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. 2010 Nov 9;103(10):1562-70.
doi: 10.1038/sj.bjc.6605926. Epub 2010 Oct 19.

Elastin-derived peptides enhance melanoma growth in vivo by upregulating the activation of Mcol-A (MMP-1) collagenase

J Devy  1 , L Duca, B Cantarelli, D Joseph-Pietras, A Scandolera, A Rusciani, L Parent, J Thevenard, S Brassart Pasco, M Tarpin, L Martiny, L Debelle
Affiliations

Elastin-derived peptides enhance melanoma growth in vivo by upregulating the activation of Mcol-A (MMP-1) collagenase

J Devy et al. Br J Cancer. .

Abstract

Background: Elastin peptides possess several biological activities and in vitro data suggest they could be involved in the early phase of melanoma growth.

Methods: Using diverse in vitro and in vivo techniques (cell proliferation, invasion and migration assays, zymography, western blots, collagen degradation assay, reverse transcription PCR, melanoma allographs and immunohistochemistry), we analysed the effect of elastin-derived peptides (EDPs) on B16F1 melanoma growth and invasion, as well as on the proteolytic systems involved.

Results: We found that EDPs dramatically promote in vivo tumour development of B16F1 melanoma, as well as their in vitro migration and invasion. The inhibition of serine proteases and matrix metalloproteinases (MMPs) activities, by aprotinin and galardin, respectively, demonstrated that these enzymes were involved in these processes. However, we found that EDPs did not increase urokinase-type plasminogen activator, tissue-type plasminogen activator or MMP-2 expression and/or activation, neither in vitro nor in vivo. Nevertheless, we observed a strong increase of pro-MMP-9 secretion in EDPs-treated tumours and, more importantly, an increase in the expression and activation of the murine counterpart of MMP-1, named murine collagenase-A (Mcol-A). Moreover, we show that plasminogen system inhibition decreases collagen degradation by this enzyme. Finally, the use of a specific blocking antibody against Mcol-A abolished EDP-induced B16F1 invasion in vitro, showing that this MMP was directly involved in this process.

Conclusion: Our data show that in vivo, EDPs are involved in melanoma growth and invasion and reinforced the concept of elastin fragmentation as a predictive factor.

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Figures

Figure 1
Figure 1
Elastin-derived peptides increase melanoma tumour growth in vivo (A) and in vitro cell proliferation (B). (A) B16F1 or EDP-pretreated B16F1 were subcutaneously injected to female syngenic C57Bl6 mice (2.5 × 105 cells per mouse) as described in the Materials and Methods section. Mice were killed at day 10. Tumour sizes were measured and statistical significance was calculated according to the Student's t-test. Data represent the average of five mice. A second experiment conducted under the same conditions gave identical results. *Significantly different at P<0.05. (B) B16F1 were seeded in 96-well plates in complete medium with or without EDPs for 72 h. Cell proliferation was determined by counting cells with a Malassez cell. Data are expressed as mean±s.e.m. values from three independent experiments, each performed in triplicate. **Significantly different at P<0.01.
Figure 2
Figure 2
Elastin-derived peptides stimulate B16F1 migration (A) and invasion (B). Invasion involves the plasminogen system (C) without inducing tPA or uPA (D). (A) B16F1 cells have been seeded in 12-well plates and a homogeneous wound was created in each well by scraping cells with a tip. Cells were stimulated or not for 48 h with 50 μg ml–1 EDPs and cell migration was evaluated by videomicroscopy. (B) Cellular invasive potential was assayed using Transwell coated with Matrigel (40 μg per well). In total, 50 × 103 cells in 100 μl of RPMI 1640 with or without EDPs (50 μg ml–1) were deposited into the upper chamber. The lower chamber contained 10% FBS and 2% of BSA. Incubation was for 40 h. Data are expressed as mean±s.e.m. values from three independent experiments, each performed in triplicate. **Significantly different at P<0.01. (C) is same as (B) except for the presence or not of aprotinin (100 μg ml–1). **Significantly different at P<0.01. NS=nonsignificantly different. (D) Upper panel: Gelatin plasminogen zymography was performed as described in the Materials and Methods section. The gels presented are representative of several in vitro (n=3) and in vivo (n=5) experiments. Lower panel: Quantification of in vitro and in vivo expressions of tPA and uPA was carried out using densitometry and calculating using Quantity One Software. C=untreated control; EDPs=EDP-treated cells or tumours; NS=nonsignificantly different.
Figure 3
Figure 3
Elastin-derived peptide-stimulated B16F1 invasion involves the MMP system (A) without upregulating MMP-2 and MMP-9 expression and/or activation (B). EDPs increase in vivo infiltrating cells in B16F1 tumours (C). (A) Cellular invasive potential was assayed using Transwell coated with Matrigel (40 μg per well). In total, 50 × 103 cells in 100 μl of RPMI 1640 with or without EDPs (50 μg ml–1) were deposited in the upper compartment, in the presence or absence of galardin (10−9M, G), aprotinin (100 μg ml–1, A) or both (A+G). The lower chamber contained 10% FBS and 2% of BSA. Incubation time was 40 h. Data are expressed as mean±s.e.m. values from three independent experiments, each performed in triplicate. **Significantly different at P<0.01. (B) Gelatin zymography was performed as described in the Materials and Methods section. The gels presented are representative of several in vitro (n=3) and in vivo (n=5) experiments. (C) Infiltrating cells in B16F1 and EDP-treated B16F1 tumours were identified by immunohistochemistry. Tumour sections were stained with an anti-monocyte/macrophage antibody. Monocyte/macrophage appears as dark-brown staining. Tumour sections were counterstained with methylene green zinc chloride double salt. The photographs presented are representative of several (n=5) experiments.
Figure 4
Figure 4
Elastin-derived peptides increase in vitro and in vivo Mcol-A expression and activation (A–C), leading to collagen degradation (D). Treatment of cells with anti-MMP-1-blocking antibody blocks EDP-stimulated B16F1 invasion (E). (A) Mcol-A expression was analysed by RT–PCR. B16F1 cells were treated with EDPs (50 μg ml–1) for 48 h and RT–PCR was conducted as described in the Material and Methods section. The gel presented is representative of several (n=3) experiments. (B) Left panel: Mcol-A secretion and activation by B16F1 and EDP-treated B16F1 cells was analysed by western blotting using an anti-MMP-1 antibody as described in the Materials and Methods section. The gel presented is representative of several (n=3) experiments. Right panel: Densitometric analyses of western blots. White bars: active form; black bars: pro-form. (C) Mcol-A expression by tumours from B16F1- and EDP-treated B16F1 was analysed by immunohistochemistry. Tumour sections were stained with an anti-Mcol-A antibody. Mcol-A appears as dark-brown staining. Tumour sections were counterstained with hematoxylin. Scale bar=200 μm. (D) B16F1-conditioned media (48 h) incubated or not with EDPs (50 μg ml–1) and/or aprotinin (100 μg ml–1) were incubated with DQ-collagen. The degradation of DQ-collagen was evaluated by fluorescence (λex=495 nm; λem=515 nm). ***Significantly different at P<0.001. (E) Cellular invasive potential was assayed using Transwell coated with Matrigel (40 μg per well). In total, 50 × 103 cells were suspended in 100 μl of RPMI 1640 with or without EDPs (50 μg ml–1) in the upper compartment and in the presence or not of anti-MMP-1-blocking antibody (10 μg ml–1). The lower chamber contained 10% FBS and 2% of BSA. Incubation was for 40 h. Data are expressed as mean±s.e.m. values from three independent experiments, each performed in triplicate. **Significantly different at P<0.01. NS=nonsignificantly different.
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