Molekularbiologische Untersuchungen zum Wirkmechanismus des ppar-gamma Agonisten Piotglitazon auf die humanen Kolonkarzinom-Zelllinien HCT-116 und HT-29

Colorectal cancer is a major global health problem with more than a million new cases diagnosed each year worldwide. The last decade has highlighted new advances in medical treatment of these carcinomas for example the activation of the peroxisome proliferatoractivated receptor γ (PPAR-γ) through specific ligands such as those belonging to the antidiabetic thiazolidinedione (TZD) class of compounds. Recent studies have demonstrated that binding on PPAR-γ leads to either terminal differentiation, inhibition of cell growth and/or apoptosis of a variety of cancers. To determine whether PPAR-γ regulates the cell growth of the colorectal tumor cell lines HCT-116 and HT-29, the expression of PPAR-γ was verified using Western-Blot analysis. The incubation with 40μM pioglitazon resulted in a clear reduction of cell growth in both cell lines. This antiproliferative effect was accompanied by a decrease of LDH – a marker of proceeded cell death - in the supernatants. Both, reduction of cell-proliferation and LDHactivity in the supernantants were antagonized using the selective PPAR-γ antagonist GW9662, indicating that the effect of pioglitazone on HCT-116 and HT-29 cells depended on PPAR-γ activation. These results were confirmed by using flow cytometric analysis which showed only a moderate alteration of apoptotic cells under pioglitazone-treatment whilst the cells in the G0/G1-phase of the cell cycle was markedly increased. This G0/G1- arrest was accompanied by a reduction the protein levels of cdk4, Cyclin D3 and p21cip1/waf1 in Western-Blot analysis. On the other hand pioglitazon had no effect on the protein levels of pro-apoptotic enzymes such as caspase 3, 9, 10 and p53. In order to determine whether PPAR-γ plays any role in TRAIL-induced apoptosis of HCT- 116 and HT-29 cells, the cells were pretreated with 40μM pioglitazone for either one or two days and afterwards their sensitivity to TRAIL-induced apoptosis was examined. TRAIL alone induced moderate apoptosis of these cells. The pretreatmen of HT-29 cells with pioglitazone increased their sensitivity to TRAIL-induced apoptosis while in HCT-116 cells such a synergism could not be found. Interestingly, Western-Blot analysis showed a reduction of the protein level of the programmed cell death 4 protein (pdcd4). Pdcd4 is a tumorsupressor protein that interacts with eIF4A thereby inhibiting translation. This report demonstrates for the first time an interaction between the PPAR-γ signaling pathway and the expression of pdcd4. To investigate if this effect can also be found in other tumors, Jurkat leukemia cells were incubated with 40μM pioglitazone and the protein level of pdcd4 was measured by Western-Blot analysis showing a reduction of the pdcd4 expression as well. The stable transfection of HCT-116 cells with siRNA against pdcd4 showed marginal reduction of cell proliferation which led to the assumption that a minimal pdcd4 level is essential for cell proliferation. Two-dimensional gelelectrophoresis of HCT-116-protein extracts were performed showing a hyperphosphorylation of cytokeratin 19. The primary function of keratins is to protect epithelial cell from mechanical and chemical stresses and provide cell integrity. Phosphorylation is an important regulatory modification of keratins and improves their solubility. Hence, hyperphosphorylation plays a crucial role in filament organisation and reorganisation during mitosis and apoptosis. This report is the first to show that the treatment of cells with the PPAR-γ ligand pioglitazone leads to a hyperphosphorylation of cytokeratin 19. In summary, this study shows that pioglitazone exerts its effect on HCT-116 cells via down-regulation of cdk4, Cyclin D3 and p21cip1/waf1 leading to a G0/G1-arrest which depended on PPAR-γ. Confronted with the need to develop further strategies for the treatment of cancer, PPAR-γ agonists like TZDs or even more potent drugs seem to be a promising tool in anti-neoplastic therapy.