Given the fact that silencing of the PPAR�� gene by siRNA had no effect on blockage of the effect of ciglitazone on PDK1 promoter activity, additional e periments are required to e plore the contribu tions of PPAR�� independent mechanisms in these processes. Interestingly, PDK1 knockdown alone did not affect cell proliferation significantly. However, inhibition of PDK1 in the setting of ciglitazone treatment resulted in largely growth inhibition. This suggests that other factors are important for control of NSCLC cell proliferation. It also suggests that the growth inhibitory effects of cigli tazone may occur by concomitant actions on pathways other then PDK1. Report shown that ciglitazone e erts effects on several other targets that were implicated in control of lung cancer growth.

In this study, we showed that activation of AMPK played a vital role in mediating the effect of ciglitazone on PDK1 e pression. In addition, activation of AMPK enhanced the effect of ciglitazone on PDK1 e pression and promoter activity. Data demonstrated that synthetic PPAR�� ligands regulated several kinase signaling pathways including AMPK in different cells. Activation or inactivation of AMPK has been shown to link synthetic PPAR�� agonists mediated signaling to the transcriptional regulation of genes that are crucial for cell growth inhib ition. Considering the recent data for the dual role of AMPK, we believed that more dedicated studies are required to further elucidate the biological function and relevant signaling of this kinase.

Having demonstrated the important role of PDK1, we further investigated whether the ciglitazone mediated downregulation of PDK1 reflected inhibition of trans activation of the PDK1 gene. Our results suggested that increased Egr 1 protein e pression and binding to the upstream areas of PDK1 gene promoter played an im portant role in mediating the effect of ciglitazone. Knockdown of Egr 1 abrogated the effect of ciglitazone on PDK1 e pression and on cell proliferation, whereas overe pression of Egr 1 had no further effect of ciglita zone on PDK1 promoter activity confirming the inhibitory property of this transcription factor. It also suggested the specificity of Egr 1 played in this process. To our know ledge, the role of Egr 1 in regulation of PDK1 e pression has never been reported. Egr 1 functions as a tumor sup pressor in many cancers.

Loss of Egr 1 e pression has been associated with invasion and anti apoptotic events, whereas overe pression of Egr 1 suppressed the tumorigenicity and metastatic potential in several cancer cells including lung. However, opposite role of Egr 1 were also found in several studies. Thus AV-951 Egr 1 is considered to play dual roles depending on the cell types and environment. One study showed that sev eral PPAR�� ligands including TZD induced the e pres sion of Egr 1 through PPAR�� independent pathway in breast cancer cells. Thus, other factors responsible for this effect need further e plor ation.