Notch Inhibitors from Calotropis gigantea That Induce Neuronal

Article pubs.acs.org/jnp

Notch Inhibitors from Calotropis gigantea That Induce Neuronal Differentiation of Neural Stem Cells Tatsuro Yoneyama,† Midori A. Arai,*,† Ryuta Akamine,† Kazune Koryudzu,† Anna Tsuchiya,† Samir K. Sadhu,‡ Firoj Ahmed,§ Motoyuki Itoh,† Ryuichi Okamoto,⊥ and Masami Ishibashi*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Pharmacy Discipline, Khulna University, Khulna 9208, Bangladesh § Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka 1000, Bangladesh ⊥ Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan ‡

S Supporting Information *

ABSTRACT: Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease occur due to loss of the structure and function of neurons. For the potential treatment of neurodegenerative diseases, accelerators of neuronal differentiation of neural stem cells (NSCs) have been focused on and a cell-based assay system for measuring Notch signaling pathway activity was constructed. Using this assay system, eight compounds isolated from Calotropis gigantea were identified as inhibitors of the Notch signaling pathway. Hes1 and Hes5 are target genes of the Notch signaling pathway, and compound 1, called uscharin, decreased the protein levels of Hes1 and Hes5 in assay cells and MEB5 cells (mouse NSCs). Furthermore, uscharin (1) enhanced the differentiation of MEB5 cells into neurons. The mechanism of uscharin (1) for the Notch signaling inhibitory activity would be acceleration of the degradation of the Notch intracellular domain (NICD) in the MEB5 cells.

N

Hairy and enhancer of split 1 (Hes1) is a member of the family of repressor-type basic helix−loop−helix (bHLH) molecules, which repress the differentiation of NSCs (Figure 1).5 Hes1 inhibits the differentiation of NSCs in at least two ways.6 First, the Hes1 homodimer binds to the N-box (CACNAG) of the activator-type bHLH transcriptional factors such as Mash1 and Neurog(Ngn)2, which activate the differentiation of NSCs, and suppresses their transcription. Second, Hes1 forms heterodimers with E47, which is necessary for acceleration of NSCs by activator-type bHLH molecules. Hes1 and Hes5 are target genes that are regulated by the Notch signaling pathway. The Notch signaling pathway is conserved evolutionarily and plays a role in various cells to control cell fate, including proliferation and differentiation of NSCs through cell−cell interaction (Figure 1).7 Direct interaction of the ligand protein Jagged or Delta with the Notch protein triggers cleavage at site 2 (S2) by ADAM/TACE metalloprotease and S3/S4 cleavage by γ-secretase8 to produce the Notch intracellular domain (NICD). After sequential processing, NICD translocates to the nucleus and forms a heterotrimer with mastermind-like protein

eurodegenerative diseases, the most common cause of dementia, are problematic worldwide. Approximately 46.8 million people worldwide were living with dementia in 2015. Degeneration of basal forebrain cholinergic neurons occurs early in Alzheimer’s disease (AD). To treat AD, the acetylcholine esterase inhibitors donepezil, galanthamine, and rivastigmine, as well as an N-methyl-D-aspartate receptor antagonist, mematine, are prescribed. Despite these AD medications, the condition of each patient tends to continues to worsen. Although multiple efforts are under way to develop new agents, no treatments are available to halt AD progression. Another strategy under investigation by many scientists for treating neurodegenerative diseases is therapy that targets neural stem cells (NSCs). NSCs have the properties of selfproliferation and pluripotency and are found in the adult human brain.1 Thus, inducing endogenous NSCs to differentiate or transplantation of NSCs may be an effective treatment for neurodegenerative diseases or brain injury. Neuronal cells derived from embryonic stem cells or induced pluripotent stem cells have been transplanted in vivo to restore the functions of neuronal systems.2−4 However, these cell types are associated with problems including the inefficient, nonselective differentiation into neural cells and ethical issues regarding embryonic stem cells. © 2017 American Chemical Society and American Society of Pharmacognosy

Received: March 31, 2017 Published: August 17, 2017 2453

DOI: 10.1021/acs.jnatprod.7b00282 J. Nat. Prod. 2017, 80, 2453−2461

Journal of Natural Products

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The natural products curcumin14 and genistein15 down-regulate Notch1. Previous studies of Notch inhibitors mostly focused on their effects as cancer therapeutic agents against T cell acute leukemia or other related cancers.16,17 On the other hand, DAPT was also reported to effect the neurogenesis of NSCs derived from human embryonic stem cells through the Notch signaling inhibition.18 There are not many reports of Notch signaling inhibitors for acceleration of neural stem cells. Our current study focused on neurogenesis via inhibition of Notch signaling.

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RESULTS AND DISCUSSION First, a cell-based reporter assay with the T-REx system to evaluate Notch signaling inhibitory activity was constructed. The assay cells were constructed based on LS174T cells (TetOn NICD1 cells),19 which produce N-terminally FLAG-tagged mouse Notch1(1704-2531) in the presence of doxycycline (Dox). Tet-On NICD1 cells were then transfected with pGL4.20-12×RBP-J binding site (12 copies of CGTGGGAA)-β-globin promoter, and stably transfected cells were selected with puromycin (final concentration 50 μg/mL). In these assay cells, in the absence of Dox, expression of Notch1(1704-2531), which is 40 amino acids longer than NICD at the N-terminus, is suppressed by TetR (Figure 3).

Figure 1. Notch signaling pathway and Hes1. Interaction of ligands such as Delta or Jagged that are expressed on the surface of the cell sending the signal with the receptor Notch triggers the cleavage of the Notch protein by ADAM/TACE metalloproteases and γ-secretase to produce NICD. NICD moves to the nucleus and forms a trimer with RBP-J and MAML to initiate transcription of target genes, such as Hes1. In the nucleus, Hes1 forms a homodimer and inhibits the transcription of Mash1 and Ngn2. NICD: Notch intracellular domain, MAML: mastermind-like protein (co-activator), Hes1: hairy and enhancer of split 1, bHLH: basic helix−loop−helix, TLE: transducinlike enhancer of split (co-repressor), Grg: Groucho-related gene.

(MAML) and DNA binding protein recombination signal binding protein for the immunoglobulin kappa J region (RBPJ) to initiate transcription of the target genes, Hes1, Hes5, Hey1, Hey2, Deltex1, and cyclin D1.9 Notch signaling regulates differentiation of NSCs via Hes1 (Figure 2).10 In NSCs, self-renewal of stem cells is up-regulated

Figure 2. Notch signaling regulates NSC differentiation. The Notch signal promotes self-renewal and induces survival signals of neural stem cells to inhibit cell death. Differentiation of glial progenitors into mature astrocytes is promoted by the Notch signal. On the other hand, the Notch signal inhibits the differentiation of neural progenitors into neurons.

Figure 3. Assay system using the T-REx system. Without Dox, expression of Notch1(1704-2531) is suppressed by TetR. Addition of Dox allows the expression of Notch1(1704-2531) due to binding with TetR. Notch1(1704-2531) is then cleaved by endogenous γ-secretase to form NICD, which translocates to the nucleus and forms a heterotrimer with RBP-J and MAML. The trimer binds to a 12× RBPJ binding site to express luciferase protein. Luciferase activity was detected as Notch signaling pathway activity. Dox: doxycycline, TetR: tetracycline repressor, Luc: luciferase.

by Notch, whereas maturation of neural progenitors into neuronal cells is inhibited. In glial progenitors, Notch activates differentiation into astrocytes. Furthermore, differentiation of oligodendrocyte precursors into mature oligodendrocytes is inhibited by Notch signaling. In the present study, Notch signaling inhibitors of natural origin that induce neuronal differentiation of NSCs were identified. Various Notch inhibitors have been reported including γ-secretase inhibitors, DAPT (N-[N-(3,5-difluorophenacetyl)- L -alanyl]-S-phenylglycine tert-butyl ester), 11 LY411,575,12 and SHAM1, a MAML1-derived peptide that inhibits the binding of full-length MAML1 to NICD1-RBP-J.13

When Dox is added, repression by TetR is arrested, and Notch1(1704-2531) is expressed. Then, Notch1(1704-2531) is cleaved by γ-secretase to give NICD, which translocates to the nucleus and forms a heterotrimer with RBP-J to contribute to luciferase transcription. Cell viability was measured by a 2454



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Figure 4. Inhibition of Notch transcriptional activity and cell viability by 1−8. Assay cells (LS174T Notch cells) were cultured in a 96-well white plate at 2 × 104 cells/well at 37 °C for 12 h. After incubation, 50 ng/mL Dox was added to each well to induce expression of exogenous mNotch1(1704-2531) protein, and cells were incubated at 37 °C for 12 h. The medium was replaced with Dox-free medium containing individual samples. After treatment at 37 °C for 12 h, luciferase activity was measured. Viability was evaluated by the FMCA method. These assays were performed in 0.1% DMSO. Error bars represent SD (n = 3).

fluorometric microculture cytotoxicity assay20 at the same time to check the cytotoxicity in the samples. Next, the assay conditions (incubation time, cell density, amount of Dox) were established. First, the concentration of Dox was determined. Assay cells (LS174T Notch cells) were seeded (2 × 104 cells/well) in 96-well plates. After 12 h, Dox at each concentration (0, 0.1, 0.5, 1, 10, 50, 100 ng/mL) was added, and luciferase activity was detected at each time point (6, 12, 18, 24 h). Assay cells (LS174T Notch cells) treated with Dox (100, 50, 10 ng/mL) were saturated at 12 h, and we used a

12 h incubation of Dox (Figure S1, Supporting Information). Next, to determine the cell density and incubation time after treatment with Dox, cells were seeded at several densities (1 × 104, 2 × 104, 5 × 104, 1 × 105 cells/well). After a 12 h incubation, Dox (50 ng/well) was added, and luciferase activities were measured after another 6, 12, 24, 36, or 48 h of incubation. Luciferase activities were saturated after 12 h at each cell density (Figure S2, Supporting Information). To assess the Notch inhibitory activity, samples should be added in the presence of NICD. Therefore, Western blotting was used, 2455



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Chart 1

and NICD was detected 6−24 h after treatment with Dox (50 ng/mL), suggesting that samples should be used at these time points (Figure S3, Supporting Information). From these results, the assay conditions were determined (cell density: 2 × 104 cells/well, Dox concentration: 50 ng/mL, sample incubation: 12 h). In the screening of a plant extract library, a Calotropis gigantea (L.) W. T. Aiton (Asclepiadaceae) extract showed strong inhibitory activity. Previously, we reported the isolation of six compounds, uscharin (1),21 asclepin (2),22 2″oxovoruscharin (3),23 calotropin (4),22 voruscharin (5),24 and uscharidin (6),24 from C. gigantea (Figure 4).25 In this study, the remaining extract of C. gigantea was analyzed to find additional Notch inhibitors. Active fractions of C. gigantea were purified by using chromatographic techniques, resulting in the isolation of calactinic acid methyl ester (7)26 and 16αhydroxyuscharin (8).27 Compounds 1−8 were identified by comparing their spectroscopic data to published values. These eight compounds showed dose-dependent Notch inhibitory activity (Figure 4 and Table 1). Comparison of the

Figure 5. Inhibition of the protein expression by 1. Assay cells (LS174T Notch cells) were seeded in a 6 cm dish at 1 × 106 cells/dish and incubated for 12 h at 37 °C. After 12 h of incubation with 50 ng/ mL Dox and washing with PBS, cells were treated with 1 for 12 h. The effect on protein expression was analyzed by Western blotting. Western blot analysis of Hes1 and Hes5 in whole lysates of assay cells (LS174T Notch cells) after a 12 h treatment with 1. β-Actin was used as internal control.

activity assay, compounds 1, 2, and 4 did not affect the activity, suggesting that these compounds do not directly affect γsecretase (data not shown). To evaluate the effect of Notch inhibitors on NSC differentiation, the conditions for the NSC differentiation assay were determined. First, it was tested whether MEB5 cells28 are responsive to Notch signaling, because no reports have been published about the relationship between Notch signaling and differentiation of MEB5 cells. To assess the responsiveness to Notch signaling, a cotransfection assay with TP-129 (RBP-J-Luc; 400 ng/well) and mNotch1-RAMIC30,31 (20 or 100 ng/well) was performed. Luciferase activity was increased according to the amount of transfected mNotch1RAMIC, suggesting that MEB5 cells express components of the Notch signaling pathway (Figure 6A). Then, Western blot analysis was performed to assess the presence of NICD in MEB5 cells. Dissociated MEB5 cells were seeded at several densities (Figure 7, [A] 2 × 104, [B] 4 × 105, and [C] 2 × 106 cells/well) and lysed at three time points ([1] soon after dissociation, [2] 12 h after seeding, and [3] 36 h after seeding). In the lowest density (2 × 104 cells/well), NICD was not produced (Figure 7A), although Notch1 was expressed

Table 1. Notch Signal Inhibitory Activity (μM) by Compounds 1−8 1

2

3

4

IC50

0.4 5

0.3 6

0.3 7

0.8 8

IC50

2

17.3

0.7

2.4

Notch inhibitory activity of compound 7 with the other compounds suggests the importance of the aglycone and sugar moiety at the C-2 and C-3 positions. Western blot analysis revealed the effect of uscharin (1) in assay cells. Uscharin (1) decreased the protein levels of the target genes Hes1 and Hes5, whereas this compound did not affect the protein level of Notch1(1704-2531) (Figure 5 and Figure S4, Supporting Information). As shown in Figure 5, uscharin (1) decreased the protein levels of Hes1 stronger than Hes5 at 0.1 and 1 μM. On the other hand, DAPT reduced Hes5 stronger than Hes1 at the same concentration (data not shown). In the γ-secretase 2456



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Figure 6. Notch reporter activity in MEB5 cells transfected with plasmids. Dissociated MEB5 cells were seeded at a density of [A] 2 × 104 cells/well or [B] 2 × 106 cells/well in 24-well plates and incubated for 12 h. MEB5 cells were transfected with [A] pRL-CMV (25 ng/well), TP-1 (400 ng/ well), and Notch1-RAMIC (20 or 100 ng/well) or [B] pRL-CMV (25 ng/well) and TP-1 (400 ng/well). After 24 h of incubation, cells were lysed to measure firefly and renilla luciferase activities. TP-1: RBP-J binding site-Luc, Notch1-RAMIC: pEF-BOSneo-mNotch1 RAMIC.

Figure 7. Western blot of MEB5 cells seeded at each cell density after dissociation. Dissociated MEB5 cells were seeded in 6 cm dishes at individual cell densities and incubated for 12 h. After washing with NeuroCult basal medium (serum-free medium), cells were incubated in differentiation medium. [A] 2 × 104 cells/well, [B] 4 × 105 cells/well, [C] 2 × 106 cells/well. [1] soon after dissociation, [2] 12 h after seeding, and [3] 36 h after seeding. [4] shows the NICD levels after treatment with 10 μM DAPT for 12 h.

secretase component proteins, Nicastrin, PS1, PS2, and PEN2, were not affected, it may be speculated that the activity of uscharin (1) involves the degradation of NICD. To evaluate the effect of this compound on degradation of NICD via the proteasome pathway, uscharin (1) and MG132, a proteasome inhibitor, were applied together. NICD was recovered by cotreatment of uscharin (1) and MG132 (Figure 9b). This result suggested that uscharin (1) inhibited the Notch signaling pathway by increasing the degradation of NICD via the proteasome pathway (Figure 9C). Notch signaling interacts with other pathways, such as Wnt signaling and Hh signaling. Wnt signaling facilitates the selfproliferation and differentiation of NSCs via the target gene Nmyc.32 Furthermore, β-catenin induces Notch signaling by binding to NICD and activates transcription of target genes.33 Carotlopin (4), which has a similar structure to uscharin (1), inhibits the Wnt signaling pathway by activating CK1α and accelerating the degradation of β-catenin.25 Uscharin (1) also decreases the protein level of β-catenin in SW480 colon cancer cells with aberrant Wnt signaling caused by mutation of adenomatous polyposis coli.34 However, uscharin (1) did not decrease the protein level of β-catenin in MEB5 cells (Figure S5, Supporting Information). These data suggested that the Notch inhibitory activity of uscharin (1) did not occur via Wnt signaling and degradation of β-catenin. When MEB5 cells were treated with uscharin (1), astrocytes were not decreased to the same level as when treated with DAPT. This may be a result of uscharin (1) acting on other pathways. Since a large decrease in astrocytes would lead to a loss of balance in the homeostasis of

(data not shown). NICD was not detected in the middle cell density (4 × 105 cells/well) except soon after dissociation [1] (Figure 7B). In the highest density (2 × 106 cells/well, Figure 7C), NICD was detected at 12 [2] and 36 h after seeding [3]. Moreover, NICD was decreased by 10 μM DAPT [4], which suggests that the activity of Notch inhibitors was detectable in these conditions. TP-1 reporter activity was detected without transfection of mNotch1-RAMIC at the highest cell density (Figure 6B). Furthermore, the protein level of NICD and the Notch reporter activity were decreased with 10 μM DAPT (Figure 6B and Figure 7C [4]). From these results, Notch signaling was activated in MEB5 cells at a cell density of 2 × 106 cells/well, and activities of Notch inhibitors were detectable. Next, the NSC differentiation assay was performed with the conditions determined above including a cell density of 2 × 106 cells/well (Figure 8). DAPT (10 μM) and uscharin (1) (1 μM) accelerated neuronal differentiation. Thus, DAPT and uscharin (1) produced Tuj-1 positive neuronal differentiation rates of 19.7% and 20.8%, which are increases of 48% and 56%, respectively, compared to the control (Figure 8B). Also, DAPT and uscharin (1) decreased the percent of astrocytes compared with control (DMSO) cultures (Figure 8A). DAPT and uscharin (1) induced neurite extension lengths of 6.2 and 5.2 μm, which are 43% and 36% increases, respectively, compared to the control (Figure 8C). Uscharin (1) exhibited comparable neural differentiation activity to DAPT at a lower concentration. In Western blot analysis with MEB5 cells treated with 1 μM uscharin (1), NICD and the Notch signaling target genes, Hes1 and Hes5, were decreased (Figure 9a). Since the γ2457



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Figure 8. Effect of 1 on NSC differentiation. (A) Fluorescent immunostaining of MEB5 cells after treatment with DAPT or 1. MEB5 cells were dissociated using the NeuroCult chemical dissociation kit and seeded at a density of 2 × 106 cells/well on a cover glass coated with poly-L-lysine and fibronectin/laminin. After incubation for 12 h, MEB5 cells were treated with DMSO, DAPT (positive control, 10 μM), or 1 (1 μM) for 4 days. Then, cells were fixed with 4% paraformaldehyde in PBS and immunostained with Tuj-1 (green) for neurons, GFAP (red) for astrocytes, and TOPRO-3 (blue) for nuclei. Slide glasses were viewed and photographed with an LSM 700 (Carl Zeiss). Four pictures were taken per well, and the assays were carried out with three individual wells per condition. (B) Effect of 1 on the number of neurons. (C) Effect of 1 on neurite length. Over 4000 cells and 670 neurites in each sample were measured.

stem cells is still limited, this finding may be important for the development of neuronal regenerative medicine.

neural cells, uscharin (1) would likely be a more potent agent for clinical use than DAPT. Although further analysis is needed to identify the target protein of uscharin (1), this compound accelerated NSC differentiation by inhibiting Notch activity via activation of NICD degradation. In conclusion, an assay system was constructed successfully using the T-REx system to evaluate Notch signaling activity. Screening of a Bangladesh plant extract library showed that C. gigantea had strong Notch inhibitory activity. Activity-guided isolation of C. gigantea led to isolation of eight compounds with Notch inhibitory activities. Uscharin (1) decreased the protein levels of target genes Hes1 and Hes5 in assay cells. Furthermore, uscharin (1) accelerated neuronal differentiation, with a 56% increase compared to the control, and decreased the protein levels of Hes1, Hes5, and NICD in MEB5 cells. Coadministration of uscharin (1) and MG132 revealed that uscharin (1) inhibited the Notch signaling pathway by increasing the degradation of NICD via the proteasome pathway in MEB5 cells. Since the number of reported Notch signaling inhibitors that accelerate the differentiation of neural

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-1020 polarimeter. NMR spectra were recorded on JEOL ECA600, ECP600, and ECZ600 NMR spectrometers with deuterated solvent (CDCl3, acetone-d6, and pyridine-d5), and the solvent chemical shifts were taken as the internal standard. ESIMS were obtained on a Shimadzu LCMS-2020 instrument. Protein concentrations were measured using a Nano Drop 2000 (Thermo Fisher Scientific). High-performance liquid chromatography (HPLC) was performed using an LC-2000 Plus series instrument (JASCO). Plant Material. An exudate of Calotropis gigantea was collected in Bangladesh in 2008, and the plant identified by one of the authors (S.K.S.). A voucher specimen (KKB69) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Extraction and Isolation. A MeOH extract of a dried exudate of C. gigantea was chromatographed as reported previously.25 In the present study, the remaining extract from the previous study was used 2458



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Figure 9. (a) Western blot analysis of NICD, RBP-J, Hes1, Hes5, Nicastrin, PS1, PS2, and PEN2 after treatment of MEB5 cells with 1. (b) Western blot analysis of NICD after cotreatment of 1 and MG132. (c) Schematic representation of the mechanism of 1. Dissociated MEB5 cells were seeded in 6 cm dishes at 2 × 106 cells/well and incubated for 12 h. After washing with NeuroCult basal medium (serum-free medium), cells were incubated in differentiation medium. zeocin, 7.5 μg/mL blasticidin, and 1 μg/mL puromycin. Assay cells were cultured in DMEM-high glucose with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco). Mouse NSCs (MEB5) were purchased from Health Science Research Resources Bank. MEB5 cells were cultured as neurospheres in proliferation medium consisting of NeuroCult basal medium (mouse, Stemcell Technologies) with 10% NeuroCult proliferation supplement (mouse, Stemcell Technologies), and 20 ng/mL epidermal growth factor (Stemcell Technologies). For neural differentiation, cells were cultured in differentiation medium consisting of NeuroCult basal medium with 10% NeuroCult differentiation supplement (mouse, Stemcell Technologies) All cultures were maintained in a humidified incubator at 37 °C in 5% CO2. Construction of the Stable Cell Line. Tet-On NICD1 cells were seeded (1 × 106 cells/6 cm dish) and cultured for 17 h, and then the pGL4.20 β-globin 12×RBP-J binding site (5.0 μg) was transfected using Lipofectamine 2000 (Invitrogen). After incubation for 24 h, the medium was changed to fresh medium, and cells were cultured for another 24 h. The cells were passaged into 10 cm dishes with fresh medium including 1 μg/mL puromycin. The cells were cultured for 23 days, and the medium was changed every 3 days. The most active transfectant in the luciferase activity was selected as the assay cells. Notch Signaling-Mediated Transcriptional Activity Assay. Assay cells (LS174T Notch cells) were cultured in a 96-well white plate (Thermo Fisher Scientific) in 100 μL of DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 2 × 104 cells/well at 37 °C for 12 h. After incubation, 50 ng/mL Dox was added to each well to induce expression of exogenous mNotch1(17042531) protein, and cells were incubated at 37 °C for 12 h. The medium was replaced with Dox-free medium containing individual samples. After treatment at 37 °C for 12 h, luciferase activity was measured in a Luminoskan Ascent microplate luminometer (Thermo

for isolation. Fr.16C (11.2 mg) was purified by ODS HPLC (Cosmosil Cholester, ϕ 10 × 250 mm; eluent MeOH−H2O (48:52), wavelength 220 nm, flow rate 3.0 mL/min) to afford 7 (Fr.17B, 6.5 mg, tR 16 min). Fr.16F (10 mg) was purified with ODS HPLC (Cosmosil Cholester, ϕ 10 × 250 mm; eluent MeOH−H2O (45:55), wavelength 220 nm, flow rate 3.0 mL/min) to give 8 (Fr.18E, 0.4 mg, tR 72 min). Fr.2D (180 mg), which was eluted with CHCl3−MeOH (95:5), was chromatographed over an ODS column (ϕ 27 × 245 mm) using H2O−MeOH (3:7−0:1) to give Fr.19A−19I. Fr.19D (112.1 mg), which was eluted with MeOH−H2O (4:6), was subjected to ODS column chromatography (ϕ 23 × 245 mm) using H2O−MeOH (4:6− 3:7) to give Fr.20A−20F. Fr.20B (49.2 mg) was purified by ODS HPLC (Cosmosil 5CN-MS, ϕ 10 × 250 mm; eluent MeOH−H2O (4:6), wavelength 210 nm, flow rate 3.0 mL/min) to give a further quantity of 7 (Fr.21E, 3.5 mg, tR 19 min). Fr.19F (10.3 mg) was purified by silica gel column chromatography (ϕ 12 × 180 mm) to an additional amount of 7 (Fr.23F, 1.1 mg). Calactinic acid methyl ester (7): white solid; [α]27D −3.0 (c 1.0, CH2Cl2); 1H and 13C NMR data, see Supporting Information; ESIMS m/z 585 [M + Na]+. 16α-Hydroxyuscharin (8): white solid; [α]27D −16.0 (c 0.4, MeOH); 1H NMR data, see Supporting Information; ESIMS m/z 604 [M + H]+. Plasmids. Initially, 12×RBP-J binding sites and the β-globin promoter region were digested with SalI and XhoI and ligated into pGL4.20 (Promega) to construct the pGL4.20 β-globin 12×RBP-J binding site. TP1-luc (pGa981-6) was obtained from RIKEN BRC. pEF-BOSneo-mNotch1 RAMIC was obtained from RIKEN DNA Bank. Cell Culture. Tet-On NICD1 cells and LS174T Notch cells were cultured in DMEM-high glucose (Wako) with 10% fetal bovine serum (FBS, Bio West), 1% antibiotic−antimycotic (Gibco), 750 μg/mL 2459



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Scientific) using the Bright-Glo luciferase assay system (Promega) according to the manufacturer’s protocol. At the same time, the viability of the sample-treated cells was measured using the Fluorometric microculture cytotoxicity assay. Assay cells were cultured in a 96-well black plate (Thermo Fisher Scientific) at 2 × 104 cells/ well at 37 °C for 24 h. Samples were added at the same time as the luciferase assay (24 h after seeding), and the cells were incubated at 37 °C for 12 h. Cell viability was determined with the fluorometric microculture cytotoxicity assay using a Fluoroskan Ascent microplate fluorometer (Thermo Scientific). Notch signaling-mediated transcriptional activity was determined, and cell viability was calculated as the ratio of viable sample-treated cells to nontreated cells. Western Blotting Analysis. Assay cells (LS174T Notch cells) were seeded in a 6 cm dish in 5 mL of DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 1 × 106 cells/dish, and incubated for 12 h at 37 °C. To activate expression of exogenous mNotch1(1704-2531) protein, 50 ng/mL Dox was added to each dish, and cells were incubated another 12 h. The medium was then removed, and fresh medium containing individual compounds was added. After a 12 h incubation, cells were washed twice with 500 μL of PBS and collected by scraping the dishes. For preparation of whole cell lysates, cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 10 mM EDTA, 1 mM sodium orthovanadate, and 0.1 mM NaF) containing 1% protease inhibitor cocktail (Nacalai Tesque). Protein lysates were subjected to 5% or 10% SDS-PAGE and subsequently transferred to PVDF membranes (Bio-Rad). Blots were blocked with 5% skim milk (Morinaga) in TBST (10 mM Tris-HCl pH 7.4, 100 mM NaCl, and 0.1% Tween 20) for 1 h and incubated at 4 °C for 12 h with primary antibodies (anti-Flag, 1:1000, Sigma; cleaved Notch1 (Val1744), 1:1000, Cell Signaling Technologies; Notch1 (C-20), 1:1000, Santa Cruz Biotechnology; Hes1 (H-20), 1:1000, Santa Cruz Biotechnology; Hes5, 1:700, Cell Signaling Technologies; RBP-J 1:2000, Santa Cruz Biotechnology; Flag, 1:1000, Sigma; β-catenin, 1:2000, Cell Signaling Technologies; CK1α, 1:500, Santa Cruz Biotechnology; and γsecretase antibody sampler kit, 1:2000, Cell Signaling Technologies). The membranes were then washed with TBST and incubated at room temperature for 1 h with secondary antibodies (anti-goat IgG, 1:10 000, Sigma; anti-rabbit IgG, 1:4000, Jackson Immuno Research; anti-mouse IgG, 1:4000, GE Healthcare/Amersham Biosciences; or anti-rat IgG, 1:1000, Santa Cruz Biotechnology). After washing with TBST, immunocomplexed bands were detected using an ECL Advance Western (GE Healthcare Biosciences) or an Immobilon Western (Millipore) detection system. For Western blotting of MEB5 cells, cells were seeded in 6 cm dishes coated with poly-L-lysine and fibronectin/laminin at individual cell densities in proliferation medium and incubated for 12 h. After washing with NeuroCult basal medium (mouse), cells were incubated with each sample in differentiation medium. After incubation for 24 h, protein lysates were prepared. Transfection. Neurospheres of MEB5 cells were collected and dissociated using the NeuroCult chemical dissociation kit (Stemcell Technologies), seeded in 24-well plates coated with poly-L-lysine and fibronectin/laminin at individual cell densities in proliferation medium, and incubated for 12 h. After washing with NeuroCult basal medium (mouse), cells were transfected with each plasmid (TP-1 400 ng/well, Notch1-RAMIC 20 or 100 ng/well, pRL-CMV 25 ng/well) with Lipofectamine 2000 (2.5 μL/well Invitrogen) in differentiation medium for 24 h. Luciferase activities were measured with the DualGlo Luciferase assay system. Neural Stem Cell Differentiation Assay. MEB5 cells were dissociated using the NeuroCult chemical dissociation kit and seeded at a density of 2 × 106 cells/well on a cover glass (Matsunami Glass) coated with poly-L-lysine and fibronectin/laminin. After incubation for 12 h, cells were washed with NeuroCult basal medium (mouse) and treated with individual compounds in differentiation medium for 4 days. Immunofluorescence Staining. After incubation for 4 days, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature (rt) and washed with 1% BSA (Merck) in PBS three times. Cells were blocked in 10% BSA in 0.3% Triton X-100 (Wako)

in PBS for 45 min at rt and incubated with primary antibodies (0.5 mg/mL anti-βIII-tubulin; neuronal class III, Mouse-Mono, 1:400, R&D Systems; anti-GFAP, 1:400, VERITAS) for 12 h at 4 °C. After washing three times with 1% BSA in PBS (500 μL/well), cells were incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG (H+L), 1:400, Life Technologies; Alexa Fluor 555 goat anti-rabbit IgG (H+L), 1:200, Life Technologies) for 1 h at rt in the dark. After washing three times with 1% BSA in PBS (500 μL/well), cells were treated with 200 μg/mL RNase (Nacalai Tesque) in PBS containing 1% BSA and 0.1% Triton X-100 for 1 h at 37 °C in the dark. Then, after washing a further three times with 500 μL/well PBS, cells were incubated in 30 μM TO-PRO-3, 1% BSA, and 0.1% Triton X-100 in PBS for 10 min at rt in the dark. The cover glass on which the cells were attached was transferred onto slide glass and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Slide glasses were viewed and photographed with an LSM 700 (Carl Zeiss). Four photographs were taken per well, and the assays were carried out with three individual wells per condition.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00282. Information regarding assay development, isolation chart, and additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +81-439226-2924. Fax: +81-439226-2924. E-mail: [email protected]. *Tel: +81-43-226-2923. Fax: +81-439226-2923. E-mail: mish@ chiba-u.jp. ORCID

Midori A. Arai: 0000-0003-0254-9550 Masami Ishibashi: 0000-0002-2839-1045 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by KAKENHI grant numbers 26305001, 26293022, 25670045, and 15H04650 from JSPS and 23102008 from MEXT (a Grant-in-Aid for Scientific Research on Innovative Areas in “Chemical Biology of Natural Products”). The study was supported also by a Workshop on Chirality at Chiba University (WCCU). We also thank Prof. T. Tomita of the University of Tokyo for assaying test compound γ-secretase inhibition and for helpful discussions. This work was inspired by the international and interdisciplinary environments of the JSPS Core-to-Core Program “Asian Chemical Biology Initiative”.

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