1.00 ± 0.24 1.00 ± 0.04 1.00 ± 0.23 1.00 ± 0.41   10 1.21 ± 0.17   1.29 ± 0.26 1.09 ± 0.11 1.40 ± 0.66 1.00 ± 0.26   50 1.81 ± 0.18**   0.60 ± 0.05 1.07 ± 0.04 3.07 ± 0.32*** 1.09 ± 0.22   100 3.34 ± 0.16***   0.49 ± 0.15* 1.42 ± 0.06*** 3.13 ± 0.11*** 0.85 ± 0.06 PC-14 (Adenocarcinoma) DMSO 1.00 ± 0.07 N.D. N.D. 1.00 ± 0.05 1.00 ± 0.05 N.D.   10 1.13 ± 0.12 find more     0.98 ± 0.11 1.29 ± 0.09**     50 1.80 ± 0.08     1.29 ± 0.47 1.39 ± 0.08**     100 4.18 ± 0.21***     1.68 ± 0.24* 1.35 ± 0.09**   A549

(Adenocarcinoma) DMSO 1.00 ± 0.05 N.D. N.D. 1.00 ± 0.12 1.00 ± 0.23 1.00 ± 0.10   10 1.06 ± 0.11     0.89 ± 0.05 1.40 ± 0.66 1.16 ± 0.28   50 1.90 ± 0.32***     1.35 ± 0.42 3.07 ± 0.32*** 1.95 ± 0.44**   100 2.10 ± 0.16***     1.04 ± 0.12 3.13 ± 0.11*** 1.36 ± 0.06 Data were normalized relative to the level of 18S rRNA, and expressed as mean (SD) of 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle control. Figure 1 Effect of TZDs on VEGF-A mRNA expression in lung cancer cell lines. RERF-LC-AI (left panel) and PC-14 (right panel) cells were treated with 0, 10, 50, or 100 μM of Emricasan troglitazone (upper panel) or ciglitazone (lower

panel). The culture medium contained 0.1% DMSO to maintain the same conditions throughout the experiments. After 24 h of treatment, Selleck LY2090314 specific mRNA was quantified using real-time PCR. Data were normalized relative to the level of 18S rRNA, and expressed as mean (SD) (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle control. To clarify the correlation between the interaction of VEGF-A and its receptor

NRP-1, and cell growth inhibition by troglitazone, PC-14 cells were used for the following experiment. Because the expressions of FLT-1 and KDR mRNA were not detected in the PC-14 cells. Western blot analysis showed that VEGF-A protein levels varied with TZD levels in a dose-dependent manner (Figure 2A). The results were consistent with those obtained by RT-PCR analysis. GW9662, a PPARγ antagonist, completely blocked the TZD-induced expression of VEGF-A mRNA through a PPARγ-dependent pathway in the PC-14 cells (Figure 2B). These results indicate that the TZDs–troglitazone and ciglitazone–induce the expression of VEGF-A mRNA and protein and that this induction depends on PPARγ activation. Figure 2 The expression of VEGF-A Dolichyl-phosphate-mannose-protein mannosyltransferase protein and PPARγ dependent pathway. A. PC-14 cells were treated with 0, 10, 50, or 100 μM troglitazone or ciglitazone and 48 h after treatment the expression of VEGF-A protein was measured by western blot analysis. B. PC-14 cells were treated with or without GW9662 (20 μM), a PPARγ inhibitor, for 1 h before they were exposed to troglitazone or ciglitazone (50 μM each). After 24 h of thiazolidinedione treatment, the relative expression of VEGF-A mRNA was evaluated using real-time PCR. Data are expressed as mean (SD) (n = 3).