Investigation of the antiproliferative mechanisms of the chemopreventive agent ursodeoxycholic acid (UDCA) in vitro [thesis]

Roser Peiró Jordán, Universitätsbibliothek Der FU Berlin, Universitätsbibliothek Der FU Berlin
Colorectal cancer is the second most common cancer and the third most common cause of cancer death in the western world. Ursodeoxycholic acid (UDCA), a bile acid used in the treatment of primary biliary cirrhosis, has been shown to prevent colon cancer in animal models. The mechanism of the chemopreventive action of UDCA is not understood. Objectives: The objective of this project was to investigate the antiproliferative mechanisms of UDCA. Materials and Methods: Four established p53wt human
more » ... cinoma cell lines were treated with UDCA (0-400 µM) for 3 days and proliferation was investigated by cell count, MTT test and BrdU incorporation. Apoptosis was determined by DAPI staining and detection of PARP cleavage by western blot. Senescence was determined by β-galactosidase staining. The cell cycle was studied by FACS using nocodazole-synchronized or non-synchronized cells. Expression of c-Myc and other cell cycle markers were analysed by western blot. Effect of UDCA on PCNA binding to chromatine was detected by inmunohistochemistry and E2F-1 transcriptional activity was investigated by luciferase reporter assay. c-Myc was suppressed by transfection by pSuper-c-Myc plasmid. Overexpression of c-Myc was induced by transient transfection of pCMV-c-Myc plasmid. Transcriptional regulation of c-Myc was investigated by luciferase reporter assay and OneStep RT-PCR. The influence on the wnt pathway was tested in an isogenic pair of wnt-proficient and wnt-deficient cell lines. Results: UDCA inhibited the growth of colon cancer cells in a partially reversible manner, the extent of inhibition was independent from the speed of growth of the cell line. UDCA did not induce apoptosis and induced senescence only in LS513 cells. S-phase population was decreased after treatment in all cell lines investigated, in HCT116 and HCT8 cells UDCA slowed down the cell cycle and induced G1-->S transition delay, and in LS513 it induced increase in G2/M population and S-phase arrest. UDCA treatment downregulated c-Myc, cyclin A, C [...]
doi:10.17169/refubium-14852 fatcat:lofff7k3wrhi5hp5senv7w4d4q