Figure 3

Effect of COX-2 inhibition on v-FLIP/K13-induced C–X–C ligands, C–C ligands, chemokines, adhesion molecules, MMP gene expression and MMP secretion levels. (A–C) Histograms depict the fold induction in gene expression of p-SIN, v-FLIP/K13-HMVECs, NS-398 or celecoxib-pretreated (4 h, 8 h and 24 h) v-FLIP/K13-HMVECs. Fold induction of chemokine (C-X-C motif) ligands (Aa, Ab, Ac, Ad), chemokine related genes (Ba, Bb, Bc, Bd, Be), chemokine (C-C motif) ligands (Ca, Cb, Cc) gene expression was calculated by considering expression in p-SIN-HMVECs as onefold. % Inhibition was calculated by considering gene expression in untreated v-FLIP/K13-HMVECs at the respective time of measurement as 100%. Each reaction was done in quadruplicate, and each bar represents the average±s.d. of four independent experiments. ^* and ^**: statistically significant at P<0.01 and P<0.005, respectively. (D) Effect of COX-2 inhibition on MMP secretion levels and invasion of v-FLIP/K13-HMVECs; activation of MMP-2, MMP-9 and MMP-10 measured by their respective assay kits in conditioned media obtained from untreated p-SIN-HMVECs and untreated, NS-398 or celecoxib-pretreated v-FLIP/K13-HMVECs. The levels of total and active MMPs in p-SIN-HMVECs were considered onefold for comparison. % Inhibition in these protease levels (total or active) upon COX-2 inhibition was calculated considering respective MMP levels (total or active) in untreated v-FLIP/K13-HMVECs as 100%. (E) COX-2 regulates v-FLIP/K13-HMVECs invasion via autocrine and paracrine mechanisms. Invasion of HMVEC cells in the presence of supernatant from pSIN or v-FLIP/K13-transduced or NS-398- or celecoxib-pretreated v-FLIP/K13-HMVECs. (a) Histogram represents the fluorescence intensity (mean±s.d. of three independent experiments) of HMVEC-d cells invaded in the presence of conditioned media obtained from pSIN or v-FLIP/K13 or NS-398/ celecoxib/ solvent control pretreated v-FLIP/K13-HMVECs. % Inhibition in invasion upon either NS-398 or celecoxib treatment was calculated considering invasion in the presence of supernatant from v-FLIP/K13-HMVECs at the indicated time point as 100%. (b) Effect of v-FLIP/K13-induced COX-2-mediated invasive potential of v-FLIP/K13-HMVECs as measured by fluorescence-based invasion assay as described in Materials and methods. Fluorescence intensity is presented and the values shown are the mean±s.d. of three independent experiments. % Inhibition in invasion by NS-398 or celecoxib treatment was calculated considering invasion of untreated v-FLIP/K13-HMVECs as 100%. Fold increase in the invasion of v-FLIP/K13-HMVECs was calculated using the invasive potential of p-SIN-HMVECs as onefold. Fluorescence associated with the invaded cells is shown and the values correspond to the mean±s.d. of three independent experiments. ^* and ^**: statistically significant at P<0.01 and P<0.005, respectively.