Figure 3

TSP1 expression by LNCaP cells after tasquinimod exposure in in vitro cell cultures. (A) Up-regulation of the TSP1 protein levels by tasquinimod (50 μM) in LNCaP cells was measured by western blot analysis (left panel (i)). Protein bands represent intact TSP1 at ~160 kD (monoclonal Ab11). TSP1 secreted into cell culture medium was measured with ELISA after 72 h (right panel (ii)). The culture medium levels of TSP1 were 50.8 ± 1.5 ng/ml for untreated cells and 80.6 ± 10.2 ng/ml for exposed cells, respectively (n = 3; p ≤ 0.05, ANOVA). (B) TSP1 secretion into cell culture medium after exposure of LNCaP cells to 10 μM tasquinimod (+) (p ≤ 0.01, ANOVA). TSP1 levels in untreated (-) cell culture medium were 22, 36.6 and 51.6 ng/ml after 6, 24 and 72 h incubation, respectively, and 6 ng/ml in the R10 medium. (C) Up-regulation of TSP1 mRNA levels occurred in the hormone independent prostate cancer cell line LNCaP19 but not in DU145, (-) untreated control and (+) 10 μM tasquinimod (p ≤ 0.0001; ANOVA). Presented data represent the mean ± SD of at least two independent experiments. (*) p ≤ 0.05 and (**) p ≤ 0.01 compared with untreated control (Bonferroni's multiple comparison test). (D) TSP1 protein levels measured by western blot analysis of prostate cancer cell lysates, (-) untreated control and (+) 10 μM tasquinimod for 72 h. PL indicates lysate prepared from human platelets and rTSP1 is recombinant TSP1.