Tetrastatin, the NC1 Domain of the α4(IV) Collagen Chain: A Novel Potent Anti-Tumor Matrikine

BACKGROUND: NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (-38%) and invasive (-52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (-80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (K(D) = 148 ± 9.54 nM). CONCLUSION/SIGNIFICANCE: Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine

AG-9, an Elastin-Derived Peptide, Increases In Vitro Oral Tongue Carcinoma Cell Invasion, through an Increase in MMP-2 Secretion and MT1-MMP Expression, in a RPSA-Dependent Manner

Oral tongue squamous cell carcinoma is one of the most prevalent head and neck cancers. During tumor progression, elastin fragments are released in the tumor microenvironment. Among them, we previously identified a nonapeptide, AG-9, that stimulates melanoma progression in vivo in a mouse melanoma model. In the present paper, we studied AG-9 effect on tongue squamous cell carcinoma invasive properties. We demonstrated that AG-9 stimulates cell invasion in vitro in a modified Boyen chamber model. It increases MMP-2 secretion, analyzed by zymography and MT1-MMP expression, studied by Western blot. The stimulatory effect was mediated through Ribosomal Protein SA (RPSA) receptor binding as demonstrated by SiRNA experiments. The green tea-derived polyphenol, (−)-epigallocatechin-3-gallate (EGCG), was previously shown to bind RPSA. Molecular docking experiments were performed to compare the preferred areas of interaction of AG-9 and EGCG with RPSA and suggested overlapping areas. This was confirmed by competition assays. EGCG abolished AG-9-induced invasion, MMP-2 secretion, and MT1-MMP expression

The YSNSG cyclopeptide derived from tumstatin inhibits tumor angiogenesis by down-regulating endothelial cell migration.

International audienceWe previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a β-turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained β-turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1-MMP at the migration front as well as a decrease in u-PA and u-PAR expression. The cyclopeptide also altered β1-integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125FAK), disorganized F-actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity

Angiogenesis Inhibition by a Short 13 Amino Acid Peptide Sequence of Tetrastatin, the α4(IV) NC1 Domain of Collagen IV

International audienceAngiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α 5 β 1 integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α 5 β 1 integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes

Conformation-dependent binding of a Tetrastatin peptide to αvβ3 integrin decreases melanoma progression through FAK/PI3K/Akt pathway inhibition

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Recombinant human NC1 α4(IV) inhibits <i>in vitro</i> melanoma cell proliferation and invasion.

<p>(A): Recombinant human NC1 α4(IV) domain obtention: Recombinant human NC1 α4(IV) domain was expressed in <i>E. coli</i> JM109, DE3 strain (1) SDS-PAGE (lane M: low molecular weight markers; lane 1: T4h crude lysate, non-induced by IPTG; lane 2: T4h crude lysate, induced by IPTG, lane 3: recombinant human NC1 α4(IV) domain purified by chromatography on a Ni-NTA superflow resin. (2) Western blot using an anti-NC1 α4(IV) antibody. Lane 1, 2, 3: same as above. (3) Western blot using an anti-His tag antibody. Lane 1, 2, 3: same as above. (B): Cell proliferation: Melanoma cells and dermal fibroblasts were incubated without or with 10 or 20 µg/mL recombinant human NC1 α4(IV) for 48 h. Cell proliferation was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. NS: Non significant. ***: p<0.001. (C): Cell invasion: UACC-903 melanoma cells were incubated without or with 20 µg/mL recombinant human NC1 α4(IV). Cell invasion was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. ***: p<0.001.</p

NC1 α4(IV) overexpression by melanoma cells decreases tumor growth in a mouse xenograft model.

<p>Mock or NC1 α4(IV)-overexpressing UACC-903 cells (5×10⁶ cells) were subcutaneously injected into the left side of athymic mice. (A): Tumor size was measured at days 10, 14, 19 and 26. Tumor volumes were determined according to v = ½ A×B², where A denotes the largest dimension of the tumor and B represents the smallest dimension, and expressed as mean±SD (n = 10). ***: p<0.001. The insert shows example of tumors obtained in each mouse series after injection of Mock or NC1 α4(IV)-overexpressing cells. (B): Immunostaining of tumor section with an anti-Ki67 antibody. Quantification was performed with Image J. Scale bar: 50 µm.</p

NC1 α4(IV) overexpression inhibits <i>in vitro</i> melanoma cell proliferation.

<p>(A): Cell proliferation was measured using WST-1 reagent after 24, 48 and 72 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Materials and Methods</a> (mean of three clones±SD. ***: p<0.001) (A). (B): Cell proliferation of the three selected clones at T 72 h.</p

Recombinant human NC1 α4(IV) binds to αvβ3 integrin.

<p>(A): Adhesion assays were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. Cells were incubated or not with 5 mM EDTA. Cells were fixed after 30, 60 or 90 min with 1.1% glutaraldehyde and stained with crystal violet. After elution with 10% acetic acid, absorbance was read at 560 nm. *: p<0.05. Adhesion was restored by the addition of 1.3 mM Ca²⁺ or 0.5 mM Mg²⁺. (B): Adhesion assays were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029587#s2" target="_blank">Material and Methods</a> section. Cells were preincubated for 30 min with an anti-αvβ3 blocking antibody or an irrelevant IgG (10 µg/mL) before seeding. Cells were fixed with 1.1% glutaraldehyde and stained with crystal violet. After elution with 10% acetic acid absorbance was read at 560 nm. *: p<0.05. (C): Surface plasmon resonance (SPR) binding assays were performed by injecting recombinant human NC1 α4(IV) (3.57 µM at 30 µL/min) over αvβ3 integrin immobilized on a CM5 sensor chip (2655 RU). Binding was expressed as resonance units.</p