (a) Dm venom Chromatogram obtained after preparative HPLC in a C18 column, eluting 4 mL/min with a gradient of acetonitrile in 0.1% TFA in water (star points collection of fractions F11 & F12 of interest) (b) Effect on 96 h—HMEC proliferation (3 assays) of 5 µL amount of each chromatographic fractions (1m L) from 50 mg of crude venom obtained via 3 min–collection and 20–fold diluted. Star points effects of the most active fraction. (c) Analytic HPLC chromatogram of fraction F27,3 obtained in the (a) conditions but at a flow rate of 1 mL/min.

Figure 2

Sequence of peptide P₁ compared to that of Lebein subunits.

Figure 3

Effect of P₁ on cell viability. (a) HMECs were incubated (2 assays) for 96h with various concentrations of P1 (in 100 µL per well), then with 0,5mg/mL MTT. After solubilisation with DMSO, the absorbance was measured at 600 nm. (b) HMECs were incubated (3 assays) for 5h with various concentration of P1 (50 µL per well) estimated through LHD release evaluated via Assay Kit TOX7.

Figure 4

HMECs (25,000 in 50 µL) were allowed to attach (a) to various ECMs, in the presence of peptide P₁ at concentration equal to 2 × 10³ ng/mL. (2 assays), (b) at various concentrations (6 assays); (c) to P₁ used as a matrix at various concentrations, assuming adhesion to fibronectin (Fn) as a control (2 assays).

Figure 5

Dose–effect of peptide P₁ on migration: (a) in a Boyden chamber of 30,000 HMECs per 100 µL– well when attracted during 5 h by ECM (2 assays) (b) checked by video–microscopy during 4 h of HMECs (10,000 /300 µL–well) after P₁ contact on 50 µg/mL fibrinogen or 5 µg/mL fibronectin ECM, measuring velocity, (c) distance to origin, (d) direction persistency, (e) cell shape (5 assays for b–e).

Figure 6

(a) Honeycomb structures formed in 6 h by HMECs (10,000 cells / 50 µL well) on Matrix BD Matrigel^TM. (b) Cell aggregates formed in the same conditions, but in presence of 400 ng/mL peptide P₁. (c) Estimation of the remaining tubule bends with Metamorph software via 5 photographs per well in presence of various P₁ amounts (3 assays).

Figure 7

Effect of peptide P₁ on adhesion (a) of K562 cells (50,000/well–2h – 3 assays) to fibronectin; (b) of α6–U87 cells (50,000/well– 30 min – 3 assays) to vitronectin et laminin; (c) of HT29D4 cells (50,000/ well – 2 h – 4 assays) to some various ECM; (d) modulation of HMECs (20,000 / well – 1 h – 4 assays) to various anti–integrins.