| Apoptosis is the tightly regulated form of cell death and is critical to embryogenesis, development and cellular homeostasis. Inhibition of apoptosis is one mechanism by which abnormal cells accumulate, leading to tumorigenesis. Therefore, triggering apoptosis in cancer cells represents a promising strategy for anticancer therapy. This dissertation research focuses on inducing apoptosis and understanding its underlying molecular mechanisms in human cholangiocarcinoma, a malignancy of the bile duct. These studies are specifically focused on apoptosis regulated by the death receptor Fas, calcium/calmodulin (Ca2+/CaM) and interferon-gamma (IFN-γ).; The data presented here demonstrate that IFN-γ enhances Fas-mediated apoptosis in cholangiocarcinoma cells by up-regulating many apoptosis-related molecules, including Fas; caspases-3, -4, -7 and -8; and Bak. Pretreatment with IFN-γ facilitates Fas-mediated cleavage of caspases, cytochrome c release and Bax translocation. This process is associated with the ability of IFN-γ to inhibit tumorigenesis in nude mice. IFN-γ also renders cholangiocarcinoma cells sensitive to calmodulin (CaM) antagonists, tamoxifen, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide. During CaM antagonist-mediated apoptosis in IFN-γ-pretreated cells, there occur cleavage of caspases-8, -9 and -3 and Bid; release of cytochrome c from the mitochondria; depolarization of the mitochondrial membrane potential; and an increase in intracellular free calcium concentration. These events also occur in Fas-mediated apoptosis.; Finally, to determine the mechanism by which CaM antagonists induce apoptosis, the interaction of CaM with Fas was investigated. CaM binds to Fas directly, and the CaM-binding motif in the Fas death domain has been identified. Selected point mutations in the CaM-binding motif cause reduced Fas-CaM binding in vitro. These data support a novel function of CaM in Fas-mediated apoptosis.; Taken together, the results of this dissertation research show that Fas- and Ca2+/CaM-mediated apoptotic pathways can be regulated by IFN-γ. The combination of IFN-γ and death receptor- or CaM-targeted strategies may present novel treatment opportunities for cholangiocarcinoma and possibly other malignancies. |
