The stained cells were analyzed by fluorescence-activated cell sorting (FACS, BD Biosciences, San Jose, CA, USA). Tumor xenograft model SW480 cells (9??106 cells/mouse) were suspended in Matrigel? (Corning, Tewksbury, MA, USA) and inoculated subcutaneously in the right flank of 5-week old female BALB/c nu/nu mice (Nara Biotech, Seoul, Republic of Korea). This is the first report on the lysosomal degradation of FoxM1 by a small molecule. DFS may be useful in treating cancers that feature the elevated expression of FoxM1. The Wnt/-catenin signaling pathway plays a primary role in cellular differentiation and proliferation. Beta-catenin forms a complex with APC/Axin/GSK3 and is degraded by the proteasome under Wnt-free conditions. However, the Wnt/-catenin pathway is constitutively activated in most sporadic and hereditary colorectal tumors caused by mutations in Wnt/-catenin pathway-related molecules, such as adenomatous polyposis coli (APC) and -catenin1. Aberrantly activated -catenin increases nuclear translocation of other oncogenes2,3 and binds to T-cell factor/lymphoid enhancer factor transcription factors to promote expression of target genes, such as cyclin D1, survivin, and c-Myc, which play key roles in cellular differentiation and proliferation4,5. Thus, aberrantly activated Wnt/-catenin signaling is regarded as a Dexamethasone target for the chemoprevention and treatment of colorectal cancer. FoxM1 is a member of the Forkhead box transcription factor family. The varied biological activities of FoxM1, include regulation of cellular proliferation, DNA damage repair, angiogenesis, apoptosis, and tumorigenesis6. From the early stage of tumor development to later metastasis, FoxM1 expression is highly elevated in a variety of cancers6,7. Elevation in FoxM1 levels promotes cancer initiation and maintenance through regulation of the progression of cancer cell cycle and proliferation6,7. For example, elevation in FoxM1 levels promotes development and proliferation of colon adenocarcinomas and depletion of FoxM1 reduces colon cancer cell growth (Betulaceae) grows in the low mountainous areas of Korea, northeast China, and Japan. It has been used in traditional oriental medicine to treat fever, hemorrhage, diarrhea, and alcoholism. Recent studies have shown that has various phytochemicals, such as diarylheptanoids, triterpenoids, and flavonoids9,10,11,12,13,14. In this study, we isolated a lignan [(?)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol, DFS] from and explored its activity against colon cancer. DFS was first reported by Nomura and Tokoroyama15 and its cytotoxic action against several cancer cell types has been described16,17,18. Presently, we describe the ability of DFS to block -catenin nuclear translocation through the lysosomal-dependent degradation of FoxM1 protein. Results DFS suppresses the -catenin pathway TOPFlash and FOPFlash reporter cell lines were Dexamethasone used to test the effects of DFS (Fig. 1A) on the Wnt/-catenin pathway. Treatment with Wnt3a-conditioned media (CM) significantly increased TOPFlash activity, and treatment with DFS suppressed Wnt3a-induced TOPFlash activity in a dose-dependent manner (Fig. 1B). To test whether GSK-3 is involved in the inhibition of -catenin transcription, we treated HEK293 cells with LiCl as an inhibitor of GSK-314. DFS suppressed LiCl-induced TOPFlash activity in a dose-dependent manner (Fig. 1C). These data indicate that DFS suppresses the -catenin pathway in a GSK-3-independent manner. Open in a separate window Figure 1 DFS suppresses the -catenin signaling pathway.The structure of DFS (A). TOPFlash or FOPFlash reporter expressed HEK293 cells were treated with the indicated concentrations of DFS in the presence of Wnt3a (B) or LiCl (C) for 16?h and TOPFlash activity was measured. SW480 and HCT116 colon cancer cells were transiently transfected with the TOPFlash plasmid and treated with the indicated concentrations of DFS for 16?h, TOPFlash activity was measured (D). Next, we tested the ability of Dexamethasone DFS to suppress the Wnt/-catenin pathway Dexamethasone in colon cancer cells. SW480 and HCT116 cells (adenomatous polyposis [APC] mutated or -catenin mutated, respectively) were transiently transfected Rabbit polyclonal to PCBP1 with the TOPFlash plasmid and treated with DFS to assess luciferase activity. DFS significantly suppressed TOPFlash activity.