Figure 1.
Lumcorin decreases melanoma growth.
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 (*, p< 0.05, **, p<0.01***, p<0.001). Scale bar in the inserts: 200µm.
Figure 2.
Lumcorin inhibits melanoma motility.
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 After 24h incubation, the culture-inserts were withdrawn and migration was monitored for 48h by computer-assisted phase contrast videomicroscopy as described in Materials and Methods.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 (**, p<0.01, ***, p<0.001).
Figure 3.
Lumcorin alters the expression and activity of MMP-14 and inhibits FAK phosphorylation at tyrosine 397.
(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 (*, p<0.05; **, p<0.01; ***, p<0.001; NS, no significant difference).
Figure 4.
Characterization of lumcorin derived L9M peptide.
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 (*, p<0.05; **, p<0.01; ***, p<0.001).