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A novel αvβ3-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis

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Abstract

The integrin αvβ3 is involved in multiple aspects of malignant cancer, including tumor angiogenesis and metastasis, which makes the receptor a key target for the development of anti-cancer therapies. We report here on the production, the characterization and the in vivo anti-angiogenic and anti-metastatic properties of a novel αvβ3-binding disintegrin, DisBa-01, isolated from a cDNA library made with RNAs from the venom gland of Bothrops alternatus. The 11,637 Da-recombinant monomeric form of DisBa-01 displayed an RGD motif and interacted with purified αvβ3 integrin in surface plasmon resonance studies, in a dose-dependent and cation sensitive manner. A three-dimensional molecular model of DisBa-01 in complex with αvβ3 predicted a large surface of contacts with the β3 subunit. DisBa-01 inhibited the adhesion of αvβ3-expressing human microvascular endothelial cell line-1 (HMEC-1) and murine melanoma cell line B16F10 to vitronectin (IC50 = 555 nM and 225 nM, respectively), and transiently inhibited their proliferation without direct cell toxicity, but did not affect the binding nor the proliferation of a human breast cancer-derived cell line (MDA-MB-231) not expressing αvβ3. In vivo, DisBa-01 dose-dependently decreased bFGF-induced angiogenesis in a matrigel plug assay in athymic nude mice (IC50 = 83 nM). When injected intravenously to C57BL/6 mice together with B16F10 melanoma cells, DisBa-01 time- and dose-dependently inhibited lung metastasis monitored by bioluminescent imaging. We conclude that DisBa-01 is a potent new inhibitor of αvβ3-dependent adherence mechanisms involved in neo-vascularization and tumor metastasis processes.

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Abbreviations

BFGF:

Basic fibroblast growth factor

ECM:

Extracellular matrix

FBS:

Foetal bovine serum

FITC:

Fluorescein isothiocyanate

GFP:

Green fluorescent protein

HMEC-1:

Human microvascular endothelial cell line-1

IPTG:

Isopropyl thio-β-d-galactopyranoside

MD:

Molecular dynamics

MIDAS:

Metal-ion-dependent adhesion site

MS:

Mass spectroscopy

PCR:

Polymerase chain reaction

PDB:

Protein data base

RGD:

Arginine-glycine-aspartic acid

RU:

Resonance units

SVMP:

Snake venom metalloproteinase

Vn:

Vitronectin

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Acknowledgments

The authors wish to thank Dr Marika Pla and Martine Chopin, Département d’Expérimentation Animale, IFR 105 -Saint Louis-Institut Universitaire d’Hématologie, Paris, France, for their skillful management of animal care facilities, and Monique Etienne from SMBH, Bobigny, France, for histological work. MRC and OHPR were the recipient of research grants provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo). This work was supported by travel grants provided in cooperation by INSERM and the Brazilian government (CNPq-INSERM collaboration), a Marie Curie European Reintegration Grant (Project MERG-CT-2004-006377) and grants from the Association pour la Recherche sur le Cancer (Project ARC-7801 and Pôle ARECA) and from the Canceropole Ile-de-France.

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Authors and Affiliations

  1. Dep. Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP, Brasil

    Oscar H. P. Ramos, Márcia R. Cominetti, Carmen L. Salla Pontes & Heloisa S. Selistre-de-Araujo

  2. INSERM, U 553, Paris, 75010, France

    Alexandre Kauskot, Iga Bechyne, Chantal Legrand, Michel Crépin & Arnaud Bonnefoy

  3. Université Paris 7-Denis Diderot, Faculté de Médecine, IFR 105 -Saint Louis-Institut Universitaire d’Hématologie, Paris, 75475, France

    Alexandre Kauskot, Iga Bechyne, Fabrice Chareyre, Jan Manent, Marco Giovannini, Chantal Legrand & Arnaud Bonnefoy

  4. INSERM, U 674, Paris, 75010, France

    Fabrice Chareyre, Jan Manent & Marco Giovannini

  5. Laboratoire de Pharmacologie Clinique et Experimentale, Université Paris 13, CNRS UMR 7033, Bobigny, 93000, France

    Roger Vassy & Michel Crépin

  6. Laboratoire d’Immunologie, Centre de Recherche du Centre Hospitalier de l’Université de Montréal-Saint-Luc, INSERM U743, 264, rue René Lévesque Est, Montreal, QC, Canada, H2X1P1

    Arnaud Bonnefoy

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  1. Oscar H. P. Ramos
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  11. Heloisa S. Selistre-de-Araujo
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  13. Arnaud Bonnefoy
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Correspondence to Arnaud Bonnefoy.

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Oscar H. P. Ramos and Alexandre Kauskot contributed equally to this work.

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Ramos, O.H.P., Kauskot, A., Cominetti, M.R. et al. A novel αvβ3-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis. Clin Exp Metastasis 25, 53–64 (2008). https://doi.org/10.1007/s10585-007-9101-y

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