Statistical analysis Data are expressed as mean (SD). Statistical analysis was performed either by one-way analysis of variance and subsequent Tukey multiple comparison procedure, or by two-way analysis of variance with subsequent Bonferroni post-test; all

Selleck Gefitinib of these were performed using the GraphPad Prism Software (version 4). P < 0.05 was considered statistically significant. Results First, we determined whether troglitazone affects the expression of VEGF-A and its receptors, fms-like tyrosine kinase (FLT-1/VEGFR1), kinase insert domain receptor 1 (KDR/VEGFR2), and neuropilin-1 (NRP-1) in the human lung cancer cell lines, RERF-LC-AI, SK-MES-1, PC-14, and A549 (Table 1). In these cell lines, we found that troglitazone had a dose-dependent effect on the expression of VEGF-A mRNA. To further prove that troglitazone YAP-TEAD Inhibitor 1 in vivo enhances VEGF-A expression in lung cancer cells, we studied the effects of ciglitazone on the expression of VEGF-A mRNA in the RERF-LC-AI and PC-14 cells. Ciglitazone enhanced the expression of

VEGF-A mRNA in both cell lines; however, it was less effective than troglitazone (Figure 1). The mRNA expression of its receptors, KDR and FLT-1, was hardly affected; however, mRNA expression of NRP-1, which is thought to be a receptor of the VEGF-A splicing variant VEGF165 [21], was affected in a dose-dependent manner. In addition, the level of FLT-1 and KDR mRNA expression in the all cell lines were extremely low (threshold cycle values of these mRNAs were around 34-37 cycles; data not next shown), or not detected (N.D.). We also investigated the mRNA expression of transcription factor HIF-1α, a known regulating factor of VEGF-A [22, 23], and

transcriptional coactivator PGC-1α (Table 1). Our results indicate that troglitazone significantly enhanced HIF-1α expression in the RERF-LC-AI, SK-MES-1, and PC-14 cells (Table 1). On the other hand, the expressions of PGC-1α mRNA in the RERF-LC-AI and SK-MES-1 cells were not affected by troglitazone, and PGC-1α mRNA in the PC-14 cells was not detected. These results indicate that, in NSCLC, troglitazone enhances VEGF-A mRNA expression by increasing HIF-1α expression, and that the VEGF-A receptor is mainly NRP-1. We hypothesize that the interactions of VEGF-A and NRP-1 directly affect cell growth, because the arrest of cell growth by TZDs has been widely reported. Table 1 Relative mRNA expression levels of VEGF-A, its receptors, transcription factor HIF-1α, and transcriptional coactivator PGC-1α. Troglitazone (μM) VEGF-A FLT-1 KDR NRP-1 HIF-1α PGC-1α RERF-LC-AI (Squamous cell carcinoma) DMSO 1.00 ± 0.28 1.00 ± 0.13 N.D. 1.00 ± 0.03 1.00 ± 0.16 1.00 ± 0.20   10 1.14 ± 0.08 1.08 ± 0.43   1.00 ± 0.18 1.24 ± 0.31 0.95 ± 0.20   50 1.39 ± 0.42 0.97 ± 0.48   1.03 ± 0.45 1.27 ± 0.23 0.82 ± 0.05   100 4.26 ± 0.74 ** 1.23 ± 0.18   5.79 ± 0.48*** 1.35 ± 0.26 0.92 ± 0.