Article pubs.acs.org/jnp
9‑Hydroxycanthin-6-one, a β‑Carboline Alkaloid from Eurycoma longifolia, Is the First Wnt Signal Inhibitor through Activation of Glycogen Synthase Kinase 3β without Depending on Casein Kinase 1α Kensuke Ohishi,† Kazufumi Toume,† Midori A. Arai,† Takashi Koyano,‡ Thaworn Kowithayakorn,§ Takamasa Mizoguchi,† Motoyuki Itoh,† and Masami Ishibashi*,† †
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164-0012, Japan § Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ‡
S Supporting Information *
ABSTRACT: Wnt signaling regulates various processes such as cell proliferation, differentiation, and embryo development. However, numerous diseases have been attributed to the aberrant transduction of Wnt signaling. We screened a plant extract library targeting TCF/βcatenin transcriptional modulating activity with a cell-based luciferase assay. Activity-guided fractionation of the MeOH extract of the E. longifolia root led to the isolation of 9-hydroxycanthin-6-one (1). Compound 1 exhibited TCF/β-catenin inhibitory activity. Compound 1 decreased the expression of Wnt signal target genes, mitf and zic2a, in zebrafish embryos. Treatment of SW480 cells with 1 decreased βcatenin and increased phosphorylated β-catenin (Ser 33, 37, Tyr 41) protein levels. The degradation of β-catenin by 1 was suppressed by GSK3β-siRNA, while compound 1 decreased β-catenin even in the presence of CK1α-siRNA. These results suggest that 1 inhibits Wnt signaling through the activation of GSK3β independent of CK1α.
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destruction complex is phosphorylated by CK1α and GSK3β at the N-terminal Ser45, Thr 41, Ser37, and Ser33 residues. Phosphorylated β-catenin at the Ser37 and Ser33 residues is known to be recognized and ubiquitinated by the F-box βtransducin repeat-containing protein (β-TrCP) and degraded by the proteasome.8 On the other hand, when Wnt ligands bind to the frizzled (Fz) receptor and low-density lipoprotein receptor-related protein5/6 (LRP5/6) co-receptor, this receptor complex inactivates the destruction complex, leading to the accumulation of β-catenin. β-Catenin that accumulated in the cytoplasm was found to translocate to the nucleus and bind to TCF/LEF transcriptional factors, which activated the expression of target genes including c-myc and cyclin D.7 The small molecules that control Wnt signaling may be used in biological studies and as candidates for medicines. Some Wnt signaling inhibitors with various mechanism of actions have already been identified, including salinomycin,9 windorphen,10 IWP-2,11 XAV939,12 and ICG-001.13,14 Salinomycin was previously reported to block phosphorylation of the Wnt coreceptor lipoprotein receptor-related protein 6 (LRP6) and
nt signaling is one of the important cell signaling transduction pathways that play crucial roles in cell proliferation, cell fate determination, embryonic development, and tissue homeostasis.1,2 However, aberrant Wnt signaling underlies a wide range of human diseases including cancer, Alzheimer’s disease, and diabetes.3 Therefore, the Wnt signaling pathway represents an attractive therapeutic target. Patients with FAP (familial adenomatous polyposis) have hundreds to thousands of colorectal tumors, which are known as adenomatous polyps. More than 85% of patients with FAP have mutations in APC (adenomatous polyposis coli), which induces the abnormal activation of Wnt signaling.4 On the other hand, Wnt signaling maintains self-renewal of neural stem cells.5 Wnt3a has been shown to promote the generation of iPS cells in the absence of c-myc, which is a risk factor of tumorigenesis. The Wnt signaling pathway is also known to be involved in the reprogramming of cells.6 β-Catenin plays a central role in the up-regulation of target genes in the Wnt/β-catenin signaling pathway. β-Catenin forms a cytoplasmic destruction complex with other components, including axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β(GSK3β), and casein kinase 1α (CK1α).7 In the absence of Wnt ligands, β-catenin in the cytoplasmic © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 13, 2015
A
DOI: 10.1021/acs.jnatprod.5b00153 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. Structures of isolated compounds from Eurycoma longifolia.
These compounds were identified as 9-hydroxycanthin-6-one (1),20 20,21,22,23-tetrahydro-23-oxoazadirone (2),21 and canthin-6-one 9-O-β-glucopyranoside (3)22 by comparing their spectral data with those in the literature (Figure 1). We investigated the effects of these isolated compounds (1− 3) on TOP activity and viability using STF/293 cells. Compounds 1−3 inhibited TOP activity in a dose-dependent manner (IC50 values, 6.8, 3.0, and 11.6 μM, respectively) with high cell viability (Figure 2). We then assessed FOP15 activity
induced its degradation, thereby down-regulating Wnt/βcatenin signaling. Windorphen has been shown to disrupt the association between β-catenin and p300 and down-regulated the expression of targeted genes. A previous study demonstrated that IWP-2 inhibited the activity of porcupine, an acyltransferase required for the production of Wnt proteins. XAV939 leads to the degradation of β-catenin by stabilizing axin. ICG-001 blocks the interaction between β-catenin and CBP (CREB-binding protein) and decreases the expression of target genes. We recently performed screening for Wnt signal inhibitors with a cell-based luciferase assay system in order to assess TCF/β-catenin transcription activity.15 Using this system, several natural products were identified as Wnt signal inhibitors, including xylogranin B from Xylocarpus granatum16 and calotropin from Calotropis gigantean.17 Xylogranin B decreased β-catenin levels in the nuclear fraction and reduced the expression of target genes. Calotropin increased CK1α and promoted the degradation of β-catenin. By further screening of our plant extract collection, we selected Eurycoma longifolia (root) from plants collected as a hit sample. We performed activity-guided isolation on E. longifolia, which led to the isolation of three active compounds, 1−3, and further investigated the effects of 9-hydroxycanthin-6-one (1) on the Wnt signaling pathway. The zebrafish (Danio rerio) has been used in developmental, cell biology, and disease research.18 Zebrafish embryos are a suitable size for culture plates (96-well or 48-well), and embryonic development is rapid; the formation of organs is almost complete in 72 hpf (hours postfertilization). Zebrafish embryos are transparent during the embryo and larval stages, which allows real-time imaging of the development of organs. Therefore, zebrafish are useful as an in vivo model for analyzing the pharmacological effects of small molecules.19 We herein describe the effects of the β-carboline alkaloid 9-hydroxycanthin-6-one (1) on Wnt signaling in vitro and in vivo using zebrafish.
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Figure 2. TCF/β-catenin inhibitory transcriptional activity of 1−3. Compounds 1−3 inhibited TCF/β-catenin transcriptional activity. Luciferase activities for TOP and FOP were evaluated in STF/293 cells and 293T cells, respectively. Viability was evaluated using STF/ 293 cells. Data are expressed as the mean ± SE (n = 3). The significance of differences was determined with Student’s t-test (*p < 0.05, **p < 0.01 significantly different from the control).
using HEK293T cells transiently transfected with SuperFOPFlash to exclude false positive samples. Since SuperFOPFlash contained six mutated TCF-binding sites (CCTTTGGCC), selective inhibitors of TCF/β-catenin transcription were unlikely to decrease FOP activity. Compounds 1−3 did not exhibit significant effect on FOP activity (Figure 2). These results suggested that 1−3 inhibited TCF/β-catenin transcriptional activity. We then examined the effects of 1−3 against the proliferation of colorectal cancer cell lines. Of the cell lines used, SW480, DLD1, and HCT116 were Wnt-dependent cell lines. SW480 and DLD1 were APC-truncated mutant colon cancer cells,23 while HCT116 is a colon cancer cell with mutant β-catenin.23,24 RKO exerted Wnt-independent cell growth25 and was used as a control for comparisons along with noncancer cells (HEK293 and HEK293T). Compounds 1 and 2 inhibited the viabilities of the Wnt-dependent cell lines (Table 1). HEK293, HEK293T, and RKO cells were not
RESULTS AND DISCUSSION
We screened plant extracts using STF/293 cells, which are 293 cells stably transfected with the luciferase reporter gene (SuperTOPFlash,15 which contains seven LEF/TCF binding sites (CCTTTGATC)), in order to assess TOP inhibitory activity. We selected the extracts of plants collected from Thailand, and the extract of E. longifolia was found to decrease TOP activity with 92% inhibition at 100 μg/mL. The MeOH extract was partitioned with hexane, EtOAc, n-BuOH, and water, and the hexane and EtOAc layers were found to decrease TOP activity (79% and 65% inhibition at 50 μg/mL, respectively). Activity-guided separation of the hexane and EtOAc layers by silica gel, ODS column chromatography, and preparative HPLC led to the isolation of three compounds. B
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reduced TOPdGFP activity without inducing excess cell death (control MO: 100%, n = 10; p53 MO: 100%, n = 10, Figure 3). These results suggest that 1 inhibits Wnt signaling in vivo. We examined the effects of 1 on the target gene of Wnt signaling in zebrafish embryos. Zic2a was previously reported to be a target gene of Wnt signaling28,29 and is required for neural development, including the midbrain tectum.28 Zebrafish embryos were injected with control MO or p53 MO at the one- or two-cell stage and then treated with 1 from 4 hpf to 24 hpf. We found using in situ hybridization that 1 reduced the expression of zic2a in the midbrain tectum (control MO: 55%, n = 6; p53 MO: 54%, n = 7, Figure 4). Microphthalmia-associated transcription factor (mitf) has also been identified as a target gene of Wnt signaling.28,30 Compound 1 also reduced the expression of mitf in neural-crest cells (NCCs) (control MO: 50%, n = 7; p53 MO: 60%, n = 9, Figure 5). Mitf is required for differentiation from NCCs to pigment cells;30 therefore, we focused on the effects of 1 on pigment cells in zebrafish embryos. Zebrafish embryos were treated with 1 from 4 hpf to 30 hpf, and 1 was found to have reduced the number of pigment cells on the tails of zebrafish embryos (100% n = 10, Figure 6). These results suggested that 1 reduced the expression of mitf and blocked the differentiation of NCCs to pigment cells. We subsequently investigated the effects of 1 on the Wnt pathway using SW480 cells, which have a mutation in APC that causes the accumulation of β-catenin. β-Catenin is a transcriptional activator in Wnt/β-catenin signaling; therefore, we examined the effects of 9-hydroxycanthin-6-one (1) on βcatenin protein levels using Western blotting. As a result, 1 decreased β-catenin in both the cytosolic and nuclear fractions (Figure 7). β-Catenin is phosphorylated at the Ser45 residue by CK1α and then at the Thr41, Ser37, and Ser33 residues by GSK3β. The phosphorylation of β-catenin at Ser33 and Ser37 creates a
Table 1. Cytotoxicities of 1−3 against Noncancer Cells and Cancer Cellsa IC50 (μM) colon cancer cells (human) noncancer cells (human embryonic kidney cells)
Wnt-independent
Wnt-dependent
compound
HEK293
HEK293T
RKO
SW480
HCT116
DLD1
1 2 3
36.7 31.2 36.7
>40 >40 >40
>40 26.4 >40
17.4 11.2 >40
30.0 24.4 >40
>40 31.7 >40
a
1 exhibited cytotoxicity against Wnt-dependent colon cancer cell lines, especially for SW480.
significantly affected by 1. These results suggested that 1 exhibited selective cytotoxicity against Wnt-dependent colon cancer cells. Although compound 2 showed more potent inhibition of TOP activity and cytotoxicity against Wntdependent cell lines than compound 1, compound 2 also exhibited cytotoxicity against Wnt-independent RKO cells, suggesting that compound 2 had less selective cytotoxicity. Therefore, compound 1 was selected in subsequent experiments. We determined whether the alkaloid 9-hydroxycanthin-6-one (1) inhibited Wnt signaling in vivo using a transgenic zebrafish line stably expressing the Wnt/β-catenin signaling reporter construct (TOPdGFP), which contained destabilized green fluorescent protein (dGFP) under the confines of a TCF/LEFresponsive element.26 To eliminate false positives in TOPdGFP data due to cell death induced by 1, p53-induced apoptosis was inhibited by injecting p53 morpholino27 antisense nucleotides (MO) with the treatment with 1. Zebrafish embryos were injected with control MO or p53 MO at the one- or two-cell stage and treated with 1 from 10 hpf to 24 hpf, and then TOPdGFP and apoptotic cells were detected. Compound 1
Figure 3. Effect of 9-hydroxycanthin-6-one (1) on TOPdGFP transgenic zebrafish embryos. Compound 1 reduced TOPdGFP activity in 24 h postfertilization (hpf) TOPdGFP transgenic zebrafish embryos, which were co-injected with control MO or p53 MO. All panels showed the left side of the zebrafish head. Top panels were bright-field (BF) images under a microscope. White dashed circles in the middle panels indicated TOPdGFP visualized in the zebrafish midbrain tectum by fluorescence microscopy. Red fluorescent signals in the bottom panels were apoptotic cells detected by the TUNEL apoptosis assay. C
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Figure 4. Effect of 9-hydroxycanthin-6-one (1) on expression of zic2a in the zebrafish embryos. Compound 1 reduced the expression of zic2a in 24 hpf zebrafish embryos, which were co-injected with control MO or p53 MO. Panels show whole mount in situ hybridization for zic2a. Red dashed lines indicate the midbrain tectum. Red arrows indicate regions of reductions.
Figure 5. Effect of 9-hydroxycanthin-6-one (1) on expression of mitf in the zebrafish embryos. Compound 1 reduced the expression of mitf in 24 hpf zebrafish embryos that were co-injected with control MO or p53 MO. Panels show whole mount in situ hybridization for mitf. Black and red arrowheads indicate the neural-crest cells (NCCs) on the ventral side. Red arrowheads indicate regions of reductions.
increased p-β-catenin at Ser 33, 37, and Thr 41 (p-βcatenin(S33/S37/T41)) protein levels. However, p-β-catenin at Ser 45 (p-β-catenin(S45)) protein levels remained unchanged by the treatment with 1, as did GSK3β and CK1α protein levels (Figure 8). These results suggested that 1 enhanced GSK3β enzyme activity.
Figure 6. Effect of 9-hydroxycanthin-6-one (1) on pigment cells in the zebrafish embryos. Compound 1 reduced the number of pigment cells in 30 hpf zebrafish embryos. Black arrowheads in the lower panel indicate pigment cells in the tail region.
Figure 8. Effects of 9-hydroxycanthin-6-one (1) on the expression of β-catenin, p-β-catenin, GSK3β, and CK1α in SW480 cells. Compound 1 decreased β-catenin protein levels and increased p-β-catenin(S33/ S37/T41).
recognition site for β-TrCP (ubiquitin ligase), which leads to degradation by the ubiquitin−proteasome system.31 Therefore, the effects of 1 on CK1α, GSK3β, and the phosphorylation of β-catenin were investigated. 1 decreased β-catenin and
Figure 7. 9-Hydroxycanthin-6-one (1) decreased β-catenin levels in SW480 cells. Cell lysates were analyzed by Western blotting. D
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This result was further supported by an examination of the effects of 1 by the knockdown of GSK3β. Among the sites phosphorylated by GSK3β, the Thr 41, Ser37, and Ser33 residues, the two phosphorylation sites of β-catenin at the Ser37 and Ser33 residues were essential for recognition by βTrCP, and this process was important for the degradation of βcatenin by the ubiquitin−proteasome system. As shown in Figure 9, 1 decreased β-catenin levels in the presence of control
by 1 was most likely mediated through the activation of GSK3β. We focused on the results shown in Figure 8. Compound 1 decreased β-catenin and increased p-β-catenin(S33/S37/T41) protein levels. On the other hand, p-β-catenin(S45) protein levels remained unchanged. Therefore, we hypothesized that 1 induced the degradation of β-catenin without the phosphorylation of β-catenin by CK1α. As shown in Figure 11, 1
Figure 9. Effects of 9-hydroxycanthin-6-one (1) on the Wnt signaling in the presence of GSK3β siRNA. SW480 cells were transfected with control siRNA or GSK3β siRNA for 3 h and then incubated with 1.
Figure 11. Effects of 9-hydroxycanthin-6-one (1) on the Wnt signaling in the presence of CK1α siRNA. SW480 cells were transfected with control siRNA or CK1α siRNA for 6 h and then incubated with 1.
siRNA. However, the reductions induced in β-catenin levels by 1 were attenuated in the presence of GSK3β siRNA. Phosphorylated GSK3β Ser 9 is an inactive form that does not promote β-catenin degradation.32 1 decreased p-GSK3β at Ser 9 (p-GSK3β(S9)) protein levels. These results suggested that 1 enhanced GSK3β activity and the phosphorylation of βcatenin at GSK3β sites, thereby leading to the degradation of βcatenin. We also examined the effects of 1 on the zebrafish when treated together with a GSK3β inhibitor. The aberrant activation of Wnt signaling induces a small eye or eyeless phenotype in zebrafish embryos because Wnt signaling is essential for brain development.10,33 BIO (6-bromoindirubin3′-oxime) is a GSK3β inhibitor (Wnt signaling activator)34 that has been shown to induce the eyeless phenotype in zebrafish embryos.10,35 Therefore, we determined whether 1 inhibited Wnt signaling mediated by GSK3β in vivo. Zebrafish embryos were treated with 1 and BIO from 4 hpf to 30 hpf. As expected, the treatment of zebrafish embryos with BIO induced the eyeless phenotype (no eyes 100% n = 13). However, the combined treatment of 1 and BIO rescued the eyeless phenotype caused by BIO (rescued eyes, 46%, n = 6, Figure 10, and slightly rescued eyes 54%, n = 7, as shown in Figure S1, respectively). These results suggested that 1 down-regulated Wnt signaling through GSK3β. Taken together, these in vitro and in vivo results suggested that the inhibition of Wnt signaling
decreased β-catenin and increased p-β-catenin(S33/S37/T41) protein levels in the presence of control siRNA. Compound 1 also decreased β-catenin and increased p-β-catenin(S33/S37/ T41) protein levels in the presence of CK1α siRNA. Compound 1 decreased p-GSK3β(Ser9) in the presence of CK1α siRNA. These results suggest that 1 inhibits Wnt signaling through the activation of GSK3β without depending on CK1α. In conclusion, activity-guided separation of the E. longifolia roots extract led to the isolation of 9-hydroxycanthin-6-one (1). Compound 1 inhibited Wnt signaling by activating GSK3β independent of CK1α. Although the expected binding protein of compound 1 was not known, a decrease of phosphorylated GSK3β (inactive form) (Figure 9) may lead to activation of the GSK3β function. CK1α is a priming kinase for the phosphorylation of β-catenin at the Ser45 residue, after which the Thr41, Ser37, and Ser33 residues of β-catenin are phosphorylated by GSK3β. Phosphorylated β-catenin is subjected to ubiquitination and degradation by the ubiquitin−proteasome system. Therefore, phosphorylation of βcatenin at the Ser45 by CK1α is essential for degradation of β-catenin. However, 1 decreased β-catenin in the presence of CK1α siRNA (Figure 8). This result suggested a novel discovery that β-catenin leads to degradation without phosphorylation by CK1α. Compound 1 is therefore not amenable to the current theory that β-catenin is sequentially phosphorylated by CK1α and GSK3β (Figure S2), implying that compound 1 is the first Wnt signal inhibitor through GSK3β activation without depending on CK1α.
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EXPERIMENTAL SECTION
General Experimental Procedure. Column chromatography was performed using silica gel PSQ100B Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan). Preparative HPLC was performed using YMC-Pack ODS-AM (YMC Co., Ltd., Kyoto, Japan). Protein concentrations were measured using a Nano Drop 2000 spectrophotometer (Thermo). Plant Materials. Eurycoma longifolia (Simaroubaceae) stems were collected from Khon Kaen, Thailand, and identified by T. Kowithayakorn. A voucher specimen (KKP315) was deposited at
Figure 10. Effects of 9-hydroxycanthin-6-one (1) on eyeless phenotype induced by BIO in the zebrafish embryos. Compound 1 rescued the eyeless phenotype in 30 hpf zebrafish embryos. As indicated, red dashed lines show rescued eyes in the right panels. E
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the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University. Activity-Guided Extraction and Isolation. The air-dried roots of E. longifolia (51.8 g) were extracted with MeOH overnight at room temperature, followed by homogenization, filtration, and evaporation under reduced pressure to obtain a crude extract (4.4 g). The extract was suspended in 10% aqueous MeOH (100 mL) and partitioned between hexane, EtOAc, and BuOH (100 mL × 3) to obtain hexane (780 mg), EtOAc (467 mg), BuOH (683 mg), and aqueous (2.4 g) soluble fractions. The hexane-soluble fraction having 79% TOP inhibitory activity at 50 μg/mL (780 mg) was subjected to silica gel PSQ100 column chromatography (⦶ 2.5 × 30 cm) eluted with the hexane/EtOAc solvent system (9:1−0:1, then washed with 1%TFA in MeOH), yielding the fractions 1A to 1E. Fraction 1D (56 mg), which was eluted with hexane/EtOAc (1:9) and had 20% TOP inhibitory activity at 25 μg/mL, was subjected to ODS column chromatography (⦶ 2.5 × 30 cm) eluted with the MeOH/H2O solvent system (6:4− 0:1, then washed with 1% TFA in MeOH) to afford fractions 2A to 2H. Fraction 2C (5.4 mg), which was eluted with MeOH/H2O (8:2) and had 24% TOP inhibitory activity at 25 μg/mL, was further separated by preparative HPLC [YMC-Pack ODS-AM, ⦶ 10 × 250 mm, MeOH/H2O (70:30), flow rate: 2.0 mL/min, RI and UV detection at 254 nm] to give 1 (8.6 mg, tR 11 min). Fraction 1C (232.0 mg), which was eluted with hexane/EtOAc (4:6 and 3:7, then washed with MeOH and 1% TFA) from the first silica gel column and had 33% TOP inhibitory activity at 25 μg/mL, was subjected to silica gel column chromatography (2 × 30 cm) eluted with a hexane/EtOAc solvent system (95:5−0:1, then washed with MeOH) to afford fractions 4A to 4H. Fraction 4F (26.6 mg), which was eluted with hexane/EtOAc (6:4 and 5:5) and had 31% TOP inhibitory activity at 25 μg/mL, was further separated by preparative HPLC [YMC-Pack ODS-AM, ⦶ 10 × 250 mm, MeOH/H2O (80:20), flow rate: 2.0 mL/ min, RI and UV detection at 254 nm] to give 2 (0.8 mg, tR 16 min). The EtOAc-soluble fraction having 65% TOP inhibitory activity at 50 μg/mL (467 mg) was subjected to ODS column chromatography (⦶ 2.5 × 30 cm) eluted with the MeOH/H2O solvent system (3:7−0:1, then washed with 1% TFA in MeOH) to afford fractions 6A to 6G. Fraction 6F (86 mg), which was eluted with MeOH/H2O (7:1 and 9:1) and had 36% TOP inhibitory activity at 20 μg/mL, was further separated by preparative HPLC [YMC-Pack ODS-AM, ⦶ 10 × 250 mm, MeOH/H2O (7:3), flow rate: 2.0 mL/min, RI and UV detection at 254 nm] to give 1 (7.4 mg, tR 11 min). Fraction 6C (140.0 mg), which was eluted with hexane/EtOAc (4:6 and 5:5), was subjected to silica gel column chromatography (2 × 20 cm) eluted with a CHCl3/ MeOH solvent system (1:0−0:1, then washed with MeOH and 1% TFA) to yield fractions 8A to 8G. Fraction 8E (10.8 mg), which was eluted with hexane/EtOAc (8:2 and 7:3) and had 40% TOP inhibitory activity at 20 μg/mL, was subjected to preparative HPLC [YMC-Pack ODS-AM, ⦶ 10 × 250 mm, MeOH/H2O, 43:57, flow rate: 2.0 mL/ min, RI and UV detection at 254 nm] to give 3 (2.0 mg, tR 13 min). Cell Cultures. RKO, HCT116, and DLD1 cells were purchased from ATCC; SW480 cells were derived from the Institute of Development, Aging, and Cancer, Tohoku University. HEK293T was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. The STF/293 cell line was a generous gift from Prof. Jeremy Nathans (John Hopkins Medical School). HEK293, STF/293, HEK293T, HCT116, SW480, and DLD1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with fetal bovine serum (10% FBS). RKO cells were cultured in Eagle’s minimal essential medium (EMEM) with fetal bovine serum (10% FBS). Cultures were maintained in a humidified incubator at 37 °C in 5% CO2/95% air. Viability Assay. Cells were seeded on a 96-well culture plate (STF/293 3 × 104 cells/well) in 200 μL of medium containing FBS. Cells were incubated at 37 °C in a 5% CO2 incubator for 24 h. Test samples at different doses dissolved in medium were added to each well. After a 24 h incubation, cells were washed with PBS, and 200 μL of PBS containing fluorescein diacetate (10 μg/mL; Wako Pure Chemical Industries, Osaka, Japan) was added to each well. The plates were incubated at 37 °C for 1 h, and fluorescence at 538 nm with
excitation at 485 nm was measured using Fluoroskan Ascent (Thermo). Luciferase Assay. Stable reporter cells (STF/293 cells 3 × 104/ well) were seeded on 96-well plates and treated 24 h later with compounds and LiCl (final conc. 15 mM). After being incubated for 24 h, cells were lysed with CCLR (20 μL/well cell culture lysis reagent; Promega, Fitchburg, WI, USA), and luciferase activity was measured with a Luciferase 1000 assay system (Promega) using a Luminoskan Ascent (Thermo Fisher, Waltham, MA, USA). Assays were performed at least in triplicate. Quercetin (27.7 μM) was used as a positive control. Transient transfection was conducted by Lipofectamine 2000 (Invitrogen, USA). 293T cells (1 × 105/well) were split into 24-well plates. After a 24 h incubation, cells were treated with 1 μg/well of the luciferase reporter construct (SuperFOPFlash) and 0.025 μg/well of pRL-CMV (for normalization, Promega) dissolved in Opti-MEM for transfection. Transfection was conducted for 4 h. After transfection, media were replaced with new medium containing compounds and 15 mM LiCl. Cells incubated for 24 h were lysed by shaking for 45 min in passive lysis buffer (Promega, 50 μL/well), and luciferase activity was measured with a PICAGENE Dual Seapansy (Toyo Ink, Tokyo, Japan) using Luminoskan Ascent (Thermo Fisher). The assay sample was stored as a 10 mM solution in DMSO, then diluted to the indicated concentrations with medium. The final concentration of DMSO was less than 0.1% (v/v). Luciferase activity was compared with that of a control to which no compound was added. Preparation of Whole Cellular, Cytosolic, and Nuclear Proteins. SW480 cells (1 × 106) were seeded on 90 mm dishes and, after a 24 h incubation, were treated with 1 for 24 h. Cells were then harvested after trypsinization. Whole cellular proteins were obtained by lysing cells in ice-cold lysis buffer containing 20 mM TrisHCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 10 mM EDTA, 1 mM Na3VO4, 0.1 mM NaF, and 1% protease inhibitor cocktail (1%; #25955-11, Nacalai Tesque, Kyoto, Japan) for 30 min. The lysates were centrifuged at 13 000 rpm at 4 °C for 30 min, and supernatants were stored at −80 °C before use. Cytosolic and nuclear proteins were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Thermo) from trypsinized cells, following the manufacturer’s protocol, and proteins were stored at −80 °C. Western Blot Analysis. Protein was mixed 4:1 with 5× sample buffer (20% 2-ME, 20% sucrose, 8% SDS, 0.02% bromophenol blue, and 0.25 mM Tris-HCl, pH 6.8), boiled (100 °C for 3 min), then loaded onto a 10% SDS-PAGE gel, and run at 200 V 20−30 mA. Proteins were transferred electrophoretically onto an Immunblot polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocking with skimmed milk (3%) or bovine serum albumin (BSA, 1%, for p-β-catenin, p-GSK3β) in TBST for 1 h, the membrane was incubated at room temperature with the primary antibodies [anti-β-catenin, 1:2000, #610153, BD Biosciences; anti-p-βcatenin(S33/S37/T41), 1:1000, #9561, Cell Signaling; anti-p-βcatenin(S45), 1:1000, #9564, Cell Signaling; anti-GSK3β, 1:1000, #sc-71186, Cell Signaling; anti-p-GSK3β(S9), 1:1000, #sc-9336, Santa Cruz Biotechnology; anti-CK1α, 1:1000, #sc-6477, Santa Cruz Biotechnology; β-actin, 1:4000, #A2228, Sigma; histone H1 (AE-4), sc-8030, #1:1000, Santa Cruz Biotechnology] for 1 h. β-Actin and histone H1 were used as internal controls. After being washed with TBST, the membrane was incubated at room temperature with HRPconjugated anti-mouse IgG (1:4000, #NA931VS, GE Healthcare), anti-rabbit IgG (1:4000, #111-035-144, Jackson ImmunoResearch), or anti-goat IgG (1:4000, #A5420, Sigma). After further washing with TBST, immunoreactive bands were detected using the ECL Advance Western detection system (GE Healthcare) or Immobilon Western chemiluminescent HRP substrate. Chemiluminescent signals were imaged with ChemiDoc XRS Plus (Bio-Rad). The quantity of each protein was normalized with respect to β-actin or histone H1. GSK3β Knock-down Experiment by GSK3β siRNA. SW480 cells (1 × 105/well) were seeded on a 24-well plate, incubated for 24 h, transfected with control siRNA (10 pmol; #sc-37007, Santa Cruz Biotechnology) or GSK3β siRNA (10 pmol; #sc-35527, Santa Cruz F
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Biotechnology) by using Lipofectamine 2000 (2 μL; Invitrogen, USA) in accordance with the manufacturer’s instructions, and incubated for 3 h. Additionally, 1 was treated with DMEM containing FBS (10%) and incubated for 24 h. Whole cellular proteins were obtained and stored by the protocol described above. CK1α Knock-down Experiment by CK1α siRNA. SW480 cells (5 × 104/well) were seeded on a 24-well plate, incubated for 24 h, transfected with control siRNA (10 pmol; #sc-37007, Santa Cruz Biotechnology) or CK1α siRNA (10 pmol; #sc-37007, Santa Cruz Biotechnology) by using Lipofectamine 2000 (2 μL; Invitrogen, USA) in accordance with the manufacturer’s instructions, and incubated for 6 h. Additionally, 1 was treated with DMEM containing FBS (10%) and incubated for 24 h. Whole cellular proteins were obtained and stored by the protocol described above. Zebrafish Lines and Maintenance. Zebrafish were raised and maintained under standard conditions with approval by the Institutional Animal Care and Use Committee at Chiba University. Zebrafish embryos were obtained from natural spawnings of the wild-type AB strain and TOPdGFP strain. Morpholino Injections. Antisense morpholino oligonucleotides (MOs) (standard control: 5′-CCTCTTACCTCAGTTACAATTTATA-3′; p53: 5′-GCGCCATTGCTTTGCAAGAATTG-3′) were obtained from Gene Tools, USA. A 5 ng sample of standard control and p53 MO27 were injected at the oneor two-cell stage. Whole-Amount in Situ Hybridization. Zebrafish embryos were injected with control MO or p53 MO at the one- or two-cell stage and incubated from 4 hpf to 24 hpf at 28.5 °C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) with 1 (300 μM). Embryos were fixed in 4% PFA O/N. Digoxigenin-labeled RNA antisense probes were prepared from templates encoding zic2a36 and mitf.37 After in situ hybridization, the expression of zic2a and mitf was detected by NBT/BCIP and imaged using reflection microscopy. Reporter Assay and TUNEL Apoptosis Assay. The Wnt/βcatenin signaling reporter construct (TOPdGFP) (Dorsky et al., 2002) was stably transfected into zebrafish embryos injected with control MO or p53 MO at 0.75 hpf and incubated from 10 hpf to 24 hpf at 28.5 °C in E3 medium with 1 (300 μM). TOPdGFP in the zebrafish midbrain tectum was visualized by fluorescence microscopy. Apoptotic cells was detected using the in situ Cell Death Detection Kit (Roche Applied Science) according to the manufacturer’s instructions. Rescued Experiments by 1 in the Zebrafish Embryos. Zebrafish embryos were treated with BIO (0.5 μM) or both BIO and 1 (100 μM) from 4 hpf to 30 hpf at 28.5 °C in E3 medium and then observed with a stereomicroscope.
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Foundation for the Advancement of Biochemistry, and the Uehara Memorial Foundation.
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Figures S1and S2. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +81-43-226-2923. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Prof. J. Nathans (John Hopkins University School of Medicine) for the STF/293 cells and Prof. R. T. Moon (University of Washington) for the SuperFOPFlash plasmid. This study was supported by JSPS KAKENHI Grant Numbers 26293022, 26305001, 25670045, 25870128, and 23404007 and MEXT KAKENHI Grant Number 23102008 (a Grant-in-Aid for Scientific Research on Innovative Areas in “Chemical Biology of Natural Products”), the Cosmetology Research Foundation, the Hamaguchi G
DOI: 10.1021/acs.jnatprod.5b00153 J. Nat. Prod. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jnatprod.5b00153 J. Nat. Prod. XXXX, XXX, XXX−XXX
