Figure 3

SC236 affects proliferation and invasive properties of SKNEP1 tumors in vivo and in vitro. (A) Quantification of phosphohistone H3 (pH3) (N = 3; 23-30 sections studied, each condition) demonstrated that SC236 reduced proliferation in vivo as compared to both control and BV-treated tumors (33.8 ± 3.2 cells/hpf vs. 53.6 ± 3.6 and 46.4 ± 2.8 cells/hpf, respectively; P < 0.005, both). Combined SC236+BV treatment also reduced proliferation versus controls (43.1 ± 2.4 cells/hpf, P < 0.02). Thus, SC236 restriction of proliferation persisted during VEGF blockade, but was not altered. (B) We assessed MMP-2 and MMP-9 activation, which can promote lung metastasis and interacts with VEGF and COX-2 status, using gelatin zymography [23–26] in each treatment group (N = 3 each). MMP-2 activity was unchanged (not shown). MMP-9 activity was significantly reduced in each treatment group as compared to controls (versus SC236 group, 35%; in BV, 34%, SC36+BV, 25%; P < 0.03, each). Thus, combined SC236 and BV did not further reduce MMP9 activity. (C) We used the MTT assay to determine whether hypoxia, SC236 concentration, or duration of exposure would reduce SKNEP1 tumor cell viability. Both hypoxia and increasing concentration suppressed activity measured by MTT, with effects enhanced by increasing duration (96 h). These results support an increased effect of SC236 in hypoxic conditions, as may occur during VEGF blockade. (D). We assessed SC236 effect on tumor cell invasion through basement membrane (Matrigel). Invasion was reduced at 50 μM SC236 by 7% in normoxia and 14% in hypoxia (P < 0.005, each).