There is a worldwide and steady increase in the incidence of cholangiocarcinoma, a malignancy with very limited treatment options and high mortality. This expecially involves racial groups where the prevalence of biliary stones are high - such as Native Americans, South American Indians, andAsians. Of all the etiologic factors, cancers of the gallbladder and the biliary tree are most highly correlated with the presence of biliary stones, and chronic bacterial infection. We have recently discovered that in patients with gallbladder and intrahepatic stones, which are almost always covered by a bacterial biofilm, that there are oxidzed products of cholesterol. Two of these oxysterol species have been unequivocally identified. The presence and abundance of oxysterols in human bile and gallstones correlated with the presence of bacterial infection, and with the amount of bacterial DMA present in the stones. Oxysterols, on short term exposure to biliary epithelial cells, exhibit profound effects on apoptosis, cell proliferation, mucin synthesis and secretion. With long term exposure, dog gallbladder epithelial exhibited features characteristic of malignant transformation. We hypothesize that biliary oxysterols originate from endogenous biliary cholesterol as a result of oxidation from leukocytic enzymes in bacterial-leukocyte interactions. In addition, oxysterols mediate inflammatory and malignant transformation of biliary epithelial cells. We also hypothesizethat the molecular mechanism of oxysterol induced carcinogenesis to mediate through lipid microdomains (rafts).
The Specific Aims Tof this proposal are: 1) To determine the origin of biliary'bxysJerol.'We propose in vitro ' experiments using human leukocytes and model bile, as well,as in vivo experiments using a rabbit model with controlled induction of bacterial infection. The1 kinetics ofioxysterol production, the intermediates and the products wilhbe determined 2a) To construct a1 molecular library of;the;composition and structural identity of the oxidative products of cholesterol in hurpan bile'using high perforirfancdjiiiquidilchromatography (HPLC), interfacing,,with '''ij':^ j mass spectometry (MS) and, IMS/MS. 2$),To characterize'ithe'physicpchemical and biological function of'these;|y', | oxysterols. 3) To use oxysterol in long-term co-cultur;e with normal human biliary (gallbladder) epithelial cells to'''/1 induce carciongenesis. Cell morphology/cancerjgrqwth pathways wiil.'be determined before,'du'ringiand after V cancer formation., _ __, 'jfv t '?, V'1''li'i !!''JU'H . . '?!;! :!''<'.! i""""""""V ? ,'i1. ?.! this proposal uses a multidisciplinary approach including structural biochemistry, cell and molecular biology, and physical chemistry methodologies. It promises to open up a new area of biliary physiology and metabolism; create a molecular library for cholesterol oxidation products in humans;add new and important understanding and insight into the etiology of cholangiocarcinoma;and guide strategic treatment and prevention of this important and vexinq disease.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Hepatobiliary Pathophysiology Study Section (HBPP)
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Knowlton, John R
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Seattle Institute for Biomedical/Clinical Research
United States
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Jusakul, Apinya; Loilome, Watcharin; Namwat, Nisana et al. (2013) Anti-apoptotic phenotypes of cholestan-3*,5*,6*-triol-resistant human cholangiocytes: characteristics contributing to the genesis of cholangiocarcinoma. J Steroid Biochem Mol Biol 138:368-75
Jusakul, Apinya; Loilome, Watcharin; Namwat, Nisana et al. (2012) Liver fluke-induced hepatic oxysterols stimulate DNA damage and apoptosis in cultured human cholangiocytes. Mutat Res 731:48-57
Kuver, Rahul (2012) Mechanisms of oxysterol-induced disease: insights from the biliary system. Clin Lipidol 7:537-548
Jusakul, Apinya; Khuntikeo, Narong; Haigh, W Geoffrey et al. (2012) Identification of biliary bile acids in patients with benign biliary diseases, hepatocellular carcinoma and cholangiocarcinoma. Asian Pac J Cancer Prev 13 Suppl:77-82
Kuver, Rahul (2011) Effects of statins on cholestasis: good, bad or indifferent? J Gastroenterol Hepatol 26:1467-9
Van Rooyen, Derrick M; Larter, Claire Z; Haigh, W Geoffrey et al. (2011) Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis. Gastroenterology 141:1393-403, 1403.e1-5
Jusakul, Apinya; Yongvanit, Puangrat; Loilome, Watcharin et al. (2011) Mechanisms of oxysterol-induced carcinogenesis. Lipids Health Dis 10:44
Kuver, Rahul (2010) Bioactive sphingolipids in the biliary tract: relevance for cholesterol gallstone disease. J Gastroenterol Hepatol 25:1020-3
Haigh, W Geoffrey; Wong, Thomas; Lee, Sum P (2006) The production of oxysterols in bile by activated human leukocytes. Biochem Biophys Res Commun 343:467-9