Snake Venom Disintegrins and Cell Migration

Cell migration is a key process for the defense of pluricellular organisms against pathogens, and it involves a set of surface receptors acting in an ordered fashion to contribute directionality to the movement. Among these receptors are the integrins, which connect the cell cytoskeleton to the extracellular matrix components, thus playing a central role in cell migration. Integrin clustering at focal adhesions drives actin polymerization along the cell leading edge, resulting in polarity of cell movement. Therefore, small integrin-binding proteins such as the snake venom disintegrins that inhibit integrin-mediated cell adhesion are expected to inhibit cell migration. Here we review the current knowledge on disintegrin and disintegrin-like protein effects on cell migration and their potential use as pharmacological tools in anti-inflammatory therapy as well as in inhibition of metastatic invasion

Inhibition of α_vβ₃ integrin induces loss of cell directionality of oral squamous carcinoma cells (OSCC)

<div><p>The connective tissue formed by extracellular matrix (ECM) rich in fibronectin and collagen consists a barrier that cancer cells have to overpass to reach blood vessels and then a metastatic site. Cell adhesion to fibronectin is mediated by α_vβ₃ and α₅β₁ integrins through an RGD motif present in this ECM protein, thus making these receptors key targets for cell migration studies. Here we investigated the effect of an RGD disintegrin, Dis<i>Ba</i>-01, on the migration of human fibroblasts (BJ) and oral squamous cancer cells (OSCC, SCC25) on a fibronectin-rich environment. Time-lapse images were acquired on fibronectin-coated glass-bottomed dishes. Migration speed and directionality analysis indicated that OSCC cells, but not fibroblasts, showed significant decrease in both parameters in the presence of Dis<i>Ba</i>-01 (1μM and 2μM). Integrin expression levels of the α₅, α_v and β₃ subunits were similar in both cell lines, while β1 subunit is present in lower levels on the cancer cells. Next, we examined whether the effects of Dis<i>Ba</i>-01 were related to changes in adhesion properties by using paxillin immunostaining and total internal reflection fluorescence TIRF microscopy. OSCCs in the presence of Dis<i>Ba</i>-01 showed increased adhesion sizes and number of maturing adhesion. The same parameters were analyzed usingβ3-GFP overexpressing cells and showed that β3 overexpression restored cell migration velocity and the number of maturing adhesion that were altered by Dis<i>Ba</i>-01. Surface plasmon resonance analysis showed that Dis<i>Ba</i>-01 has 100x higher affinity for α_vβ₃ integrin than forα5β1 integrin. In conclusion, our results suggest that the α_vβ₃ integrin is the main receptor involved in cell directionality and its blockage may be an interesting alternative against metastasis.</p></div

Fibronectin-related integrin receptors show differential expression according to the differentiation level of the tumor cell.

<p>Cells lysates of fibroblasts (BJ), highly invasive OSCC (SCC25) or poor invasive OSCC (Cal27 were submitted to western blot for analysis of fibronectin-related integrins. BJ and SCC25 contain α_v, β₃ and α₅ integrin subunits in similar amounts while β₁ is present in smaller amounts on SCC25 cells. E-cadherin and N-cadherin are differentiation markers.</p

Dis<i>Ba</i>-01 inhibits the migration of OSCC cell line.

<p>(A) Dis<i>Ba</i>-01 significantly inhibited the migration speed of Oral Squamous Cell Carcinoma cells (SCC25) cells but not in fibroblasts (BJ) on both conditions tested (Media = DisBa-01 in the media; Subs = DisBa-01 in the substrate). Results were calculated as % of control. Statistical analysis was performed using ANOVA (one-way) followed by Tukey’s post-test, p<0.0001. (B) Migration tracks of SCC25 cells treated with Dis<i>Ba</i>-01 in the concentrations of 2μM indicates a loss of directionality when compared to the control tracks. Each individual line represents a cell path translated to a common origin.</p

DisBa-01 interacts specifically with integrin α_Vβ_3.

<p>(A) Response of increasing concentrations of Dis<i>Ba</i>-01 interacting with the α_Vβ₃ integrin immobilized to the sensor chip. Starting at the time “0” and increasing its affinity as DisBa-01 concentration increases. (B) Response of increasing concentrations of Dis<i>Ba</i>-01 interacting with the α₅β₁. As the concentration of Dis<i>Ba</i>-01 increases, the affinity curves show very little displacement. (Single column- print in color)</p

OSCCs treated with Dis<i>Ba</i>-01 show an increase in adhesions area and turnover.

<p>Cells were allowed to spread for 3 hours on fibronectin (2μg/ml) coated dishes, subsequently fixed, stained for paxillin and analyzed by confocal microscopy. <b>(</b>A) Fibroblasts show a large number of small adhesions (red arrow) with no differences between cells with or without Dis<i>Ba</i>-01 treatment, while SSC25 treated cells show larger (red arrow) adhesions and rounded shape morphology in the presence of Dis<i>Ba</i>-01 (2μM). Actin staining and negative control are shown on the right. (B) Percentage of maturing adhesions on TIRF time-lapse movies in the absence or presence of Dis<i>Ba</i>-01, (C) in the adhesion area (D) and in the protrusions area (p<0.05). Data was obtained from 3 independent experiments resulting in the analysis of 13–15 protrusions and 64–84 adhesions per experimental condition.</p

Overexpression of β3 subunit recovered DisBa-01 effects on migration speed and adhesion dynamics of OSCC cells.

<p>(A) SCC25 cells were transfected with GFP-β₃ 0.5 and 1μg of plasmid. Protein bands show the levels of GFP-β₃ and endogenous β₃ expressed by SCC25 cells. (B) Migrations speed analysis shows no differences between control and Dis<i>Ba</i>-01 treated cells. (C) Representative images of β₃ overexpressing cells (indicated with arrows) stained for paxillin. (D) β₃ overexpressing cells with Dis<i>Ba</i>-01 reduced the number of adhesions maturation, similar to the controls, when compared with the previous TIRF experiment. (E) DisBa-01 effect over the area of adhesions and (F) area of protrusion was also was reverted on β₃ overexpressing cells treated with Dis<i>Ba</i>-01.</p