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

Devy J, Duca L, Cantarelli B, Joseph-Pietras D, Scandolera A, Rusciani A, Parent L, Thevenard J, Pasco SB, Tarpin M, Martiny L, Debelle L - Br. J. Cancer (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Université de Reims Champagne Ardenne (URCA), Laboratoire SiRMa, UMR CNRS 6237, IFR 53 Interactions cellules microenvironment, UFR Sciences Exactes et Naturelles, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France.

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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Related in: MedlinePlus

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.
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fig2: 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.

Mentions: B16F1 cells were tested for their ability to migrate following EDPs stimulation in an in vitro system. Briefly, B16F1 were seeded on plastic dishes then scrapped at the centre of the dish. The migration of the cells was evaluated by videomicroscopy using their ability to fill the gap. These experiments were performed using 2.5% FBS, a concentration for which EDPs has no effect on cell proliferation. We found that EDPs enhanced B16F1 migration as the artificially created wound was more filled compared with control (Figure 2A). B16F1 cells were then tested for their ability to migrate through Matrigel-coated (40 μg per well) membranes for 40 h in the presence or absence of EDPs. The incubation of melanoma cells with EDPs led to a significant increase in their invasive properties (+36%) comparatively to control cells (Figure 2B). These results demonstrated that EDPs increased melanoma cell invasion and showed that, in addition to trigger in vivo growth, they enhance cell invasion. These results support the detrimental role of EDPs in in vivo melanoma development.


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

Devy J, Duca L, Cantarelli B, Joseph-Pietras D, Scandolera A, Rusciani A, Parent L, Thevenard J, Pasco SB, Tarpin M, Martiny L, Debelle L - Br. J. Cancer (2010)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: B16F1 cells were tested for their ability to migrate following EDPs stimulation in an in vitro system. Briefly, B16F1 were seeded on plastic dishes then scrapped at the centre of the dish. The migration of the cells was evaluated by videomicroscopy using their ability to fill the gap. These experiments were performed using 2.5% FBS, a concentration for which EDPs has no effect on cell proliferation. We found that EDPs enhanced B16F1 migration as the artificially created wound was more filled compared with control (Figure 2A). B16F1 cells were then tested for their ability to migrate through Matrigel-coated (40 μg per well) membranes for 40 h in the presence or absence of EDPs. The incubation of melanoma cells with EDPs led to a significant increase in their invasive properties (+36%) comparatively to control cells (Figure 2B). These results demonstrated that EDPs increased melanoma cell invasion and showed that, in addition to trigger in vivo growth, they enhance cell invasion. These results support the detrimental role of EDPs in in vivo melanoma development.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Université de Reims Champagne Ardenne (URCA), Laboratoire SiRMa, UMR CNRS 6237, IFR 53 Interactions cellules microenvironment, UFR Sciences Exactes et Naturelles, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France.

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.

Show MeSH
Related in: MedlinePlus