Fig 1.
DisBa-01 inhibits the migration of OSCC cell line.
(A) DisBa-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 DisBa-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.
Fig 2.
OSCCs treated with DisBa-01 show an increase in adhesions area and turnover.
Cells were allowed to spread for 3 hours on fibronectin (2μg/ml) coated dishes, subsequently fixed, stained for paxillin and analyzed by confocal microscopy. (A) Fibroblasts show a large number of small adhesions (red arrow) with no differences between cells with or without DisBa-01 treatment, while SSC25 treated cells show larger (red arrow) adhesions and rounded shape morphology in the presence of DisBa-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 DisBa-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.
Fig 3.
Fibronectin-related integrin receptors show differential expression according to the differentiation level of the tumor cell.
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 αv, 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.
Fig 4.
DisBa-01 interacts specifically with integrin αVβ3.
(A) Response of increasing concentrations of DisBa-01 interacting with the αVβ3 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 DisBa-01 interacting with the α5β1. As the concentration of DisBa-01 increases, the affinity curves show very little displacement. (Single column- print in color)
Fig 5.
Overexpression of β3 subunit recovered DisBa-01 effects on migration speed and adhesion dynamics of OSCC cells.
(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 DisBa-01 treated cells. (C) Representative images of β3 overexpressing cells (indicated with arrows) stained for paxillin. (D) β3 overexpressing cells with DisBa-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 DisBa-01.