Figure 1

(A) Chemical structure of cyclopeptide glucoside RA-XII. (B) Cytotoxic effects of RA-XII at 25 to 1600 nM on 4T1cells after 24 or 48 hours by MTT assay. Data were expressed as the mean fold of untreated controls (mean ± SD of 3 independent experiments with 5 replicates each). (C,D) Effects of RA-XII on breast cancer cell adhesion. (C) In ECM-adhesion assay, 4T1 cells treated with or without RA-XII at 100 or 500 nM were added to wells precoated with ECM proteins for 2 hours. Results were expressed as the mean fold of the untreated controls (mean + SD of 3 independent experiments in duplicates). (D) Representative flow cytometric histograms showed the effect of RA-XII (50 or 100 nM treated for 24 hours) on the expressions of adhesion molecules (integrins, VCAM-1 and ICAM-1) on 4T1 cell surface. Cells were stained with FITC- or PE-conjugated antibodies. The histograms on the right panel were expressed as fold of untreated controls (mean + SD of 3-4 independent experiments). (E) Effects of RA-XII on VCAM-1 at mRNA levels. 4T1 cells were treated with RA-XII at 50 or 100 nM for 24 hours. The RNA content of cells was isolated and was subjected to real-time PCR. The histograms were expressed as fold of untreated controls (mean + SD of 3 independent experiments with 3 replicates each). (F) Effects of RA-XII and/or integrin antagonist on fibronectin- and laminin-cell adhesion assays in 4T1 cells. Cells added to the fibronectin-coated or laminin-coated wells were treated with RA-XII (500 nM) and/or integrin antagonist (10 μM) for 2 hours. BSA-coated wells used as negative control. The intensity of coloured product extracted from the stained bound cells represents cell adhesion to fibronectin or laminin. Results were expressed as the percentage of the control (mean + SD of 3 to 4 independent experiments with 2 replicates each). Statistical differences were determined by One-way ANOVA, followed by Dunnett Test, with *p < 0.05, **p < 0.01, ***p < 0.001 against untreated controls.