Lumcorin alters the expression and activity of MMP-14 and inhibits FAK phosphorylation at tyrosine 397.

<p>(A) Expression of MMP-14 in B16F1 cells incubated 48h with 100µM lumcorin or scrambled peptide was analyzed by Western immunoblotting using polyclonal rabbit antibody and probing with anti-β-actin. (B, C) Activity of MMP-14 in B16F1 (B) and SK-MEL-28 (C) cells incubated 48h in the presence of 100µM lumcorin or its scrambled peptide, measured using fluorimetric SensoLyte® 520 MMP-14 Assay Kit as described in Materials and Methods. (D) Activity of MMP-14 in B16F1 cell extract pre-incubated 15 min or 60 min before assay with 100µM lumcorin or scrambled peptide. (E) Recombinant human MMP-14 activity pre-incubated 15 min with 100µM lumcorin or its scrambled peptide or DCN LRR9 or Fmod LRR9. Data are presented as mean ± S.D from three independent experiments. (F) Phospho-FAK (pY397) and total FAK expression in B16F1 cells incubated 15 min with 100µM lumcorin or scrambled peptide analyzed by Western immunoblotting using monoclonal mouse antibody against pFAK (pY397) and probing with polyclonal rabbit anti-total FAK antibody. After densitometric analysis of the intensity of the bands, the resulting ratio of pFAK to total FAK intensity was calculated and presented on the graph. Data are presented as mean values ± S.D from three independent experiments (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; NS, no significant difference).</p

Lumcorin decreases melanoma growth.

<p>Cell growth assay (A, B) and colony formation assay (C, D) of B16F1 cells (A, C) and SK-MEL-28 cells (B, D). For growth studies, cells were grown for 24, 48 and 72h in presence of 100 µM lumcorin or its scrambled (SCR) peptide. Cell growth was measured by MTT colorimetric test at 560nm, as described in Materials and Methods. Results were reported as mean ± S.D of sextuplicate values from three independent experiments. For colony formation, 1.2x10³cells were cultured in 0.3% agar for 14 days in presence of 100µM lumcorin or scrambled peptide as described in Materials and Methods. Representative images of cell colonies are displayed at the inserts. The quantification of the colony diameter was done using Image Tool software. Graphs represent the mean size of 100 colonies ± S.D from three independent experiments (*, <i>p</i>< 0.05, **, <i>p</i><0.01***, <i>p</i><0.001). Scale bar in the inserts: 200µm.</p

Lumican – Derived Peptides Inhibit Melanoma Cell Growth and Migration

<div><p>Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth <i>in vitro</i> and <i>in vivo</i>. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican <i>in vitro</i>. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.</p> </div

Lumcorin inhibits melanoma motility.

<p>Migration of B16F1 cells (A) and SK-MEL-28 cells (B) in the presence of lumcorin. Cells were plated on 24-well plate, 3x10⁴ cells per chamber of culture-insert. After 24h incubation, the culture-inserts were withdrawn and migration was monitored for 48h by computer-assisted phase contrast videomicroscopy as described in Materials and Methods. Representative images of cell positions after 48h of migration in the absence of peptide (a), in presence of 100µM lumcorin (b) or of its scrambled peptide (c) are displayed on left panels. Migration was quantified as percent of area colonized by cells. Graphs represent the mean value ± S.D calculated from 4 microscopic fields per insert. The experiment was done in triplicate (**, <i>p</i><0.01, ***, <i>p</i><0.001).</p

Characterization of lumcorin derived L9M peptide.

<p>Cell growth assay (A, B) and colony formation assay (C, D) of B16F1 cells (A, C) and SK-MEL-28 cells (B, D) cultured in the presence of 100 µM L9M or appropriate scrambled peptide (L9M SCR). Cell growth was measured as described in Figure 1. Results were reported as mean ± S.D of sextuplicate values from three independent experiments. For colony formation 1.2x10³cells were cultured in 0.3% agar for 14 days in presence of 100µM L9M or L9M SCR as described in Materials and Methods. Representative images of cell colonies are displayed at the inserts. The quantification of the colony diameter was done using Image Tool software. Graphs represent the mean size of 100 colonies ± S.D from three independent experiments. Scale bar in the inserts: 200µm. (E, F) Migration of B16F1 cells (E) and SK-MEL-28 cells (F) in presence of L9M peptide. Cells were plated on 24-well plate at with 3x10⁴ cells per chamber of culture-insert. After 24h of incubation, the culture-inserts were withdrawn and migration was monitored for 48h by computer-assisted phase contrast videomicroscopy. Representative images of cell positions after 48h of migration in the absence of peptide (a), in presence of 100µM L9M peptide (b) or its scrambled peptide (c) are displayed on left panels. Migration was quantified as percent of area colonized by cells. Graphs represent the mean value ± S.D calculated from 4 microscopic fields per insert. (G, H) Effect of L9M peptide on MMP-14 activity in B16F1 cells (G) and SK-MEL-28 cells (H). MMP-14 activity in cells incubated 48h in the presence of 100µM L9M or its scrambled peptide was measured using fluorimetric SensoLyte® 520 MMP-14 Assay Kit. The experiment was done in triplicate (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001).</p

Effect of Lumican on the Migration of Human Mesenchymal Stem Cells and Endothelial Progenitor Cells: Involvement of Matrix Metalloproteinase-14

<div><h3>Background</h3><p>Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful <em>in vivo</em> administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).</p> <h3>Methodology/Principal Findings</h3><p>Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.</p> <h3>Conclusion/Significance</h3><p>Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration <em>via</em> mechanisms that involve a decrease of MMP-14 expression and activity.</p> </div

Lumican inhibits MMP-14 expression and activity in MSC.

<p>(A): Lumican effect on MMP-14 expression in MSC and EPC analyzed by Western blotting using a mouse anti-MMP-14 monoclonal antibody directed against the catalytic domain of MMP-14 and probing with anti-β-actin. Semi-confluent MSC and EPC were seeded on plastic or in presence of lumican coating as described in Materials and Methods section. Inactive and active MMP-14 forms were detected in positive control cell lysates of fibrosarcoma cell line (HT-1080). Active MMP-14 was not detected in negative control cell lysates of HeLa cells. (B): MMP activity was measured using a SensoLyte® 520 MMP-14 Assay Kit as described in the Materials and Methods section. The activity of MMP-14 of cell lysates of MSC and EPC seeded on plastic or lumican coating (30 µg/cm²) was measured from four different donors in two independent experiments. Data presented as mean ± S.D. **<i>p</i><0.01. (C): MMP-2 and MMP-9 expression and activity in MSC and EPC. MMP-2 and MMP-9 expression and activity in cell lysates (left panel) and conditioned cell media (right panel) were analyzed by zymography.</p

Lumican inhibits melanoma primary tumor growth of Snail overexpressing B16F1 cells <i>in vivo</i>.

<p><i>Lum</i>^+/+ and <i>Lum</i>^-/- mice on the C57Bl/6 background were injected subcutaneously with 2.5 x 10⁵ of B16F1 cells, mock or Snail overexpressing B16F1 cells (clone Snail19-B16F1). The kinetic of melanoma primary tumor growth is displayed in volume (mm³) till day 15 when mice were sacrificed. * <i>p</i> < 0.05: significantly different from mock-transfected B16F1 cell tumors, Open circle: wild type C57Bl/6 mice injected with mock-B16F1 cells (n = 10); Diamond: lumican deleted C57Bl/6 mice injected with mock B16F1 cells (n = 10); Open square: wild type C57Bl/6 mice injected with Snail overexpressing B16F1 cells (n = 10); Triangle: Lumican deleted C57Bl/6 mice injected with Snail overexpressing B16F1 cells (n = 10).</p

Lumican inhibits Snail overexpressing B16F1 cell functional properties.

<p>(A) Cell migration of Snail overexpressing B16F1 cells (clone Snail19-B16F1) in presence or absence of lumican (100 nM) after 24h and 48h; n = 2, ***<i>p</i> < 0.001 (B) Colony formation assay of mock and Snail overexpressing B16F1 cells (clone Snail19-B16F1) in absence or presence of lumican (100 nM); Representative images of cell colonies are displayed at the inserts 14 days after seeding. The quantification of the colony diameter was performed using Image Tool software. Graphs represent the mean size of 100 colonies ± SD from two independent experiments. (**<i>p</i> < 0.01, ***<i>p</i>< 0.001). Scale bar inserts: 200 μm.</p

Lumican inhibits MSC migration and invasion.

<p>MSC (A) or EPC (B) were plated on 12-well plates at 15 000 cells per chamber of culture-insert. Cells were incubated at 37°C and 5% CO₂ for 24 h. After withdrawal of the culture-insert, lumican (100 nM) was added to the serum-free cell culture medium. The migration speed of MSC and EPC was determined by means of computer-assisted phase contrast videomicroscopy during 48 hours as described in Materials and Methods. Representative images of cell positions after 48 h of migration are displayed on the right panels. The data are representative of three independent analyses. *<i>p</i><0.05. (C) MSC and EPC (D) invasion in presence of lumican. Cells were seeded on inserts as described in the Materials and Methods section. Medium with 10% or 2% FBS was used as a chemotactic agent for MSC or EPC, respectively, and the cells were cultured for a further 48 h. Negative control medium contained 2% BSA. Medium containing 0.5% BSA and 100 nM lumican was added to the upper chamber at the time of seeding. Invading cell nuclei were stained with Hoechst 33342 (5 µg/mL) and counted using fluorescence microscopy. Representative fluorescence images of invading cells are displayed on the upper panel. Results are expressed as the percentage of control (cells invading toward chemotactic medium) (mean ± S.D.) from at least three independent experiments with triplicate inserts. Scale bar: 100 µm. **<i>p</i><0.01; (E, upper panel): Lysates from transiently transfected MSC with GFP (mock) or MMP-14-GFP constructs were analyzed for MMP-14 overexpression by Western blotting with an antibody raised against MMP-14 after 48 h of transfection. Non-transfected MSC lysate is also included as control. (E, bottom panel): Overexpression of MMP-14 in MSC induces MMP-14 activity as shown by the enhancement of active MMP-2 band by zymography. (F): Migration of GFP- (mock) or MMP-14-GFP-transfected MSC was determined 48 h post-transfection using cell culture-insert assay. The migration speed of GFP-positive MSC, was determined by means of computer-assisted phase contrast and fluorescence videomicroscopy during 24 hours as described in supplemental methods section. The data are representative of two independent analyses. **<i>p</i><0.01. (G): GFP- (mock) or MMP-14-GFP-transfected MSC invasion in presence of lumican. Cells were cultured and inserts seeded as described in the supplemental methods section. Medium with 10% FBS was used as a chemotactic agent for MSC, and the cells were cultured for a further 48 h. Negative control medium contained 2% BSA. Medium containing 0.5% BSA and 100 nM lumican was added to the upper chamber at the time of seeding. Invading cell nuclei were stained with Hoechst 33342 (5 µg/mL) and counted using fluorescence microscopy. Representative fluorescence images of invading cells are displayed on the upper panel. Results are expressed as the percentage of control (mock-transfected cells invading toward chemotactic medium) (mean ± S.D.) from two independent experiments with triplicate inserts. Scale bar: 100 µm. *<i>p</i><0.05. ***<i>p</i><0.001.</p