We found that ciglitazone inhibited PDK1 protein expression in a time and dose dependent manner, with an effective response of 20 uM at 24 h in H1650 cells. Reduction of PDK1 protein expression by ciglitazone was also found in other NSCLC cell lines. We then tested whether the effects of ciglitazone on PDK1 were mediated through the activation of PPAR. We showed that, while ciglitazone increased Dorsomorphin ALK the PPRE luciferase activity. the effects of ciglitazone on PDK1 expression were not eliminated in the presence of GW9662, a specific PPAR antagonist and in cells silencing of PPAR. The result suggests that PPAR independent signals mediate the effect of ciglita zone on PDK1 protein expression. Next, to test whether ciglitazone affects Inhibitors,Modulators,Libraries cell growth through PDK1 mediated signals, we blocked the PDK1 gene using PDK1 siRNA.

We showed that knockdown of PDK1 significantly reduced PDK1 production, while the control siRNA had no effect. Cells exposed to PDK1 siRNA showed a slight reduction in cell proliferation Inhibitors,Modulators,Libraries at baseline. however, they showed significant reduction in growth in the presence of ciglita zone as determined by cell viability assay. Overexpression of PDK1 Inhibitors,Modulators,Libraries has been reported to correlate with tumor progression. We found that overexpression of PDK1 abrogated the effect of ciglitazone on cell growth and caspase 3 7 activity. Transfection with PDK1 expression vector was confirmed by Western blot. Together, this suggested that ciglitazone not only inhibited growth but also increased apoptosis of lung cancer cells through, at least in part, the inhibition of PDK1.

The role of AMPK and SAPK JNK in mediating the effect of ciglitazone on PDK1 protein expression Studies by this group and others also demonstrated Inhibitors,Modulators,Libraries a role for AMPK in mediating the effect of PPAR ligands, such as thiazolinediones compounds, in different cell systems. We showed that ciglitazone increased phosphorylation of AMPK and SAPK JNK with maximal effect observed at 2 4 h in H1650 cells. Inhibitors,Modulators,Libraries Interestingly, the inhibitors of AMPK, compound C, but not of SAPK JNK, SP600125, blocked the inhibitory ef fect of ciglitazone on PDK1 protein expression in both H1650 and H1299 cells. Similarly, silencing of AMPK abrogated the effect of ciglitazone on PDK1 protein. This indicates the specificity of AMPK activation in this process. Interestingly, com bination treatment of ciglitazone and metformin, an ac tivator of AMPK, further reduced the PDK1 protein expression.

Ciglitazone decreases PDK1 promoter activity independent of PPAR activation We also examined if the effects of ciglitazone on PDK1 expression occurred at the transcriptional level. As shown in Figure 4A, the PDK1 gene promoter contains multiple transcription http://www.selleckchem.com/products/Temsirolimus.html factor binding sites including PPRE, Egr 1, nuclear factor ��B and p53, among others.