Investigation of the antiproliferative mechanisms of the chemopreventive agent ursodeoxycholic acid (UDCA) in vitro
Year: 2011
Type: dissertation
Abstract: Introduction: 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 carcinoma 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, CDK2, CDK6 and Rb, upregulated p21 and induced Rb hypophosporylation. UDCA decreased E2F-1 transcriptional activity and inhibited PCNA binding to chromatine. The G1-->S transition delay by UDCA was concomitant with effect on c-Myc, p21, and cyclin A expression and Rb phosphorylation. c-Myc suppression was sufficient to inhibit proliferation, induce p21 upregulation, CDK2 and CDK6 downregulation and Rb hypophosphorylation. c-Myc downregulation was not transcriptional and it was concomitant with increase in phosphorylation at threonine 58 and a persistant ERK phosphorylation. Cells overexpressing c-Myc did not proliferate and the wnt pathway deficiency did not affect the inhibition of proliferation by UDCA. Conclusion: UDCA decreased the proliferation of colon cancer cells by delaying the G1-->S transition and slowing down the cell cycle. Suppression of c-Myc contributed to the inhibition of proliferation. Together with p21 overexpression, Rb hypophosphorylation and cyclin A downregulation, it could play a role in the delay of the G1-->S transition and inhibition of proliferation.
Author: Peiró Jordán, Roser
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1810822577