Cyclooxygenase-2–Derived Prostaglandin E2 Activates B-Catenin in Human Cholangiocarcinoma Cells: Evidence for Inhibition of These Signaling Pathways by W3 Polyunsaturated Fatty Acids

Cholangiocarcinoma is a highly malignant neoplasm of the biliary tree. It has a high rate of mortality, and currently, there is no effective chemoprevention and treatment. This study was designed to investigate the potential effect of W3 polyunsaturated fatty acids (W3-PUFA) on human cholangiocarcinoma cell growth and to determine their mechanisms of actions. Treatment of three human cholangiocarcinoma cells (CCLP1, HuCCT1, SG231) with two W3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for 12 to 72 h resulted in a doseand time-dependent inhibition of cell growth; in contrast, arachidonic acid, a W6-PUFA, had no significant effect. The W3-PUFA effect is due to the induction of apoptosis, given that DHA induced the cleaved form of PARP, caspase-3, and caspase-9. DHA and EPA treatment caused dephosphorylation (and hence, the activation) of glycogen synthase kinase-3B (GSK-3B) with a decline of B-catenin protein. Accordingly, DHA treatment also decreased the B-catenin–mediated T cell factor/lymphoid enhancer factor (TCF/LEF) reporter activity, and inhibited the expression of c-Met, a B-catenin–controlled downstream gene implicated in cholangiocarcinogenesis. The GSK-3B inhibitor, SB216763, partially prevented DHA-induced reduction of B-catenin protein and TCF/LEF reporter activity, and restored cell growth, suggesting the involvement of GSK-3B dephosphorylation in W3-PUFA–induced B-catenin degradation. In parallel, DHA treatment also induced the formation of the B-catenin/Axin/GSK-3B binding complex, further leading to B-catenin degradation. Moreover, DHA inhibited the expression of cyclooxygenase-2 (COX-2) and enhanced the expression of 15-hydroxyprostaglandin dehydrogenase, a physiologic COX-2 antagonist, in human cholangiocarcinoma cells. These findings suggest that W3-PUFAs block cholangiocarcinoma cell growth at least in part through inhibition of Wnt/B-catenin and COX-2 signaling pathways. Thus, utilization of W3-PUFAs may represent an effective and safe therapeutic approach for the chemoprevention and treatment of human cholangiocarcinoma. [Cancer Res 2008;68(2):553–60] Introduction Cholangiocarcinoma is a malignant epithelial neoplasm of the biliary tree with a high rate of mortality (1–6). Early diagnosis of cholangiocarcinoma is difficult, and currently, there is no effective chemoprevention or treatment. The tumor often arises from background conditions that cause long-standing inflammation, injury, and reparative biliary epithelial cell proliferation, such as primary sclerosing cholangitis, clonorchiasis, hepatolithiasis, or complicated fibropolycystic diseases (1–6). Consistent with the strong association between bile duct chronic inflammation and cholangiocarcinoma, recent studies have documented an important role of cyclooxygenase-2 (COX-2)–derived prostaglandin E2 (PGE2), a potent lipid inflammatory mediator, in cholangiocarcinogenesis (1, 2, 4, 6). For example, increased COX-2 expression has been documented in cholangiocarcinoma cells and precancerous bile duct lesions but not in normal bile duct epithelial cells (BEC; refs. 7–9). Overexpression of COX-2 in cultured human cholangiocarcinoma cells enhances PGE2 production and promotes tumor growth, whereas depletion of COX-2 attenuates growth (10, 11). Treatment of cholangiocarcinoma cells with exogenous PGE2 increases tumor cell growth and prevents apoptosis (10–15). Consistent with these findings, selective COX-2 inhibitors prevent cholangiocarcinoma cell growth and invasion, in vitro and in nude mice (4, 10, 11, 14–16), although their effect may be mediated through COX-2–dependent and -independent mechanisms. Recent evidence suggests that alteration of other growthregulatory molecules such as Wnt/h-catenin pathway is also implicated in cholangiocarcinogenesis (17–20). h-Catenin is a key mediator in Wnt regulation of multiple cellular functions in embryogenesis and tumorigenesis (21–24). In adult tissues, h-catenin is a component of stable cell adherent complexes whereas its free form functions as a coactivator for a family of transcription factors termed T cell factor/lymphoid enhancer factor (TCF/LEF). Wnt proteins comprise a family of highly conserved secreted proteins that signal through the Frizzled receptors (21–24). In the absence of a Wnt signal, h-catenin exists within a cytoplasmic complex (h-catenin destruction complex) along with glycogen synthase kinase-3h (GSK-3h), adenomatous polyposis coli, and Axin, where it is phosphorylated and targeted for degradation by the proteasome. Activation of Wnt signaling perturbs this destruction complex, leading to cytoplasmic accumulation of h-catenin and allowing its translocation into the cell nucleus. In the nucleus, h-catenin associates with TCF/LEF that stimulate the transcription of target genes important for proliferation, differentiation, and apoptosis (21–24). Recent studies have shown that accumulation of nuclear h-catenin is induced by PGE2, in addition to the canonical Wnt/Frizzled signaling, in human colon cancer cells. Castellone et al. reported that PGE2 activates its Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Tong Wu, Department of Pathology, University of Pittsburgh School of Medicine, MUH E-740, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: 412-647-9504; Fax: 412-647-5237; E-mail: [email protected] or Kyu Lim, Department of Biochemistry, College of Medicine, Chungnam National University, Korea. Phone: 42-580-8223; Fax: 42-580-8121; E-mail: [email protected]. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-2295 www.aacrjournals.org 553 Cancer Res 2008; 68: (2). January 15, 2008 Research Article American Association for Cancer Research Copyright © 2008 on February 21, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-07-2295