A Novel Mechanism for UDCA-induced Growth Suppression
ProQuest, 2008 - 192 pages
Bile acids have been studied for many years for their role in either promoting (Deoxycholic Acid) or suppressing (Ursodeoxycholic Acid) colon tumor development in animal models. However, the molecular mechanisms of both DCA's and UDCA's biological effects in colon tumorigenesis is still unclear. The cholesterol-like composition of bile acids and evidence of deregulating signal transduction pathways, such as the p42/44 MAP kinase cascade, led us to identify the plasma membrane as a target for bile acid-mediated effects. Specifically, plasma membrane microdomains such as lipid rafts and caveolae are particularly capable of altering mitogenic signaling due to have their role as platforms to concentrate receptors and assemble signal transduction machinery. In this study I tested the hypothesis that the growth suppressive effects of UDCA are mediated by stimulating membrane microdomains to activate protein degradation machinery to facilitate the down-regulation of receptor tyrosine kinase activity. We found that UDCA suppresses EGF-induced ERK activation, promotes interactions between EGFR and Caveolin-1 membrane fractions, whereas DCA causes redistribution. EGFR proteins that are localized to membrane fractions in the UDCA treated cells are extensively ubiquitinylated and we present evidence that this yields recruitment of the ubiquitin ligase c-Cbl to membrane fractions. UDCA increases the rate of EGFR degradation, whereas DCA sustains its' stability. I present evidence that UDCA's growth inhibitory effects on colon cancer cells may be mediated by recruitment of protein degradation machinery to membrane domains that are enriched with signaling receptors, a mechanism which has not been previously described. Importantly, I demonstrate for the first time a novel mechanism by which UDCA promotes growth inhibition, through increasing the rates of degradation of EGFR, thereby down-regulating mitogenic signaling in the cell. These experiments show exciting insights into the mechanism of bile acids and represent potential mechanisms for other chemopreventive agents.
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LIST OF FIGURES
MATERIALS AND METHODS
EGFR and cav1 121
250 µM UDCA activation antibody apoptosis apoptotic assay bilayer bile acids biological effects cancer cells carcinogen cav-1 and EGFR cav-1 expression caveolae Caveolin Caveolin-1 cell lines cell lysates cells were grown cellular chemopreventive agent cholesterol clathrin clathrin coated pits co-localization colon cancer colon tumors colorectal cancer crypt DCA and UDCA DCA or UDCA degradation dietary down-regulation early endosome effects of UDCA EGFR endocytic endocytosis Figure gene genetic H7 cells HOMUR cells HT29 cells hydrophobic immunoblotted immunoprecipitated increased incubated induced inhibition interactions internalization IPTG late endosomes lipid rafts MAP-K signaling mechanism mediated membrane microdomains mitogenic mitogenic signaling molecular mutations phosphorylation plasma membrane polyps proteins raft fractions regulation resistance response to UDCA role SDS-PAGE shown signal transduction signaling molecules siRNA studies targeted transfected treated with 250 tumor promoting tumorigenesis ubiquitin ligase ubiquitinylated UDCA UDCA overnight UDCA suppresses UDCA treatment UDCA-induced growth suppression UDCA’s untreated vesicle