u-0126 and ciglitazone

In this study, we examined whether the peroxisome proliferator-activated receptor γ (PPARγ) agonists, ciglitazone (CGZ) and troglitazone (TGZ), induce cell death in human cervical cancer HeLa cells. The cells were treated with a range of CGZ or TGZ doses for 24 or 48 h. Low concentrations of CGZ (≤10 μM) or TGZ (≤20 μM) had no effect on cell viability whereas higher doses induced cell death in a time- and dose-dependent manner as evidenced by the detection of activated caspase-3 and PARP cleavage. Treatment with the PPARγ antagonist GW9662 followed by PPARγ agonists did not increase CGZ- or TGZ-induced cell death, indicating that PPARγ agonists induced HeLa cell death independently of PPARγ. Moreover, ERK1/2 activation was observed at a CGZ concentration of 25 μM and a TGZ concentration of 35 μM, both of which induced cell death. To elucidate the role of ERK1/2 activated by the two PPARγ agonists, the effect of U0126, an inhibitor of ERK1/2, on PPARγ-agonist-induced cell death was examined. Treatment with 10 or 20 μM U0126 followed by CGZ or TGZ induced the down-regulation of ERK1/2 activity and a decrease in Bcl-2 expression accompanied by the collapse of mitochondrial membrane potential, which in turn significantly enhanced CGZ- or TGZ-induced apoptotic cell death. Our results suggest that PPARγ agonists are capable of inducing apoptotic cell death in HeLa cells independently of PPARγ and that inhibition of ERK1/2 activity offers a strategy to enhance the cytotoxicity of PPARγ agonists in the treatment of cervical cancer.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Caspase 3; Cell Survival; Cervix Uteri; Chromans; Drug Synergism; Enzyme Inhibitors; Female; HeLa Cells; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; PPAR gamma; Thiazolidinediones; Troglitazone; Uterine Cervical Neoplasms

We have previously demonstrated that the PPARgamma ligand, ciglitazone, increases p27kip1 protein levels in HT-29 colon cancer cells through both inhibition of proteasome associated degradation and activation of transcriptional activity. [F. Chen, L.E. Harrison, Cell Signal. 17 (2005) 809] The purpose of this investigation was to further elucidate the mechanism of ciglitazone-induced activation of p27 gene transcription. We observed that the region -774/-462 of the p27 promoter plays a key role in ciglitazone-induced gene transcriptional activity and this region contains two Sp1 binding sites. When the p27PF-luc reporter was co-transfected with Sp1 expression plasmids, ciglitazone-induced p27PF-luc activity significantly increased, while mithramycin A, a Sp1 inhibitor, was able to abrogate its effects. Ciglitazone exposure increased both Sp1 protein expression and Sp1-DNA binding, which was also associated with a decrease of Erk1/2 phosphorylation. A similar increase of Sp1-DNA binding was observed when phosphorylation of Erk1/2 was inhibited by pretreatment with the MAP kinase inhibitor, U0126. In addition, a significant increase of p27PF-luc reporter luciferase activity was noted after MAP kinase inhibition, which could be abolished with co-treatment with mithramycin A. Based on these data, we postulate that ciglitazone induces p27 gene transcription through increased Sp1 binding to its promoter region, which in turn is mediated through increased Sp1 protein levels and decreased inhibitory regulation by the MAP kinase pathway.

Topics: Binding Sites; Butadienes; Cyclin-Dependent Kinase Inhibitor p27; Flavonoids; Gene Expression Regulation; HT29 Cells; Humans; Luciferases; MAP Kinase Signaling System; Nitriles; Phosphorylation; Plicamycin; Promoter Regions, Genetic; Sp1 Transcription Factor; Thiazolidinediones; Time Factors; Transcription, Genetic; Transfection