Discussion In this study, we showed that TZDs increase the mRNA expression of VEGF-A and NRP-1 but not that of FLT-1 and KDR in NSCLC cells. We also showed that GW9662, a PPARγ antagonist, completely reverted the TZD-induced expression of VEGF-A mRNA to the original level and that this was accompanied by the expression of transcriptional factor HIF-1α. VEGF-A expression
has been reported to be regulated by transcription factor HIF-1α [22, 23]. Recently, it has been reported that the transcriptional coactivator PGC-1α regulates VEGF expression by an HIF-1α independent pathway [25]. Our results indicate that troglitazone significantly Kinase Inhibitor Library price enhances VEGF-A expression in a HIF-1α-dependent manner. Western blot analysis showed that the level of VEGF-A proteins also increased in the presence of TZDs. Therefore, we
also studied the effect of the VEGF inhibitor Je-11. Recently, it has been reported that anti-VEGF monoclonal antibodies significantly arrest cell growth in SK-MES-1, a squamous cell carcinoma cell line from the lung [26]. However, an interesting finding of our study was that the inhibition of VEGF by Je-11 partially blocked the troglitazone-induced growth inhibition in NSCLC cells, whereas FLT-1 and KDR are still present albeit in very small amounts. Because NRP-1 binds only to the VEGF-A isoform VEGF165 [22], these results suggest that growth is arrested by the interaction of VEGF165 and NRP-1. In addition, our results showed that troglitazone see more reduces phosphorylated-JNK levels and inhibiting the phosphorylation of JNK is necessary for inducing the expression of VEGF-A mRNA. Similarly, it has been reported that TZD inhibits the proliferation of human NSCLC NCI-H23 cells and that these effects are associated with ERK1/2 activation
and SAPK/JNK deactivation [27]. Although we did not detect ERK1/2 activation, JNK deactivation was observed at 24 h after TZD treatment (Figure 5). These differences might be attributed to the concentration of TZD and the type of cell line. Further, JNK inhibitors upregulated the expression of VEGF mRNA at all time points after treatment, but MEK inhibitor and p38 inhibitor did not affect the expression of VEGF-A mRNA at 24 h after treatment, as compared to the expression old in the vehicle control. Taken together, these results indicate that TZD-induced VEGF-A expression is negatively regulated mainly by the JNK pathway. VEGF is a major angiogenic factor that stimulates the proliferation and migration of endothelial cells. Four VEGF isoforms composed of 121, 165, 189, and 206 amino acids can be synthesized by alternative splicing of VEGF mRNA. The larger isoforms (VEGF189 and VEGF206) are cell-associated and bind to glycosaminoglycans, whereas the smaller isoforms (VEGF121 and VEGF165) are secreted into the extracellular matrix [23].