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. 2012 Jan 2;287(1):170-182.
doi: 10.1074/jbc.M111.263996. Epub 2011 Oct 28.

Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways

Cláudia Alessandra Andrade de Paula  1 , Vivien Jane Coulson-Thomas  1 , Joana Gasperazzo Ferreira  1 , Paloma Korehisa Maza  2 , Erika Suzuki  2 , Adriana Miti Nakahata  1 , Helena Bonciani Nader  1 , Misako Uemura Sampaio  1 , Maria Luiza V Oliva  3
Affiliations

Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways

Cláudia Alessandra Andrade de Paula et al. J Biol Chem. .

Abstract

Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin β1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.

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Figures

FIGURE 1.
FIGURE 1.
Effect of EcTI on cell viability. Fibroblast (A and B), Hs746T (C and D), and MKN28 cells (E and F) were treated with increasing concentrations (0–150 μm) of EcTI for 24 and 48 h. Cell viability was measured using the MTT assay. Controls consisted of treating cells with medium with 7 mm HEPES, pH 7.4 (EcTI vehicle). The percentage of viable cells was calculated as the ratio of treated cells to control cells. Error bars indicate S.D. of triplicate samples. *, p < 0.05 versus control (0 μm EcTI; vehicle only).
FIGURE 2.
FIGURE 2.
Effect of EcTI on cell adhesion. Inhibitory effect of EcTI on cell-matrix adhesion. Fibroblast (A) or Hs746T (B) cells were pretreated with increasing concentrations of EcTI for 30 min and then plated on wells precoated with matrix molecules for 4 h. Controls consisted of treating cells with medium with 7 mm HEPES, pH 7.4 (EcTI vehicle). The adhered cells were stained with 1% toluidine blue and solubilized with 1% SDS, and the absorbance was measured at 540 nm. The percentage of adhered cells was calculated as the ratio of treated cells to control cells. The experiment was performed in triplicate. Error bars indicate S.D. of triplicate samples. *, p < 0.05 versus control (0 μm EcTI; vehicle only).
FIGURE 3.
FIGURE 3.
Effect of EcTI on Hs746T cell migration and invasion. A, scratch wound cell motility assay. Monolayers of Hs746T cells treated with EcTI were scratched, photographed, and rephotographed after 24 h. B and C, invasion assays were performed using collagen I- or Matrigel-coated chambers with 10% FCS as a chemoattractant. Hs746T cells were seeded at a density of 2 × 105 cells/well on top of control or coated inserts. The percentage of invasion through collagen I (B) or Matrigel (C) was calculated relative to invasion through the control insert (cells treated with vehicle only). The percentage of cell migration and invasion was calculated as the ratio of treated cells to control cells. Controls consisted of treating cells with medium with 7 mm HEPES, pH 7.4 (EcTI vehicle). D, confocal images of invasion of untreated GFP-positive Hs746T cells (panels i and ii) and GFP-positive Hs746T cells treated with EcTI (panels iii and iv). Shown are overlay images (panels i and iii) of GFP-positive Hs746T cells and fibroblasts and YZ sections of these cells (panels ii and iv) showing the invasion event. Note that in the GFP-positive Hs746T cells treated with EcTI (panel iv) the green cells have not invaded the matrix. Green, GFP-positive Hs746T cells; red, α-actin in the three-dimensional matrix formed by the fibroblasts. The nuclei (blue) were stained with DAPI. Error bars indicate S.D. of triplicate samples. *, p < 0.05 versus control (0 μm EcTI; vehicle only).
FIGURE 4.
FIGURE 4.
Confocal images. Shown is the immunolocalization of integrin β1, MT1-MMP, cortactin, N-WASP, and α-actin after treatment of Hs746T cells with 100 μm EcTI. Cells were seeded on collagen I, and monolayer scratch wounds were generated. Cells were allowed to migrate for 6 h, and immunofluorescence images of migrating cells at the wound edge were captured using confocal microscopy. Controls consisted of treating cells with medium with 7 mm HEPES, pH 7.4 (EcTI vehicle). A, integrin β1 (green) and MT1-MMP (red). B, cortactin (red). C, N-WASP (green) and α-actin (red). The nuclei (blue) were stained with DAPI. Co-localization of images is also shown (Merge). Scale bars represent 20 μm. D, the relative fluorescence levels of proteins were determined by densitometric analysis and are represented as a percentage of controls. *, p < 0.05 versus control (0 μm EcTI; vehicle only). Error bars indicate S.D. of five fields.
FIGURE 5.
FIGURE 5.
Effect of EcTI on expression of integrin β1, cortactin, N-WASP, and MT1-MMP and activity of MMP-2. A, Hs746T cells pretreated with EcTI for 30 min were plated on tissue culture plastic (No coat) or on collagen I-coated culture dishes for 1 h, and lysate proteins were separated by 10% SDS-PAGE and electrotransferred to PVDF membrane. Membranes were blocked and incubated with anti-integrin β1, anti-cortactin, anti-N-WASP, anti-MT1-MMP, and anti-β-actin (loading control). Antibody binding was visualized by chemiluminescence, and the relative levels of these proteins were determined by densitometric analysis (B). C, cells plated on tissue culture plastic or on collagen I were treated with EcTI for 6 h, and the activity of MMP-2 in supernatant was revealed by gelatin zymography. D, densitometric quantitation analysis of the active MMP-2 band. The percentage was calculated as the ratio of treated cells to control cells. The effect of EcTI on gene expression levels of integrin β1 (E) and MT1-MMP (F) is shown. Hs746T cells were pretreated with 100 μm EcTI for 30 min and seeded on collagen I. After 6 h, RNA was extracted for analysis by quantitative RT-PCR. Cells seeded without EcTI were used as controls. Gene expression was normalized against ribosomal protein S29 (RPS29). Controls consisted of treating cells with medium with 7 mm HEPES, pH 7.4 (EcTI vehicle). col I, collagen I. Error bars indicate S.D. of triplicate samples. *, p < 0.05 versus control (0 μm EcTI; vehicle only).
FIGURE 6.
FIGURE 6.
Effect of EcTI on Src-FAK signaling and effect of PP2 on invadopodia. A, Hs746T cells pretreated with EcTI for 30 min were plated on a tissue culture plastic (No coat) or on collagen I for 1 h, and lysate proteins were separated by 10% SDS-PAGE and electrotransferred to PVDF membrane. Membranes were blocked and incubated with anti-phospho-Src, anti-phospho-FAK, anti-Src, anti-FAK, and anti-β-actin (loading control) antibodies. Antibody binding was visualized by chemiluminescence, and the relative phosphorylation levels of these proteins were determined by densitometric analysis (B). C, immunolocalization of cortactin after treatment of Hs746T cells with 10 μm PP2. Cells were seeded on collagen I and allowed to migrate for 18 h. The immunofluorescence images of cells migrating were captured using confocal microscopy. Controls consisted of treating cells with medium with 0.1% DMSO (PP2 vehicle). Cortactin staining is red. Nuclei (blue) were stained with DAPI. Co-localization of images is also shown (Merge). The scale bar represents 20 μm. D, the relative fluorescence levels of cortactin were determined by densitometric analysis. Error bars indicate S.D. of five fields. *, p < 0.05 versus control (PP2 vehicle). E, cells plated on collagen I were treated with 10 μm PP2 for 18 h, and the activity of MMP-2 in the supernatant was revealed by gelatin zymography. F, densitometric quantitation analysis of the active MMP-2 band. The percentage was calculated as the ratio of treated cells to control cells. col I, collagen I.
FIGURE 7.
FIGURE 7.
Schematic mechanism of action of EcTI on invadopodium formation. A, the integrin activation leads to invadopodium formation through FAKTyr-397 and SrcTyr-416 signaling pathways. B, the inhibition of activated integrin β1 expression by EcTI leads to a decrease of FAKTyr-397 activation followed by a decrease of SrcTyr-416 activation, preventing the recruitment of proteins important for the formation of invadopodia, such as cortactin and N-WASP. Because the formation of invadopodia is impaired, the proteolytic activity of the cell is decreased, which is reflected in the inhibition of ECM degradation.
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