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cGMP signalling
Food Chemistry 129 (2011) 1559–1566
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a- and c-Mangostin inhibit the proliferation of colon cancer cells via b-catenin gene regulation in Wnt/cGMP signalling Ji-Hye Yoo a, Kyungsu Kang a, Eun Hye Jho a, Young-Won Chin b, Jinwoong Kim c, Chu Won Nho a,⇑ a
Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 210-340, Republic of Korea College of Pharmacy, Dongguk University-Seoul, Seoul 100-715, Republic of Korea c College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 23 February 2011 Received in revised form 31 May 2011 Accepted 5 June 2011 Available online 12 June 2011 Keywords: Mangostins b-Catenin Transcriptional regulation cGMP PKG
a b s t r a c t Aberrant activation of Wnt/b-catenin signalling via genetic errors within b-catenin or APC has a crucial role in carcinogenesis. Here, we studied two xanthones, a- and c-mangostin, as inhibitors of Wnt/b-catenin signalling. The mangostins inhibited TCF/b-catenin transcriptional activity. The mangostins also inhibited protein expression of b-catenin in colon cancer cells, but the inhibition was independent of the phosphorylation and degradation of b-catenin. Instead, the mangostins increased the levels of cGMP and cGMP-dependent kinase, indicating that the inhibition of b-catenin resulted from b-catenin gene regulation. Our results indicate that mangostins could be potential candidates for preventing colon cancer through a novel mechanism of inhibiting Wnt/b-catenin signalling. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Colorectal cancer is the third most common cancer among women and men worldwide, with 1.2 million cases and 630,000 deaths in 2007 (Global Cancer Facts, 2007; Jemal et al., 2008). Because the progression from normal epithelium to invasive cancer takes approximately 10–15 years, early diagnosis and treatment of colorectal cancer are important to prevent the development of malignant tumours. Colorectal cancer arises as a result of an accumulation of genetic errors that affect cell proliferation and survival (Davies, Miller, & Coleman, 2005; Miyaki et al., 1994; Watson, 2006). Among several genetic defects, mutation of APC is often the initiating lesion in the development of colorectal cancer. The absence of active APC causes aberrant activation of Wnt/b-catenin signalling, which plays a crucial role in the generation of various human cancers and has been found in 90% of colorectal cancer cases (Huang & He, 2008; Shitashige, Hirohashi, & Yamada, 2008). Thus, the agents inhibiting Wnt/b-catenin signalling are potential therapies for the treatment and prevention of colorectal cancer. Wnt/b-catenin signalling, a system conserved throughout evolution, has an essential role in development and tissue regenera⇑ Corresponding author. Address: Natural Products Research Center, Korea Institute of Science and Technology, 290 Daejeon-dong, Gangneung 210-340, Republic of Korea. Tel.: +82 33 650 3651; fax: +82 33 650 3679. E-mail address: [email protected] (C.W. Nho). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.06.007
tion (Huang & He, 2008; Miyaki et al., 1994; Shitashige et al., 2008). b-Catenin, a key regulator in Wnt/b-catenin signalling, is highly expressed in cancers, and the mechanism controlling the levels of b-catenin has been reported in numerous articles. In the canonical Wnt/b-catenin pathway, the APC complex induces the phosphorylation and degradation of b-catenin protein in a proteasome-dependent manner (Barker & Clevers, 2006; Klaus & Birchmeier, 2008). In another recently reported pathway, b-catenin was shown to be degraded by Siah-1 and the F-box protein Ebi without phosphorylation (Polakis, 2001). In both pathways, b-catenin levels are tightly controlled by the degradation of b-catenin proteins through ubiquitination to inhibit Wnt/b-catenin signalling. Wnt/b-catenin signalling can sometimes escape this regulation due to genetic defects, causing diseases including cancers; Wnt/b-catenin signalling is activated, and this constant signalling results in abnormal cell proliferation and tissue outgrowth (Huang & He, 2008; Miyaki et al., 1994; Shitashige et al., 2008). Mutations within b-catenin or APC have been identified in various colorectal cancers (Ilyas, Tomlinson, Rowan, Pignatelli, & Bodmer, 1997; Morin et al., 1997). APC mutations are very frequent in colorectal cancers, and most cases involve truncated APC with a subsequent loss of b-catenin regulatory activity. The b-catenin mutations are amino acid substitutions or in-frame deletions. Because these mutations within b-catenin or APC interfere with the normal regulation of Wnt/b-catenin signalling, known inhibitors of Wnt/b-catenin signalling that regulate phosphorylation or degradation of b-catenin have limitations in preventing or treating
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colorectal cancers. Recently, Kwon and his colleagues have reported that cGMP-dependent kinase (PKG) blocks transcription of b-catenin via a mechanism not related to phosphorylation of b-catenin (Kwon et al., 2010). As transcriptional regulation is one of the major biological mechanisms to control molecular factors necessary for maintaining the living organism, searching for inhibitors that regulate mRNA expression of b-catenin via PKG could be considered a novel approach to find anti-cancer agents that inhibit Wnt/b-catenin signalling. For this aim, we chose two human colon cancer cell lines, HCT116 and SW480 cells. The HCT116 cell line contains wild-type APC and a mutated b-catenin that is missing a site phosphorylated by GSK3b (Ilyas et al., 1997; Morin et al., 1997). Thus, b-catenin phosphorylation does not occur, despite the presence of normal APC complex in HCT116 cells. The SW480 cell line has a truncated APC with normal b-catenin (Ilyas et al., 1997; Morin et al., 1997). As a result, the degradation of b-catenin by the APC complex is mostly blocked, but regulation of b-catenin can occur by other pathways. Here, we report two xanthones, a- and c-mangostin (Fig. 1A), isolated from Garcinia mangostana L, that are known apoptotic agents against various cancer cells and that have anti-inflammatory and anti-microbial effects (Akao, Nakagawa, Iinuma, & Nozawa, 2008; Azebaze et al., 2006; Gopalakrishnan, Banumathi, & Suresh, 1997). These compounds showed significant anti-proliferative effects on human colon cancer cells, but their mechanisms of action are not clear because they have little influence on the canonical, caspase-dependant apoptotic pathway (Nakagawa, Iinuma, Naoe, Nozawa, & Akao, 2007). In this report, we suggest that the inhibitory effects of a- and c-mangostin against Wnt/bcatenin signalling occur through inhibition of b-catenin mRNA and protein levels. These effects were found to be independent of the degradation and phosphorylation of b-catenin but dependent on PKG activation.
relative to the 0.15% DMSO-treated cells and is defined as [(A450 nm treated cells)/(A450 nm untreated cells)] 100. 2.4. Luciferase assay HCT116 and SW480 cells (6 104 cells/well) were seeded in 24-well plates. Cells were transiently transfected with TOPFlash or FOPFlash using FuGENE 6 reagent (Roche Applied Science, Indianapolis, IN). pRL-CMV vectors (Promega, Madison, WI) were co-transfected as an internal reporter. The transfected cells were treated with the proper concentration of a- or c-mangostin for 48 h. After the cells were lysed, luciferase assays were performed using a dual-luciferase assay system (Promega), by following the recommended protocol. Transcriptional activity values are expressed as arbitrary units using a Renilla reporter for internal normalisation. 2.5. Western blot analysis After treatment with either the compounds or DMSO, cells were harvested and lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail and 1 mM PMSF. Proteins were separated on a 10% Bis–Tris gel (BioRad, Hercules, CA) and transferred to a PVDF membrane (Amersham Pharmacia Biotech, Amersham, UK). Primary antibodies against b-catenin (Cell Signaling Technology), phospho-b-catenin (Cell Signaling Technology), cGMP-dependent kinase-1 (PKG-1, Cell Signaling Technology) and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used at a 1:3000 dilution. Following primary antibody incubation, the blots were incubated with anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology) and visualised using the ECL or ECL advanced system (GE Healthcare, Hatfield, UK). 2.6. Quantification of cGMP
2. Materials and methods 2.1. Reagents
a- and c-Mangostin were isolated from G. Mangostana L. (Clusiaceae) (Quan et al., 2010). MG-132 was purchased from Calbiochem, and LiCl was obtained from Sigma–Aldrich (St. Louis, MO). Mangostins and MG-132 were dissolved in 100% DMSO (Sigma–Aldrich). LiCl was dissolved in deionised water (DIW).
After treatment with mangostins, cells were lysed with 0.1 M HCl and centrifuged at 1000g for 10 min. The supernatants were transferred to estimate intracellular cGMP level, using the colorimetric cGMP EIA Kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer’s protocol. Absorbance was evaluated at 405 nm using a microplate reader (Bio-Tek Instruments). 2.7. Microarray and real-time PCR analysis
2.2. Cell culture HCT116 cells (ATCC, Manassas, VA) were cultured in MEM with 10% (w/v) FBS and 1% (w/v) penicillin–streptomycin (P/S) obtained from Hyclone (Logan, UT). SW480 cells (ATCC) were cultured in RPMI 1640 containing 200 mM HEPES (Hyclone) with 10% (w/v) heat-inactivated FBS and 1% (w/v) P/S. 2.3. Cell proliferation assay HCT116 and SW480 cells were seeded at a concentration of 6 103 cells/well in 96-well flat-bottomed plates (Nunc; Thermo, Waltham, MA). The cells were treated with a- and c-mangostins at the appropriate concentrations or 0.15% DMSO. After 24, 48 or 72 h, the media with drugs or DMSO were replaced with media containing 10% EZ-Cytox solution (Daeil Labservice, Korea). After 1 h incubation at 37 °C, cell proliferation was monitored at 450 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT). All assays were performed in triplicate. The cytotoxic effect of each treatment was expressed as a percentage of cell viability
Total RNA was isolated using an RNeasy mini kit or midi kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Microarray analysis was performed by Genomictree (Daejon, Korea). The two labelled cDNAs were mixed and then hybridised to an Agilent Human whole genome 4 44 K (Agilent Technologies, Santa Clara, CA). The data were analysed using Agilent’s GeneSpring software. The cDNA for real-time PCR analysis was synthesised from 2 lg of total RNA using PrimeScript RT Master (Takara Bio Inc., Shiga, Japan) with 100 ng random primer, 500 lM dNTPs, 10 mM DTT and 2 U/lL RNase inhibitor. The reaction was incubated at 70 °C for 5 min, then at 25 °C for 10 min, followed by 42 °C for 50 min and 70 °C for 15 min. For real-time PCR, the cDNAs were amplified in a LightCyclerÒ 480 instrument (Roche Applied Science) using the LightCyclerÒ 480 Probes Master mix (Roche Applied Science). The sequences of the primer pairs were: CTNNB1, 5’-AGCTGACCAGCTCTCTCTTCA-3’ and 5’-CAATATCAAGTCCAAGATCAGC-3’; WNT5A, 5’-ATTGTACTGCAGGTGTACCTTAAAAC-3’ and 5’-CCCCC TTATAAATGCAACTGTTC-3’; FZD2, 5’-GGTGTCGGTGGCCTACAT-3’ and 5’-GAGAAGCGCTCGTTGCAC-3’; PDE5A, 5’-GCTCTAAAAGCA
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Fig. 1. Inhibition of cell proliferation and TCF/b-catenin-dependent transcriptional activity by a- and c-mangostin. (A) Structures of a- and c-mangostin. Human colon cancer cells, HCT116 cells (B) and SW480 cells (C), were treated with a- and c-mangostin to estimate the inhibitory effect on cell proliferation in a dose-dependent and timedependent manner. (D) Cells transfected by TOPFlash or FOPFlash were treated with mangostins. Luciferase activity was estimated by luminometer. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄ p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s t-test (n = 3).
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GGCAAAATTC-3’ and 5’-CAGCAATCAG CAATGCAA GT-3’; GUCY2F, 5’-TCCAACATAGCGATTTATGAGG-3’ and 5’-CCAA AATCTCCAAGGGAGAAC-3’; and GAPDH (used as the internal control), 5’AGCCACATCGCTCAGACAC -3’ and 5’-GCCCAATACGACCA AATCC-3’. 2.8. Statistical analysis The data are expressed throughout as the mean ± SD, which was calculated from at least three different experiments. The statistical significance among the test groups was determined by Student’s t-test. A p-value of less than 0.05 was considered significant. 3. Results 3.1. Mangostins inhibit the proliferation and the transcriptional activity of TCF/b-catenin The chemical structures of a- and c-mangostin are shown in Fig. 1A. The two compounds have similar structures, except for one methoxyl group. To investigate the cytotoxicity of a- and cmangostin and identify the inhibitory effect on cell proliferation, we treated SW480 and HCT116 cells with mangostins for 24, 48 and 72 h. As a- and c-mangostin have previously reported cytotoxicity (Azebaze et al., 2006), they showed significant inhibitory effect on the proliferation of colon cancer cells (Fig. 1B and C). Both mangostins showed a similar IC50 (15 lM), but no timedependent increase in the toxic effect of mangostins was observed. To understand how a- and c-mangostin control cell proliferation, we evaluated the suppression of Wnt/b-catenin signalling by measuring transcriptional activation via TCF/b-catenin (Fig. 1D). As described in the materials and methods, we co-transfected TOPFlash or FOPFlash vectors with pRL-CMV Renilla constructs into HCT116 and SW480 cells. The 48-h treatment of mangostins resulted in a reduction of TCF/b-catenin transcriptional activity in a dose-dependent manner. Despite low cytotoxicity, a low dose of mangostins significantly suppressed luciferase activity in HCT116 and SW480 cells; the effect was more clearly shown in HCT116 cells than SW480 cells. Taken together, these results indicate that mangostins have an inhibitory effect on colon cancer cell proliferation and are potential inhibitors of Wnt/b-catenin signalling. 3.2. Mangostins decrease mRNA and protein expression of b-catenin To confirm the inhibitory effect of mangostins on Wnt/b-catenin signalling, we examined changes in the levels of Wnt-related proteins. As b-catenin is the key regulator of Wnt/b-catenin signalling, we measured b-catenin levels using western blot analysis. Both mangostins decreased the protein levels of b-catenin in a dose- and time-dependent manner in both cell lines (Fig. 2A–C). To further confirm the inhibition of b-catenin, we investigated the transcriptional expression of the b-catenin gene, CTNNB1 (Fig. 2D and E). Except for the 24-h treatment with c-mangostin in SW480 cells, mangostins significantly decreased the mRNA levels of b-catenin in both cell types. The effect was more significant in HCT116 cells, especially in a dose-dependent manner, suggesting that the regulation of b-catenin by mangostins accompanied the transcriptional regulation of b-catenin. 3.3. The inhibitory effect of mangostins on Wnt/b-catenin signalling is not dependent on the degradation of b-catenin It is known that controlling the levels of b-catenin involves bcatenin phosphorylation, followed by its degradation (Barker &
Clevers, 2006; Klaus & Birchmeier, 2008; Polakis, 2001). Thus, we examined the levels of phosphorylated b-catenin by western blot analysis using a cytosolic fraction of SW480 cells. As shown in Fig. 3A and B and supplementary Fig. 1, there are no changes in the levels of phosphorylated b-catenin observed after mangostin treatment with decrease of nuclear b-catenin in SW480 cells, suggesting the effect of mangostins was not dependent on the phosphorylation of b-catenin. Another pathway for the degradation of b-catenin is controlled by Siah-1, which promotes the ubiquitination of b-catenin. To determine the changes in ubiquitination of b-catenin caused by treatment with mangostins, we examined these changes in the presence of MG-132, a proteosome inhibitor (Lee & Goldberg, 1998; Fig. 3C and D). MG-132 treatment did not alter the effect of mangostins against TCF/b-catenin transcriptional activity. To confirm these data, we attempted to compare the effect of LiCl, a Gsk3b inhibitor, with the effect of mangostins on the decrease of b-catenin (Stambolic, Ruel, & Woodgett, 1996, Fig. 3E and F). As shown in Fig. 3E and F, Gsk3b activation for phosphorylation of b-catenin was also not involved in the effect of mangostins on Wnt/b-catenin signalling. Both MG-132 and LiCl had no effects on the changes to the levels of b-catenin induced by mangostins, suggesting the degradation of b-catenin by mangostins does not occur through the classical pathway of Wnt/ b-catenin signalling. 3.4. The inhibitory effect of mangostins on Wnt/b-catenin signalling involves the regulation of c-GMP signalling Because PKG has recently been reported to downregulate bcatenin mRNA (Ilyas et al., 1997), we examined changes in the levels of PKG and cGMP, an activator of PKG, induced by treatment with mangostins. As shown in Fig. 4A and B, the levels of PKG and cGMP were noticeably increased after treatment with either mangostin. Microarray data using cDNAs from mangostin-treated SW480 cells showed the change of genes related to cGMP signalling (Tables 1 and 2). Expression of PDE5A mRNA, a protein that degrades cGMP (Ma & Wang, 2006), was significantly decreased, and the expression of GUYC2F, an enzyme that produces cGMP (Browning, 2008), was remarkably increased. Expression of the Wnt-related genes WNT5A, the FZD family genes and CTNNB1 were also reduced. We validated the data with real-time PCR analysis, and the mRNA levels of cGMP-related genes were decreased to similar levels observed in the microarray data (Fig. 4C). 4. Discussion
a- and c-Mangostin, xanthones from G. mangostana L, have been reported to have various biological activities, such as inducing apoptosis and suppressing inflammation (Akao et al., 2008; Azebaze et al., 2006; Gopalakrishnan et al., 1997). To our knowledge, the mechanism mangostins use to exert their anti-cancer effects has not yet been elucidated. In the present study, we demonstrated that the mangostins have anti-cancer effects via regulation of Wnt/b-catenin signalling in human colorectal cancer cells. Aberrant activation of Wnt/b-catenin signalling is common and seen in over 85% of sporadic cases of colorectal cancer. Two cell lines having different genetic defects were selected for this study; HCT116 cells have a mutated b-catenin with normal APC, while SW480 cells have a wild-type b-catenin with truncated APC. We expected that the differences caused by these mutations between HCT116 and SW480 cells would indicate how mangostins act on Wnt/b-catenin signalling. Our data demonstrate that mangostins inhibit cell proliferation and TCF/b-catenin transcriptional activity
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Fig. 2. The changes in b-catenin protein and mRNA expression levels after treatment with a- and c-mangostin. Various concentrations of mangostins were treated for 48 h for dose-dependency and 15 lM mangostins were treated for time-dependency in HCT116 and SW480 cells. Western blot analysis was performed with cell lysates after the treatment of mangostins in a dose-dependent (A) and time-dependent manner in HCT116 (B) and SW480 (C) cells. Expression of b-catenin mRNA was analysed by real-time PCR with cDNA from HCT116 (D) and SW480 (E) cells treated with mangostins. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s ttest (n = 3).
in both HCT116 and SW480 colon cancer cells (Fig. 1). Interestingly, mangostins showed similar inhibitory effects on both
HCT116 and SW480 cells, regardless of the different genetic defects related to Wnt/b-catenin signalling. Considering the different
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Fig. 3. The effect of a- and c-mangostin on the post-translational regulation of b-catenin. Mangostins (15 lM) were treated in HCT116 and SW480 cells. Changes in the levels of b-catenin phosphorylation after treatment with a-mangostin (A) and c-mangostin (B) were estimated by western blot analysis of the proteins from SW480 cells. b-Catenin protein levels after treatment with mangostins were estimated with or without 10 lM of MG-132, a proteasome inhibitor, in HCT116 (C) and SW480 cells (D). Luciferase activity using TOPFlash after the co-treatment of mangostins and 15 lM of LiCl, a specific Gsk3b inhibitor, was investigated in HCT116 cells (E) and SW480 cells (F). ⁄p < 0.05; ⁄⁄ p < 0.01; ⁄⁄⁄p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s t-test (n = 3).
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Fig. 4. Regulation of cGMP signalling by a- and c-mangostin. Mangostins (15 lM) were treated in SW480 cells. (A) The levels of PKG were evaluated after treatment with mangostins using western blot analysis. (B) cGMP levels were estimated by enzyme-linked immunosorbent assay. (C) Real-time PCR analysis of the genes related to cGMP signalling. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s t-test (n = 3).
mutations between the cell lines, the effect of mangostin-treatment suggests that mangostins can regulate Wnt/b-catenin signalling irrespective of mutations in either b-catenin or APC. Using western blot analysis and real-time PCR, we also found that mangostins decreased the protein level and mRNA expression of b-catenin (Fig. 2). Our results clearly demonstrate that mangostins regulate Wnt/b-catenin signalling through the inhibition of b-catenin levels. In particular, the decrease of b-catenin mRNA expression may suggest two possible mechanisms: mangostins affect the degradation of b-catenin or inhibit transcription of the b-catenin gene, CTNNB1. We first investigated degradation of b-catenin, but mangostins did not have any effect on either the phosphorylation or degradation of b-catenin (Fig. 3). Although a variety of articles have reported that the regulation of b-catenin results from phosphorylation at the Ser45 and Ser33/37/Thr41 sites (Amit et al., 2002), a change in b-catenin phosphorylation after treatment with mangostins was not observed (Fig. 3A and B). Since the nuclear b-catenin is a key factor for the transcriptional activity, we examined the nuclear b-catenin levels after mangostin treatment. We found that the nuclear b-catenin markedly decreased
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at 24 h in SW480 cells, confirming that b-catenin mainly contributes to the transcriptional regulation by mangostins in the Wnt/ b-catenin signalling (Supplementary Fig. 1). Furthermore, the inhibition of b-catenin by mangostins was not changed by MG-132 treatment (Fig. 3C and D). As MG-132 is a proteosome inhibitor (Lee & Goldberg, 1998), these results indicate that mangostin treatments decreased b-catenin levels without the action of proteosomes. We confirmed these data with LiCl, a specific inhibitor of Gsk3b (Stambolic, Ruel, & Woodgett, 1996). LiCl treatment did not change the effect of mangostins (Fig. 3E and F), suggesting mangostins have no influence on the degradation of b-catenin via its phosphorylation, which is a leading mechanism of b-catenin regulation. Secondly, we examined whether the inhibitory effect of mangostins on Wnt/b-catenin signalling involves transcriptional regulation of b-catenin. Although there are no reports about agents that inhibit Wnt/b-catenin signalling without degradation of b-catenin, one recent article has reported that PKG, an upstream regulator of b-catenin, represses the mRNA levels and not the protein levels of b-catenin (Kwon et al., 2010). Therefore, we hypothesised that the inhibitory effect of mangostins on b-catenin could be due to regulation of mRNA levels through changes in PKG and cGMP, a PKG activator (Fig. 4). PKG-1 expression and cGMP levels were increased by mangostin treatment in SW480 cells, although a-mangostin showed a more evident effect than c-mangostin. Recent studies have reported that PKG-elevating agents can be potential chemotherapeutics in colorectal cancers (Browning, 2008; Browning, Kwon, & Wang, 2010). Because PKG-1 activation exerts anti-cancer effects that are mediated by regulation of b-catenin, both mangostins could be good candidates for the treatment of colorectal cancers as PKG activators. To find other specific mechanisms, cDNA microarray was performed (Tables 1 and 2). The changes of Wnt/b-catenin-signalling and cGMP-signalling related genes were observed, and the data were confirmed using real-time PCR analysis (Fig. 4C). The data indicate that mangostins inhibited Wnt/b-catenin signalling through cGMP signalling. In particular, expression of the Wnt/bcatenin-signalling related genes WNT5A and FZD2 were lowered by mangostins, and the mRNA levels of the cGMP-signalling related genes PDE5A and GUYC2F were significantly altered. In many reports, WNT5A and FZD2 are key regulators of Wnt/Ca2+ signalling, which is a non-canonical pathway of Wnt/b-catenin signalling (Ma & Wang, 2006; Sato, Yamamoto, Sakane, Koyama, & Kikuchi, 2010; Wang & Malbon, 2003). In Wnt/Ca2+ signalling, activation of Fzd2 by binding to Wnt5A decreases the level of cGMP via activation of cGMP-specific phosphodiesterase (PDE5A protein), resulting in inactivation of PKG. Our data show that mangostins significantly inhibit the mRNA levels of PDE5A. Decreased levels of PDE5A lead to an elevation in cGMP, followed by PKG-1, and results in the inhibition of b-catenin via transcriptional regulation. One of the therapeutic approaches now used by the pharmaceutical industry is the development of PDE inhibitors for blocking cGMP degradation, thereby having anti-cancer effects, such as apoptosis, differentiation, and anti-angiogenesis (Boswell-Smith, Spina, & Page, 2006). In conclusion, we found that mangostins are potential candidates as inhibitory agents against Wnt/b-catenin signalling. These compounds inhibited the cell proliferation of human colon cancer cells and the transcriptional activity of TCF/b-catenin via inhibition of b-catenin, regardless of the mutational status of b-catenin. The mangostins reduced mRNA expression and protein levels of b-catenin without the phosphorylation and degradation of b-catenin by the typical proteasomal pathway. Rather, the mangostins decreased mRNA levels of b-catenin, through PKG activation that resulted from lowered Wnt5A and elevated cGMP. These novel findings could provide clues to explain which inhibitors of Wnt/ b-catenin signalling can exert anti-cancer effects through the transcriptional regulation of b-catenin that results from activation of
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Table 1 A list of Wnt/cGMP signaling-related genes downregulated in SW480 cells treated with mangostins. Description
Genbank accession number
Gene symbol
Wingless-type MMTV integration site family, member 5A Frizzled homolog 2 (Drosophila) Frizzled homolog 3 (Drosophila) Frizzled homolog 4 (Drosophila) Frizzled homolog 7 (Drosophila) Frizzled homolog 8 (Drosophila) Frizzled homolog 9 (Drosophila) Catenin (cadherin-associated protein), beta 1, 88 kDa Lymphoid enhancer-binding factor 1 Cyclin D1 SMAD family member 2 CXXC finger 4 Calcium binding protein P22 Phosphodiesterase 5A, cGMP-specific
NM_003392 NM_001466 NM_017412 NM_012193 NM_003507 NM_031866 NM_003508 NM_001904 NM_016269 NM_053056 AY134745 NM_025212 NM_007236 NM_001083
WNT5A FZD2 FZD3 FZD4 FZD7 FZD8 FZD9 CTNNB1 LEF1 CCND1 SMAD2 CXXC4
a-Mangostin
c-Mangostin
Average
STDEV
p value
Average
STDEV
p value
0.486 0.238 0.531 0.465 0.392 0.069 0.680 0.502 0.555 0.314 0.245 0.198 0.502 0.423
0.116 0.132 0.107 0.141 0.068 0.021 0.043 0.637 0.330 0.071 0.101 0.227 0.508 0.240
7.83E-04 2.80E-04 7.99E-04 1.37E-03 5.13E-05 8.20E-08 1.01E-04 1.23E-01 3.98E-02 3.67E-05 1.02E-04 1.81E-03 8.21E-02 7.12E-03
0.392 0.172 0.401 0.386 0.451 0.240 0.697 0.962 0.414 0.481 0.415 0.083 0.730 0.897
0.108 0.139 0.111 0.095 0.117 0.259 0.050 1.462 0.318 0.231 0.186 0.064 0.741 1.282
3.07E-04 2.52E-04 3.69E-04 1.81E-04 6.33E-04 3.52E-03 2.40E-04 4.83E-01 1.65E-02 8.85E-03 2.74E-03 7.72E-06 2.81E-01 4.48E-01
Table 2 A list of Wnt/cGMP signaling-related genes upregulated in SW480 cells treated with mangostins. Description
E1A binding protein p300 Guanylate cyclase 2F, retinal Guanylate cyclase activator 1B (retina) Guanylate cyclase activator 1C
Genbank accession number
NM_001429 NM_001522 NM_002098 NM_005459
Gene symbol
EP300 GUCA1B
cGMP signalling. This may also offer more opportunities to find effective chemotherapeutic agents for treating human colorectal cancers by controlling non-classical Wnt/b-catenin signalling. Acknowledgements This work was supported by a KIST-Gangneung Institute intramural research Grant(2Z03480). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2011.06.007. References Akao, Y., Nakagawa, Y., Iinuma, M., & Nozawa, Y. (2008). Anti-cancer effects of xanthones from pericarps of mangosteen. International Journal of Molecular Sciences, 9, 355–370. Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E., Mann, M., et al. (2002). Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: A molecular switch for the Wnt pathway. Genes & Development, 16, 1066–1076. Azebaze, A. G., Meyer, M., Valentin, A., Nguemfo, E. L., Fomum, Z. T., & Nkengfack, A. E. (2006). Prenylated xanthone derivatives with antiplasmodial activity from Allanblackia monticola STANER L.C. Chemical & Pharmaceutical Bulletin (Tokyo), 54, 111–113. Barker, N., & Clevers, H. (2006). Mining the Wnt pathway for cancer therapeutics. Nature Reviews. Drug Discovery, 5, 997–1014. Boswell-Smith, V., Spina, D., & Page, C. P. (2006). Phosphodiesterase inhibitors. British Journal of Pharmacology, 147(Suppl 1), S252–S257. Browning, D. D. (2008). Protein kinase G as a therapeutic target for the treatment of metastatic colorectal cancer. Expert Opinion on Therapeutic Targets, 12, 367–376. Browning, D. D., Kwon, I. K., & Wang, R. (2010). CGMP-dependent protein kinases as potential targets for colon cancer prevention and treatment. Future Medicinal Chemistry, 2, 65–80. Davies, R. J., Miller, R., & Coleman, N. (2005). Colorectal cancer screening: Prospects for molecular stool analysis. Nature Reviews. Cancer, 5, 199–209. Global Cancer Facts & Figures 2007. www.cancer.org. Gopalakrishnan, G., Banumathi, B., & Suresh, G. (1997). Evaluation of the antifungal activity of natural xanthones from Garcinia mangostana and their synthetic derivatives. Journal of Natural Products, 60, 519–524.
a-Mangostin
c-Mangostin
Average
STDEV
p value
Average
STDEV
p value
3.458 8.709 0.827 2.933
5.945 11.545 0.215 1.745
2.57E-01 1.47E-01 1.18E-01 6.37E-02
1.271 23.795 0.579 1.785
1.804 33.011 0.020 1.202
4.04E-01 1.41E-01 1.77E-06 1.61E-01
Huang, H., & He, X. (2008). Wnt/beta-catenin signalling: New (and old) players and new insights. Current Opinion in Cell Biology, 20, 119–125. Ilyas, M., Tomlinson, I. P., Rowan, A., Pignatelli, M., & Bodmer, W. F. (1997). Betacatenin mutations in cell lines established from human colorectal cancers. Proceedings of the National Academy of Sciences of the United States of America, 94, 10330–10334. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Murray, T., et al. (2008). Cancer statistics, 2008. CA: A Cancer Journal for Clinicians, 58, 71–96. Klaus, A., & Birchmeier, W. (2008). Wnt signalling and its impact on development and cancer. Nature Reviews. Cancer, 8, 387–398. Kwon, I. K., Wang, R., Thangaraju, M., Shuang, H., Liu, K., Dashwood, R., et al. (2010). PKG inhibits TCF signalling in colon cancer cells by blocking beta-catenin expression and activating FOXO4. Oncogene, 29, 3423–3434. Lee, D. H., & Goldberg, A. L. (1998). Proteasome inhibitors: Valuable new tools for cell biologists. Trends in Cell Biology, 8, 397–403. Ma, L., & Wang, H. Y. (2006). Suppression of cyclic GMP-dependent protein kinase is essential to the Wnt/cGMP/Ca2+ pathway. Journal of Biological Chemistry, 281, 30990–31001. Miyaki, M., Konishi, M., Kikuchi-Yanoshita, R., Enomoto, M., Igari, T., Tanaka, K., et al. (1994). Characteristics of somatic mutation of the adenomatous polyposis coli gene in colorectal tumours. Cancer Research, 54, 3011–3020. Morin, P. J., Sparks, A. B., Korinek, V., Barker, N., Clevers, H., Vogelstein, B., et al. (1997). Activation of beta-catenin-Tcf signalling in colon cancer by mutations in beta-catenin or APC. Science, 275, 1787–1790. Nakagawa, Y., Iinuma, M., Naoe, T., Nozawa, Y., & Akao, Y. (2007). Characterized mechanism of alpha-mangostin-induced cell death: Caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells. Bioorganic & Medicinal Chemistry, 15, 5620–5628. Polakis, P. (2001). More than one way to skin a catenin. Cell, 105, 563–566. Quan, G. H., Oh, S. R., Kim, J. H., Lee, H. K., Kinghorn, A. D., & Chin, Y. W. (2010). Xanthone constituents of the fruits of Garcinia mangostana with anticomplement activity. Phytotherapy Research, 24, 1575–1577. Sato, A., Yamamoto, H., Sakane, H., Koyama, H., & Kikuchi, A. (2010). Wnt5a regulates distinct signalling pathways by binding to Frizzled2. The EMBO Journal, 29, 41–54. Shitashige, M., Hirohashi, S., & Yamada, T. (2008). Wnt signalling inside the nucleus. Cancer Science, 99, 631–637. Stambolic, V., Ruel, L., & Woodgett, J. R. (1996). Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Current Biology, 6, 1664–1668. Wang, H. Y., & Malbon, C. C. (2003). Wnt signalling, Ca2+, and cyclic GMP: Visualizing Frizzled functions. Science, 300, 1529–1530. Watson, A. J. (2006). An overview of apoptosis and the prevention of colorectal cancer. Critical Reviews in Oncology/Haematology, 57, 107–121.
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a- and c-Mangostin inhibit the proliferation of colon cancer cells via b-catenin gene regulation in Wnt/cGMP signalling Ji-Hye Yoo a, Kyungsu Kang a, Eun Hye Jho a, Young-Won Chin b, Jinwoong Kim c, Chu Won Nho a,⇑ a
Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 210-340, Republic of Korea College of Pharmacy, Dongguk University-Seoul, Seoul 100-715, Republic of Korea c College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 23 February 2011 Received in revised form 31 May 2011 Accepted 5 June 2011 Available online 12 June 2011 Keywords: Mangostins b-Catenin Transcriptional regulation cGMP PKG
a b s t r a c t Aberrant activation of Wnt/b-catenin signalling via genetic errors within b-catenin or APC has a crucial role in carcinogenesis. Here, we studied two xanthones, a- and c-mangostin, as inhibitors of Wnt/b-catenin signalling. The mangostins inhibited TCF/b-catenin transcriptional activity. The mangostins also inhibited protein expression of b-catenin in colon cancer cells, but the inhibition was independent of the phosphorylation and degradation of b-catenin. Instead, the mangostins increased the levels of cGMP and cGMP-dependent kinase, indicating that the inhibition of b-catenin resulted from b-catenin gene regulation. Our results indicate that mangostins could be potential candidates for preventing colon cancer through a novel mechanism of inhibiting Wnt/b-catenin signalling. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Colorectal cancer is the third most common cancer among women and men worldwide, with 1.2 million cases and 630,000 deaths in 2007 (Global Cancer Facts, 2007; Jemal et al., 2008). Because the progression from normal epithelium to invasive cancer takes approximately 10–15 years, early diagnosis and treatment of colorectal cancer are important to prevent the development of malignant tumours. Colorectal cancer arises as a result of an accumulation of genetic errors that affect cell proliferation and survival (Davies, Miller, & Coleman, 2005; Miyaki et al., 1994; Watson, 2006). Among several genetic defects, mutation of APC is often the initiating lesion in the development of colorectal cancer. The absence of active APC causes aberrant activation of Wnt/b-catenin signalling, which plays a crucial role in the generation of various human cancers and has been found in 90% of colorectal cancer cases (Huang & He, 2008; Shitashige, Hirohashi, & Yamada, 2008). Thus, the agents inhibiting Wnt/b-catenin signalling are potential therapies for the treatment and prevention of colorectal cancer. Wnt/b-catenin signalling, a system conserved throughout evolution, has an essential role in development and tissue regenera⇑ Corresponding author. Address: Natural Products Research Center, Korea Institute of Science and Technology, 290 Daejeon-dong, Gangneung 210-340, Republic of Korea. Tel.: +82 33 650 3651; fax: +82 33 650 3679. E-mail address: [email protected] (C.W. Nho). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.06.007
tion (Huang & He, 2008; Miyaki et al., 1994; Shitashige et al., 2008). b-Catenin, a key regulator in Wnt/b-catenin signalling, is highly expressed in cancers, and the mechanism controlling the levels of b-catenin has been reported in numerous articles. In the canonical Wnt/b-catenin pathway, the APC complex induces the phosphorylation and degradation of b-catenin protein in a proteasome-dependent manner (Barker & Clevers, 2006; Klaus & Birchmeier, 2008). In another recently reported pathway, b-catenin was shown to be degraded by Siah-1 and the F-box protein Ebi without phosphorylation (Polakis, 2001). In both pathways, b-catenin levels are tightly controlled by the degradation of b-catenin proteins through ubiquitination to inhibit Wnt/b-catenin signalling. Wnt/b-catenin signalling can sometimes escape this regulation due to genetic defects, causing diseases including cancers; Wnt/b-catenin signalling is activated, and this constant signalling results in abnormal cell proliferation and tissue outgrowth (Huang & He, 2008; Miyaki et al., 1994; Shitashige et al., 2008). Mutations within b-catenin or APC have been identified in various colorectal cancers (Ilyas, Tomlinson, Rowan, Pignatelli, & Bodmer, 1997; Morin et al., 1997). APC mutations are very frequent in colorectal cancers, and most cases involve truncated APC with a subsequent loss of b-catenin regulatory activity. The b-catenin mutations are amino acid substitutions or in-frame deletions. Because these mutations within b-catenin or APC interfere with the normal regulation of Wnt/b-catenin signalling, known inhibitors of Wnt/b-catenin signalling that regulate phosphorylation or degradation of b-catenin have limitations in preventing or treating
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colorectal cancers. Recently, Kwon and his colleagues have reported that cGMP-dependent kinase (PKG) blocks transcription of b-catenin via a mechanism not related to phosphorylation of b-catenin (Kwon et al., 2010). As transcriptional regulation is one of the major biological mechanisms to control molecular factors necessary for maintaining the living organism, searching for inhibitors that regulate mRNA expression of b-catenin via PKG could be considered a novel approach to find anti-cancer agents that inhibit Wnt/b-catenin signalling. For this aim, we chose two human colon cancer cell lines, HCT116 and SW480 cells. The HCT116 cell line contains wild-type APC and a mutated b-catenin that is missing a site phosphorylated by GSK3b (Ilyas et al., 1997; Morin et al., 1997). Thus, b-catenin phosphorylation does not occur, despite the presence of normal APC complex in HCT116 cells. The SW480 cell line has a truncated APC with normal b-catenin (Ilyas et al., 1997; Morin et al., 1997). As a result, the degradation of b-catenin by the APC complex is mostly blocked, but regulation of b-catenin can occur by other pathways. Here, we report two xanthones, a- and c-mangostin (Fig. 1A), isolated from Garcinia mangostana L, that are known apoptotic agents against various cancer cells and that have anti-inflammatory and anti-microbial effects (Akao, Nakagawa, Iinuma, & Nozawa, 2008; Azebaze et al., 2006; Gopalakrishnan, Banumathi, & Suresh, 1997). These compounds showed significant anti-proliferative effects on human colon cancer cells, but their mechanisms of action are not clear because they have little influence on the canonical, caspase-dependant apoptotic pathway (Nakagawa, Iinuma, Naoe, Nozawa, & Akao, 2007). In this report, we suggest that the inhibitory effects of a- and c-mangostin against Wnt/bcatenin signalling occur through inhibition of b-catenin mRNA and protein levels. These effects were found to be independent of the degradation and phosphorylation of b-catenin but dependent on PKG activation.
relative to the 0.15% DMSO-treated cells and is defined as [(A450 nm treated cells)/(A450 nm untreated cells)] 100. 2.4. Luciferase assay HCT116 and SW480 cells (6 104 cells/well) were seeded in 24-well plates. Cells were transiently transfected with TOPFlash or FOPFlash using FuGENE 6 reagent (Roche Applied Science, Indianapolis, IN). pRL-CMV vectors (Promega, Madison, WI) were co-transfected as an internal reporter. The transfected cells were treated with the proper concentration of a- or c-mangostin for 48 h. After the cells were lysed, luciferase assays were performed using a dual-luciferase assay system (Promega), by following the recommended protocol. Transcriptional activity values are expressed as arbitrary units using a Renilla reporter for internal normalisation. 2.5. Western blot analysis After treatment with either the compounds or DMSO, cells were harvested and lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail and 1 mM PMSF. Proteins were separated on a 10% Bis–Tris gel (BioRad, Hercules, CA) and transferred to a PVDF membrane (Amersham Pharmacia Biotech, Amersham, UK). Primary antibodies against b-catenin (Cell Signaling Technology), phospho-b-catenin (Cell Signaling Technology), cGMP-dependent kinase-1 (PKG-1, Cell Signaling Technology) and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used at a 1:3000 dilution. Following primary antibody incubation, the blots were incubated with anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology) and visualised using the ECL or ECL advanced system (GE Healthcare, Hatfield, UK). 2.6. Quantification of cGMP
2. Materials and methods 2.1. Reagents
a- and c-Mangostin were isolated from G. Mangostana L. (Clusiaceae) (Quan et al., 2010). MG-132 was purchased from Calbiochem, and LiCl was obtained from Sigma–Aldrich (St. Louis, MO). Mangostins and MG-132 were dissolved in 100% DMSO (Sigma–Aldrich). LiCl was dissolved in deionised water (DIW).
After treatment with mangostins, cells were lysed with 0.1 M HCl and centrifuged at 1000g for 10 min. The supernatants were transferred to estimate intracellular cGMP level, using the colorimetric cGMP EIA Kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer’s protocol. Absorbance was evaluated at 405 nm using a microplate reader (Bio-Tek Instruments). 2.7. Microarray and real-time PCR analysis
2.2. Cell culture HCT116 cells (ATCC, Manassas, VA) were cultured in MEM with 10% (w/v) FBS and 1% (w/v) penicillin–streptomycin (P/S) obtained from Hyclone (Logan, UT). SW480 cells (ATCC) were cultured in RPMI 1640 containing 200 mM HEPES (Hyclone) with 10% (w/v) heat-inactivated FBS and 1% (w/v) P/S. 2.3. Cell proliferation assay HCT116 and SW480 cells were seeded at a concentration of 6 103 cells/well in 96-well flat-bottomed plates (Nunc; Thermo, Waltham, MA). The cells were treated with a- and c-mangostins at the appropriate concentrations or 0.15% DMSO. After 24, 48 or 72 h, the media with drugs or DMSO were replaced with media containing 10% EZ-Cytox solution (Daeil Labservice, Korea). After 1 h incubation at 37 °C, cell proliferation was monitored at 450 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT). All assays were performed in triplicate. The cytotoxic effect of each treatment was expressed as a percentage of cell viability
Total RNA was isolated using an RNeasy mini kit or midi kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Microarray analysis was performed by Genomictree (Daejon, Korea). The two labelled cDNAs were mixed and then hybridised to an Agilent Human whole genome 4 44 K (Agilent Technologies, Santa Clara, CA). The data were analysed using Agilent’s GeneSpring software. The cDNA for real-time PCR analysis was synthesised from 2 lg of total RNA using PrimeScript RT Master (Takara Bio Inc., Shiga, Japan) with 100 ng random primer, 500 lM dNTPs, 10 mM DTT and 2 U/lL RNase inhibitor. The reaction was incubated at 70 °C for 5 min, then at 25 °C for 10 min, followed by 42 °C for 50 min and 70 °C for 15 min. For real-time PCR, the cDNAs were amplified in a LightCyclerÒ 480 instrument (Roche Applied Science) using the LightCyclerÒ 480 Probes Master mix (Roche Applied Science). The sequences of the primer pairs were: CTNNB1, 5’-AGCTGACCAGCTCTCTCTTCA-3’ and 5’-CAATATCAAGTCCAAGATCAGC-3’; WNT5A, 5’-ATTGTACTGCAGGTGTACCTTAAAAC-3’ and 5’-CCCCC TTATAAATGCAACTGTTC-3’; FZD2, 5’-GGTGTCGGTGGCCTACAT-3’ and 5’-GAGAAGCGCTCGTTGCAC-3’; PDE5A, 5’-GCTCTAAAAGCA
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Fig. 1. Inhibition of cell proliferation and TCF/b-catenin-dependent transcriptional activity by a- and c-mangostin. (A) Structures of a- and c-mangostin. Human colon cancer cells, HCT116 cells (B) and SW480 cells (C), were treated with a- and c-mangostin to estimate the inhibitory effect on cell proliferation in a dose-dependent and timedependent manner. (D) Cells transfected by TOPFlash or FOPFlash were treated with mangostins. Luciferase activity was estimated by luminometer. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄ p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s t-test (n = 3).
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GGCAAAATTC-3’ and 5’-CAGCAATCAG CAATGCAA GT-3’; GUCY2F, 5’-TCCAACATAGCGATTTATGAGG-3’ and 5’-CCAA AATCTCCAAGGGAGAAC-3’; and GAPDH (used as the internal control), 5’AGCCACATCGCTCAGACAC -3’ and 5’-GCCCAATACGACCA AATCC-3’. 2.8. Statistical analysis The data are expressed throughout as the mean ± SD, which was calculated from at least three different experiments. The statistical significance among the test groups was determined by Student’s t-test. A p-value of less than 0.05 was considered significant. 3. Results 3.1. Mangostins inhibit the proliferation and the transcriptional activity of TCF/b-catenin The chemical structures of a- and c-mangostin are shown in Fig. 1A. The two compounds have similar structures, except for one methoxyl group. To investigate the cytotoxicity of a- and cmangostin and identify the inhibitory effect on cell proliferation, we treated SW480 and HCT116 cells with mangostins for 24, 48 and 72 h. As a- and c-mangostin have previously reported cytotoxicity (Azebaze et al., 2006), they showed significant inhibitory effect on the proliferation of colon cancer cells (Fig. 1B and C). Both mangostins showed a similar IC50 (15 lM), but no timedependent increase in the toxic effect of mangostins was observed. To understand how a- and c-mangostin control cell proliferation, we evaluated the suppression of Wnt/b-catenin signalling by measuring transcriptional activation via TCF/b-catenin (Fig. 1D). As described in the materials and methods, we co-transfected TOPFlash or FOPFlash vectors with pRL-CMV Renilla constructs into HCT116 and SW480 cells. The 48-h treatment of mangostins resulted in a reduction of TCF/b-catenin transcriptional activity in a dose-dependent manner. Despite low cytotoxicity, a low dose of mangostins significantly suppressed luciferase activity in HCT116 and SW480 cells; the effect was more clearly shown in HCT116 cells than SW480 cells. Taken together, these results indicate that mangostins have an inhibitory effect on colon cancer cell proliferation and are potential inhibitors of Wnt/b-catenin signalling. 3.2. Mangostins decrease mRNA and protein expression of b-catenin To confirm the inhibitory effect of mangostins on Wnt/b-catenin signalling, we examined changes in the levels of Wnt-related proteins. As b-catenin is the key regulator of Wnt/b-catenin signalling, we measured b-catenin levels using western blot analysis. Both mangostins decreased the protein levels of b-catenin in a dose- and time-dependent manner in both cell lines (Fig. 2A–C). To further confirm the inhibition of b-catenin, we investigated the transcriptional expression of the b-catenin gene, CTNNB1 (Fig. 2D and E). Except for the 24-h treatment with c-mangostin in SW480 cells, mangostins significantly decreased the mRNA levels of b-catenin in both cell types. The effect was more significant in HCT116 cells, especially in a dose-dependent manner, suggesting that the regulation of b-catenin by mangostins accompanied the transcriptional regulation of b-catenin. 3.3. The inhibitory effect of mangostins on Wnt/b-catenin signalling is not dependent on the degradation of b-catenin It is known that controlling the levels of b-catenin involves bcatenin phosphorylation, followed by its degradation (Barker &
Clevers, 2006; Klaus & Birchmeier, 2008; Polakis, 2001). Thus, we examined the levels of phosphorylated b-catenin by western blot analysis using a cytosolic fraction of SW480 cells. As shown in Fig. 3A and B and supplementary Fig. 1, there are no changes in the levels of phosphorylated b-catenin observed after mangostin treatment with decrease of nuclear b-catenin in SW480 cells, suggesting the effect of mangostins was not dependent on the phosphorylation of b-catenin. Another pathway for the degradation of b-catenin is controlled by Siah-1, which promotes the ubiquitination of b-catenin. To determine the changes in ubiquitination of b-catenin caused by treatment with mangostins, we examined these changes in the presence of MG-132, a proteosome inhibitor (Lee & Goldberg, 1998; Fig. 3C and D). MG-132 treatment did not alter the effect of mangostins against TCF/b-catenin transcriptional activity. To confirm these data, we attempted to compare the effect of LiCl, a Gsk3b inhibitor, with the effect of mangostins on the decrease of b-catenin (Stambolic, Ruel, & Woodgett, 1996, Fig. 3E and F). As shown in Fig. 3E and F, Gsk3b activation for phosphorylation of b-catenin was also not involved in the effect of mangostins on Wnt/b-catenin signalling. Both MG-132 and LiCl had no effects on the changes to the levels of b-catenin induced by mangostins, suggesting the degradation of b-catenin by mangostins does not occur through the classical pathway of Wnt/ b-catenin signalling. 3.4. The inhibitory effect of mangostins on Wnt/b-catenin signalling involves the regulation of c-GMP signalling Because PKG has recently been reported to downregulate bcatenin mRNA (Ilyas et al., 1997), we examined changes in the levels of PKG and cGMP, an activator of PKG, induced by treatment with mangostins. As shown in Fig. 4A and B, the levels of PKG and cGMP were noticeably increased after treatment with either mangostin. Microarray data using cDNAs from mangostin-treated SW480 cells showed the change of genes related to cGMP signalling (Tables 1 and 2). Expression of PDE5A mRNA, a protein that degrades cGMP (Ma & Wang, 2006), was significantly decreased, and the expression of GUYC2F, an enzyme that produces cGMP (Browning, 2008), was remarkably increased. Expression of the Wnt-related genes WNT5A, the FZD family genes and CTNNB1 were also reduced. We validated the data with real-time PCR analysis, and the mRNA levels of cGMP-related genes were decreased to similar levels observed in the microarray data (Fig. 4C). 4. Discussion
a- and c-Mangostin, xanthones from G. mangostana L, have been reported to have various biological activities, such as inducing apoptosis and suppressing inflammation (Akao et al., 2008; Azebaze et al., 2006; Gopalakrishnan et al., 1997). To our knowledge, the mechanism mangostins use to exert their anti-cancer effects has not yet been elucidated. In the present study, we demonstrated that the mangostins have anti-cancer effects via regulation of Wnt/b-catenin signalling in human colorectal cancer cells. Aberrant activation of Wnt/b-catenin signalling is common and seen in over 85% of sporadic cases of colorectal cancer. Two cell lines having different genetic defects were selected for this study; HCT116 cells have a mutated b-catenin with normal APC, while SW480 cells have a wild-type b-catenin with truncated APC. We expected that the differences caused by these mutations between HCT116 and SW480 cells would indicate how mangostins act on Wnt/b-catenin signalling. Our data demonstrate that mangostins inhibit cell proliferation and TCF/b-catenin transcriptional activity
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Fig. 2. The changes in b-catenin protein and mRNA expression levels after treatment with a- and c-mangostin. Various concentrations of mangostins were treated for 48 h for dose-dependency and 15 lM mangostins were treated for time-dependency in HCT116 and SW480 cells. Western blot analysis was performed with cell lysates after the treatment of mangostins in a dose-dependent (A) and time-dependent manner in HCT116 (B) and SW480 (C) cells. Expression of b-catenin mRNA was analysed by real-time PCR with cDNA from HCT116 (D) and SW480 (E) cells treated with mangostins. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s ttest (n = 3).
in both HCT116 and SW480 colon cancer cells (Fig. 1). Interestingly, mangostins showed similar inhibitory effects on both
HCT116 and SW480 cells, regardless of the different genetic defects related to Wnt/b-catenin signalling. Considering the different
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Fig. 3. The effect of a- and c-mangostin on the post-translational regulation of b-catenin. Mangostins (15 lM) were treated in HCT116 and SW480 cells. Changes in the levels of b-catenin phosphorylation after treatment with a-mangostin (A) and c-mangostin (B) were estimated by western blot analysis of the proteins from SW480 cells. b-Catenin protein levels after treatment with mangostins were estimated with or without 10 lM of MG-132, a proteasome inhibitor, in HCT116 (C) and SW480 cells (D). Luciferase activity using TOPFlash after the co-treatment of mangostins and 15 lM of LiCl, a specific Gsk3b inhibitor, was investigated in HCT116 cells (E) and SW480 cells (F). ⁄p < 0.05; ⁄⁄ p < 0.01; ⁄⁄⁄p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s t-test (n = 3).
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Fig. 4. Regulation of cGMP signalling by a- and c-mangostin. Mangostins (15 lM) were treated in SW480 cells. (A) The levels of PKG were evaluated after treatment with mangostins using western blot analysis. (B) cGMP levels were estimated by enzyme-linked immunosorbent assay. (C) Real-time PCR analysis of the genes related to cGMP signalling. ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001; ⁄⁄⁄⁄p < 0.001 compared to control cells using the Student’s t-test (n = 3).
mutations between the cell lines, the effect of mangostin-treatment suggests that mangostins can regulate Wnt/b-catenin signalling irrespective of mutations in either b-catenin or APC. Using western blot analysis and real-time PCR, we also found that mangostins decreased the protein level and mRNA expression of b-catenin (Fig. 2). Our results clearly demonstrate that mangostins regulate Wnt/b-catenin signalling through the inhibition of b-catenin levels. In particular, the decrease of b-catenin mRNA expression may suggest two possible mechanisms: mangostins affect the degradation of b-catenin or inhibit transcription of the b-catenin gene, CTNNB1. We first investigated degradation of b-catenin, but mangostins did not have any effect on either the phosphorylation or degradation of b-catenin (Fig. 3). Although a variety of articles have reported that the regulation of b-catenin results from phosphorylation at the Ser45 and Ser33/37/Thr41 sites (Amit et al., 2002), a change in b-catenin phosphorylation after treatment with mangostins was not observed (Fig. 3A and B). Since the nuclear b-catenin is a key factor for the transcriptional activity, we examined the nuclear b-catenin levels after mangostin treatment. We found that the nuclear b-catenin markedly decreased
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at 24 h in SW480 cells, confirming that b-catenin mainly contributes to the transcriptional regulation by mangostins in the Wnt/ b-catenin signalling (Supplementary Fig. 1). Furthermore, the inhibition of b-catenin by mangostins was not changed by MG-132 treatment (Fig. 3C and D). As MG-132 is a proteosome inhibitor (Lee & Goldberg, 1998), these results indicate that mangostin treatments decreased b-catenin levels without the action of proteosomes. We confirmed these data with LiCl, a specific inhibitor of Gsk3b (Stambolic, Ruel, & Woodgett, 1996). LiCl treatment did not change the effect of mangostins (Fig. 3E and F), suggesting mangostins have no influence on the degradation of b-catenin via its phosphorylation, which is a leading mechanism of b-catenin regulation. Secondly, we examined whether the inhibitory effect of mangostins on Wnt/b-catenin signalling involves transcriptional regulation of b-catenin. Although there are no reports about agents that inhibit Wnt/b-catenin signalling without degradation of b-catenin, one recent article has reported that PKG, an upstream regulator of b-catenin, represses the mRNA levels and not the protein levels of b-catenin (Kwon et al., 2010). Therefore, we hypothesised that the inhibitory effect of mangostins on b-catenin could be due to regulation of mRNA levels through changes in PKG and cGMP, a PKG activator (Fig. 4). PKG-1 expression and cGMP levels were increased by mangostin treatment in SW480 cells, although a-mangostin showed a more evident effect than c-mangostin. Recent studies have reported that PKG-elevating agents can be potential chemotherapeutics in colorectal cancers (Browning, 2008; Browning, Kwon, & Wang, 2010). Because PKG-1 activation exerts anti-cancer effects that are mediated by regulation of b-catenin, both mangostins could be good candidates for the treatment of colorectal cancers as PKG activators. To find other specific mechanisms, cDNA microarray was performed (Tables 1 and 2). The changes of Wnt/b-catenin-signalling and cGMP-signalling related genes were observed, and the data were confirmed using real-time PCR analysis (Fig. 4C). The data indicate that mangostins inhibited Wnt/b-catenin signalling through cGMP signalling. In particular, expression of the Wnt/bcatenin-signalling related genes WNT5A and FZD2 were lowered by mangostins, and the mRNA levels of the cGMP-signalling related genes PDE5A and GUYC2F were significantly altered. In many reports, WNT5A and FZD2 are key regulators of Wnt/Ca2+ signalling, which is a non-canonical pathway of Wnt/b-catenin signalling (Ma & Wang, 2006; Sato, Yamamoto, Sakane, Koyama, & Kikuchi, 2010; Wang & Malbon, 2003). In Wnt/Ca2+ signalling, activation of Fzd2 by binding to Wnt5A decreases the level of cGMP via activation of cGMP-specific phosphodiesterase (PDE5A protein), resulting in inactivation of PKG. Our data show that mangostins significantly inhibit the mRNA levels of PDE5A. Decreased levels of PDE5A lead to an elevation in cGMP, followed by PKG-1, and results in the inhibition of b-catenin via transcriptional regulation. One of the therapeutic approaches now used by the pharmaceutical industry is the development of PDE inhibitors for blocking cGMP degradation, thereby having anti-cancer effects, such as apoptosis, differentiation, and anti-angiogenesis (Boswell-Smith, Spina, & Page, 2006). In conclusion, we found that mangostins are potential candidates as inhibitory agents against Wnt/b-catenin signalling. These compounds inhibited the cell proliferation of human colon cancer cells and the transcriptional activity of TCF/b-catenin via inhibition of b-catenin, regardless of the mutational status of b-catenin. The mangostins reduced mRNA expression and protein levels of b-catenin without the phosphorylation and degradation of b-catenin by the typical proteasomal pathway. Rather, the mangostins decreased mRNA levels of b-catenin, through PKG activation that resulted from lowered Wnt5A and elevated cGMP. These novel findings could provide clues to explain which inhibitors of Wnt/ b-catenin signalling can exert anti-cancer effects through the transcriptional regulation of b-catenin that results from activation of
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Table 1 A list of Wnt/cGMP signaling-related genes downregulated in SW480 cells treated with mangostins. Description
Genbank accession number
Gene symbol
Wingless-type MMTV integration site family, member 5A Frizzled homolog 2 (Drosophila) Frizzled homolog 3 (Drosophila) Frizzled homolog 4 (Drosophila) Frizzled homolog 7 (Drosophila) Frizzled homolog 8 (Drosophila) Frizzled homolog 9 (Drosophila) Catenin (cadherin-associated protein), beta 1, 88 kDa Lymphoid enhancer-binding factor 1 Cyclin D1 SMAD family member 2 CXXC finger 4 Calcium binding protein P22 Phosphodiesterase 5A, cGMP-specific
NM_003392 NM_001466 NM_017412 NM_012193 NM_003507 NM_031866 NM_003508 NM_001904 NM_016269 NM_053056 AY134745 NM_025212 NM_007236 NM_001083
WNT5A FZD2 FZD3 FZD4 FZD7 FZD8 FZD9 CTNNB1 LEF1 CCND1 SMAD2 CXXC4
a-Mangostin
c-Mangostin
Average
STDEV
p value
Average
STDEV
p value
0.486 0.238 0.531 0.465 0.392 0.069 0.680 0.502 0.555 0.314 0.245 0.198 0.502 0.423
0.116 0.132 0.107 0.141 0.068 0.021 0.043 0.637 0.330 0.071 0.101 0.227 0.508 0.240
7.83E-04 2.80E-04 7.99E-04 1.37E-03 5.13E-05 8.20E-08 1.01E-04 1.23E-01 3.98E-02 3.67E-05 1.02E-04 1.81E-03 8.21E-02 7.12E-03
0.392 0.172 0.401 0.386 0.451 0.240 0.697 0.962 0.414 0.481 0.415 0.083 0.730 0.897
0.108 0.139 0.111 0.095 0.117 0.259 0.050 1.462 0.318 0.231 0.186 0.064 0.741 1.282
3.07E-04 2.52E-04 3.69E-04 1.81E-04 6.33E-04 3.52E-03 2.40E-04 4.83E-01 1.65E-02 8.85E-03 2.74E-03 7.72E-06 2.81E-01 4.48E-01
Table 2 A list of Wnt/cGMP signaling-related genes upregulated in SW480 cells treated with mangostins. Description
E1A binding protein p300 Guanylate cyclase 2F, retinal Guanylate cyclase activator 1B (retina) Guanylate cyclase activator 1C
Genbank accession number
NM_001429 NM_001522 NM_002098 NM_005459
Gene symbol
EP300 GUCA1B
cGMP signalling. This may also offer more opportunities to find effective chemotherapeutic agents for treating human colorectal cancers by controlling non-classical Wnt/b-catenin signalling. Acknowledgements This work was supported by a KIST-Gangneung Institute intramural research Grant(2Z03480). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2011.06.007. References Akao, Y., Nakagawa, Y., Iinuma, M., & Nozawa, Y. (2008). Anti-cancer effects of xanthones from pericarps of mangosteen. International Journal of Molecular Sciences, 9, 355–370. Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E., Mann, M., et al. (2002). Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: A molecular switch for the Wnt pathway. Genes & Development, 16, 1066–1076. Azebaze, A. G., Meyer, M., Valentin, A., Nguemfo, E. L., Fomum, Z. T., & Nkengfack, A. E. (2006). Prenylated xanthone derivatives with antiplasmodial activity from Allanblackia monticola STANER L.C. Chemical & Pharmaceutical Bulletin (Tokyo), 54, 111–113. Barker, N., & Clevers, H. (2006). Mining the Wnt pathway for cancer therapeutics. Nature Reviews. Drug Discovery, 5, 997–1014. Boswell-Smith, V., Spina, D., & Page, C. P. (2006). Phosphodiesterase inhibitors. British Journal of Pharmacology, 147(Suppl 1), S252–S257. Browning, D. D. (2008). Protein kinase G as a therapeutic target for the treatment of metastatic colorectal cancer. Expert Opinion on Therapeutic Targets, 12, 367–376. Browning, D. D., Kwon, I. K., & Wang, R. (2010). CGMP-dependent protein kinases as potential targets for colon cancer prevention and treatment. Future Medicinal Chemistry, 2, 65–80. Davies, R. J., Miller, R., & Coleman, N. (2005). Colorectal cancer screening: Prospects for molecular stool analysis. Nature Reviews. Cancer, 5, 199–209. Global Cancer Facts & Figures 2007. www.cancer.org. Gopalakrishnan, G., Banumathi, B., & Suresh, G. (1997). Evaluation of the antifungal activity of natural xanthones from Garcinia mangostana and their synthetic derivatives. Journal of Natural Products, 60, 519–524.
a-Mangostin
c-Mangostin
Average
STDEV
p value
Average
STDEV
p value
3.458 8.709 0.827 2.933
5.945 11.545 0.215 1.745
2.57E-01 1.47E-01 1.18E-01 6.37E-02
1.271 23.795 0.579 1.785
1.804 33.011 0.020 1.202
4.04E-01 1.41E-01 1.77E-06 1.61E-01
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