Limonoids with Wnt signal inhibitory activity isolated from the fruits of Azadirachta excelsa Rolly G. Fuentes a,d , Kazufumi Toume a , Midori A. Arai a , Samir K. Sadhu b , Firoj Ahmed c , Masami Ishibashi a, * a Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan b Pharmacy Discipline, Life Science School, Khulna University, Khulna 9208, Bangladesh c Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka 1000, Bangladesh d University of the Philippines Visayas Tacloban College, Tacloban City 6500, Philippines 1. Introduction The Wnt signaling pathway is involved in various embryonic developmental and cellular processes (Clevers and Nusse, 2012). In the absence of the Wnt protein, b-catenin levels in normal cells are regulated by the action of a destruction complex consisting of glycogen synthase kinase 3b (GSK3b), casein kinase 1a (CK1a), and adenomatous polyposis coli (APC). GSK3b and CK1a phosphorylate b-catenin (Liu et al., 2002) which is then degraded via the ubiquitin-proteasome pathway (Aberle et al., 1997). However, inactivation of this destruction complex due to either stimulation of the Wnt protein or a mutation in the components of the destruction complex has been shown to result in the stabilization and accumulation of b-catenin (Li et al., 2012). Accumulated b-catenin enters the nucleus and forms a complex with TCF and other co-activators, such as Bcl9, pygopus, and CREBB-binding protein (CBP), to activate the transcription of important proliferation genes (Clevers and Nusse, 2012). However, the deregulated activation of the Wnt signal has been associated with several human diseases such as cancer (Clevers and Nusse, 2012). Most colorectal cancer cells have truncated adenomatous polyposis coli (APC) or mutated b-catenin, which stabilizes b- catenin and activates the Wnt signal (Morin et al., 1997). Thus, inhibiting the accumulation of b-catenin accumulation or forma- tion of the TCF/b-catenin complex could represent a good strategy for preventing these diseases. Several compounds had been shown to inhibit the Wnt signal, some of which were derived from natural resources. The flavonoid quercetin (Park et al., 2005), phenolic compound curcumin (Ryu et al., 2008), and carbazole alkaloid murrayafoline A (Choi et al., 2010) are some of the plant-derived compounds that inhibit the Wnt signal. Our group also had previously described a naturally derived compound that inhibited the Wnt signal and also exhibited cytotoxicity against Wnt-dependent cancer cells (Park et al., 2014; Toume et al., 2013). In our continuous work to search for Wnt signal inhibitors from natural resources, we identified Azadirachta excelsa as a potential source from our screening study. A. excelsa belongs to the family Meliaceae, which is known to be a source of limonoid-type compounds. Meliaceous limonoids have been shown to possess antineoplastic, antimicrobial, and antiprotozoal Phytochemistry Letters 11 (2015) 280–285 A R T I C L E I N F O Article history: Received 19 November 2014 Received in revised form 15 January 2015 Accepted 20 January 2015 Available online 31 January 2015 Keywords: b-Catenin Limonoids Adenomatous polyposis coli Colorectal cancer A B S T R A C T The Wnt signal regulates various biological processes, and its aberrant activation is associated with the development of diseases. Thus, inhibiting the Wnt signal provides a promising strategy to treat these diseases. Our cell-based luciferase assay system, which targets the Wnt signal (TOP assay), revealed that Azadirachta excelsa inhibited the Wnt signal. The activity-guided isolation of the MeOH fruit extract of A. excelsa provided one new (1) and seven known (2–8) limonoids. Their structures were elucidated based on their spectroscopic data, and their NMR data were compared with those in the literature. Compounds 3–6 potently inhibited the Wnt signal with IC 50 values of 127 nM, 300 nM, 252 nM, and 121 nM, respectively. Compound 4 exhibited selective cytotoxicity against AGS and HCT116. Western blot analysis showed that 4 did not affect the level or localization of b-catenin, but downregulated the level of c-myc. Our results suggested that 4 may have inhibited the Wnt signal by affecting the components downstream of b-catenin. ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +81 43 226 2923; fax: +81 43 226 2923. E-mail address: mish@chiba-u.jp (M. Ishibashi). Contents lists available at ScienceDirect Phytochemistry Letters jo u rn al h om ep ag e: ww w.els evier.c o m/lo c ate/p hyt ol http://dx.doi.org/10.1016/j.phytol.2015.01.015 1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.