The Notch Inhibitors Isolated from

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. 2018, 81, 1235−1240

The Notch Inhibitors Isolated from Nerium indicum Midori A. Arai,*,† Ryuta Akamine,† Narumi Hayashi,† Takashi Koyano,‡ Thaworn Kowithayakorn,§ and Masami Ishibashi*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Temko Corporation, Tokyo 168-0062, Japan § Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ‡

S Supporting Information *

ABSTRACT: Notch signaling plays a crucial role in differentiation and cell maintenance, but once aberrantly activated, it contributes to cancer progression. Notch inhibitors were isolated from plant extracts and tested using an originally constructed cell-based assay system. We isolated eight compounds from Nerium indicum that showed inhibition of the Notch signaling pathway. HES1 and HES5 are target genes of the Notch signaling pathway, and oleandrin (1) decreased the protein levels of HES1 and HES5 in assay cells. Oleandrin (1) showed potent cytotoxicity against HPB-ALL cells and decreased HES1 and the Notch intracellular domain in these cells. The main mechanism of action of 1 appears to be inhibition of Notch signaling by acceleration of Notch intracellular domain degradation.

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immunoglobulin kappa J region (RBP-J) to initiate transcription of target genes. Many reports have described various Notch inhibitors including γ-secretase inhibitors, DAPT (N-[N-(3,5-difluorophenacetyl)- L -alanyl]-S-phenylglycine tert-butyl ester), 13 RO4929097,14 MK-0752,15 and PF0384014.16 SHAM1, a MAML1-derived peptide that inhibits the binding of full-length MAML1 to NICD1-RBP-J, was also reported as a potent Notch signaling inhibitor.17 The natural product inhibitors curcumin18 and genistein19 downregulate Notch1. Several clinical trials have been conducted to develop Notch signaling inhibitors as clinical agents for chemotherapeutics to treat cancer.20 The development of Notch inhibitors from natural products has been limited but is eagerly anticipated because natural products have contributed to the development of new drugs.21 Here, we report the search for new Notch signaling inhibitors from natural resources and their testing using a recently constructed cell-based reporter assay that was developed by our group.

otch signaling plays a crucial role in differentiation at various stages during embryonic and adult development and in regulation of many basic cellular processes such as proliferation and stem cell maintenance.1,2 An important direct output of Notch activity is upregulation of basic helix−loop− helix transcriptional repressors such as hairy and enhancer of split 1 (HES1), HES5, and HES-related family genes. The progression of neural stem cell differentiation into neurons can be inhibited by Notch signaling, which thus plays an important role in neural stem cell differentiation.3,4 Aberrant upregulation of Notch signaling is oncogenic for multiple hematologic and solid malignancies.5−7 Activating mutations of the transmembrane receptor Notch1 play a role in the etiology of human T-cell acute lymphoblastic leukemia (T-ALL).8 Many lines of evidence show that Notch functions as an oncoprotein in melanocytes,9 prostate cancer cells,10 and breast cancer cells.11 Therefore, the Notch signaling pathway has been identified as a potential therapeutic target in many cancers.12 Notch signaling is activated by interaction between the ligand-expressing cell and the signal-receiving cell (Figure 1). When the ligand proteins Jagged or Delta bind to the Notch, a conformational change is induced in the Notch receptor, exposing the S2 cleavage site to a metalloproteinase-containing protein called ADAM17/TACE (a disintegrin and metalloprotease domain-containing protein 17/TNFα-converting enzyme). After S2 cleavage, the subsequent S3 cleavage is mediated by γ-secretase, which consists of presenilin 1, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1. The Notch intracellular domain (NICD) is released after S3 cleavage. NICD translocates to the nucleus and forms a heterotrimer with mastermind-like protein (MAML), the DNA binding protein, and recombination signal binding protein for © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION We recently constructed a cell-based reporter assay system with the T-REx system to search for Notch signaling inhibitors (Figure 2).22,23 In the absence of doxycycline (Dox), the tetracycline repressor (TetR) suppresses the expression of Nterminal FLAG-tagged mouse Notch1(1704−2531), which is 40 a.a. longer than NICD at the N-terminus. In the presence of Dox, TetR binds Dox and releases the suppression of expression of Notch1(1704−2531) mediated by TetR. Then, Notch1(1704−2531) is cleaved by γ-secretase to give NICD. Received: December 6, 2017 Published: April 25, 2018 1235

DOI: 10.1021/acs.jnatprod.7b01031 J. Nat. Prod. 2018, 81, 1235−1240

Journal of Natural Products

Article

Figure 1. Notch signaling pathway. NICD: Notch intracellular domain, MAML: mastermind-like protein (coactivator), HES1: hairy and enhancer of split 1, RBP-J: recombination signal binding protein for immunoglobulin kappa J region, ADAM17/TACE: a disintegrin and metalloprotease domain-containing protein 17/TNFα-converting enzyme.

ques (silica gel and ODS), resulting in isolation of oleandrin (1),25 nerigoside (2),26 odoroside A (3),26 16-anhydrodeacetylnerigoside (4),25 deacetyloleandrin (5),27 digitoxigenin (6),28 8β-hydroxyodoroside A (7),27 and beaumontoside (8).29 These compounds are classified as cardiotonic steroids with a steroid nucleus. A subgroup of cardioactive steroids that contain sugar residues are called cardiac glycosides. Cardiac glycosides, such as oleandrin, are well known as inhibitors of the Na/K-ATPase pump. These compounds have been used to treat heart failure, and recently, many biological activities including anticancer activity have been reported.30 Chart 1

Figure 2. Assay system using the T-REx system. Without Dox, expression of Notch1(1704−2531) is suppressed. Addition of Dox allows the expression of Notch1(1704−2531) due to binding with TetR. Notch1(1704−2531) is then cleaved by 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× RBP-J binding site to express luciferase protein. Dox: doxycycline, TetR: tetracycline repressor, Luc.: luciferase.

NICD translocates to the nucleus and forms a heterotrimer with RBP-J and MAML to contribute to luciferase transcription. The assay cells (LS174T cells) include the stably incorporated pGL4.20 luciferase plasmid, which has a 12× RBP-J binding site (12 copies of CGTGGGAA) β-globin promoter. To remove false positive hits, cell viability is measured with a fluorometric microculture cytotoxicity assay24 to assess the cytotoxicity in the samples. Screening of our plant extract library showed that a crude MeOH extract of Nerium indicum includes Notch inhibitors. This MeOH extract (100 μg/mL) reduced luciferase activity to 15% with cell viability of 47%. The N. indicum MeOH extract was subjected to Diaion HP-20, resulting in three fractions (fr. 1A−C). The active fraction (fr. 1A) was partitioned successively with EtOAc, nBuOH, and water. The active EtOAc layer was investigated using chromatographic techni-

Next, we investigated the inhibitory activity of the eight isolated compounds on Notch signaling (Figure 3). Compounds 1, 2, 3, 5, and 8 showed potent inhibitory activity with IC50 values less than 5 μM (0.12, 2.7, 4.2, 2.5, and 2.7 μM, respectively). Compounds 4, 6, and 7 were inactive. Comparison of the activity of 3 and 6 revealed that the sugar moiety at the C-3 position was important for activity. When a double bond at C-16 and C-17 was introduced, the activity was dramatically decreased (comparison of 3 and 4). The OH group at C-8 in compound 7 also reduced the activity compared to that of 3. Regarding the sugar species, L1236



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Figure 3. Inhibition of Notch transcriptional activity and cell viability by 1−8. These assays were performed in 0.1% DMSO (n = 3). Error bars represent SD.

Figure 4. Inhibition of protein expression by 1 in assay cells. [A] Western blot analysis of HES1 and HES5 in assay cells after treatment with 1. [B] Western blot analysis of Notch1(1704−2531) and NICD in assay cells after treatment with 1. [C] Effect of MG132 on the decrease in NICD.

inhibitor MG132, which restored the amount of NICD (Figure 4C). This result indicated that the mechanism of action of 1 was inhibition of Notch signaling by acceleration of the NICD degradation system. To investigate the effect of 1 on cancer cells, HPB-ALL cells, which are T-ALL cells, were used. The cytotoxicity of 1 on HPB-ALL cells was evaluated (Figure 5A). Oleandrin (1) reduced the cell viability of HPB-ALL cells at 25 nM. Although cell viability of a normal cell line, human embryonic kidney 293 cells, was also affected by the addition of 1, we found a difference in the IC50 values between HPB-ALL cells and 293 cells (Figure 5B). The IC50 values of 1 were 14 nM (HPB-ALL cells) and 28 nM (293 cells). Oleandrin (1) showed

oleandrose seems to show higher activity than D-diginose (comparison of 1 and 2). In addition, the importance of the acetate was found in the comparison of 1, 5, and 8 or 2 and 7. The removal of the acetate group led to the decrease of the activity. We focused on the most active compound, oleandrin (1). The effect of 1 on protein expression in the assay cells was investigated. When the cells were treated with 1, the Notch signal-related proteins, HES1 and HES5, were reduced (Figure 4A). Notch1(1704−2531) and NICD were reduced in a concentration-dependent manner (Figure 4B). Next, we speculated that 1 accelerated the degradation of NICD. To test this hypothesis, cells were incubated with the proteasome 1237



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Figure 5. Effect of 1 on HPB-ALL cells. [A] Cytotoxicity of 1 on 293 and HPB-ALL cells. [B] IC50 values of 1 for cytotoxicity. [C] Western blot analysis of NICD and HES1 in HPB-ALL cells after treatment with 1. [D] The main mechanism of action of 1 for inhibition of Notch signaling is acceleration of degradation of NICD. whose chemical shift was taken as an internal standard. Column chromatography was performed using Chromatorex PSQ100B and Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan). Preparative HPLC was performed using Cosmosil πNAP (ϕ 10 × 250 mm) (Nacalai Tesque, Inc. Kyoto, Japan) and YMC-Pack ODS-AM (YMC Co., Ltd., Kyoto, Japan). Protein concentrations were measured using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Plant Materials. Nerium indicum (Apocynaceae) was collected from Thailand in 2013. A voucher specimen (KKP411) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University. Extraction and Isolation. The MeOH extract (29.6 g) of N. indicum (leaves; 146 g) was subjected to Diaion HP-20 (ϕ 80 × 230 mm) with the MeOH−acetone solvent system (1:0−0:1) to afford fractions 1A to 1C. The active fr. 1A (22.8 g) was suspended in 10% aqueous MeOH (250 mL) and partitioned between EtOAc and BuOH (250 mL × 3) to obtain EtOAc (6.4 g), BuOH (6.3 g), and aqueous (7.4 g) soluble fractions. An active EtOAc-soluble fraction was subjected to silica gel column chromatography (ϕ 48 × 210 mm; CH3Cl−MeOH, 50:1−0:1, then washed with 0.1% TFA in MeOH) to afford fractions 2A to 2F. Fr. 2C (481.4 mg) was subjected to ODS column chromatography (ϕ 25 × 200 mm; H2O−MeOH, 35:65−0:1) to afford fractions 3A to 3L. Recrystallization of fr. 3C (25.8 mg) from MeOH−H2O (60:40) gave compound 1 as crystals (10.8 mg). The rest of fr. 3C was subjected to ODS HPLC (YMC-Pack ODS-AM; MeOH−H2O, 65:35, flow rate 1.5 mL/min) to give compound 4 (0.5 mg, tR 35.6 min), compound 2 (1.0 mg, tR 38.2 min), compound 1 (4.0 mg, tR 56.4 min), and compound 3 (0.5 mg, tR 60.6 min). Fr. 3D (25.6 mg) was subjected to HPLC (Cosmosil πNAP; MeOH−H2O, 80:20, flow rate 4.0 mL/min) to give compound 1 (3.3 mg, tR 11.6 min) and compound 3 (3.0 mg, tR 14.6 min). Fr. 2D (1876.6 mg) was

cytotoxicity against 293 cells, but at the low concentration of 25 nM, a remarkable difference was seen between these two cell types in terms of cytotoxicity. The Notch signaling pathway is aberrantly activated in HPB-ALL cells, and Notch signal dependency in the growth of HPB-ALL cells was reported.31 This would be the reason that there is the difference in cytotoxicity between these cells. The amounts of NICD and HES1 were reduced in HPB-ALL cells in a concentrationdependent manner (Figure 5C). We also examined the effect of oleandrin (1) (0.5 μM) against HPB-ALL cells (12 h treatment, Supporting Information, Figure S3). Oleandrin (1) decreased Notch1 and NICD. When the cells were treated with MG132 (18.5 μM), NICD was partially recovered and Notch1 was not recovered. We also checked Notch1 mRNA expression after 12 h of treatment of oleandrin (1). The mRNA expression of Notch1 was not affected by addition of oleandrin (1). Therefore, the main mechanism of oleandrin (1) would be acceleration of degradation of NICD by proteasome (Figure 5D). In conclusion, we report here the isolation of eight compounds and evaluation of their effects as Notch signaling inhibitors with our original cell-based assay. Oleandrin (1) is a strong Notch inhibitor (IC50 = 0.12 μM). The main mechanism of action of 1 for inhibition of Notch signaling is acceleration of degradation of NICD.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on JEOL ECP600 and ECZ600 spectrometers in a deuterated solvent 1238



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subjected to ODS column chromatography (ϕ 40 × 200 mm; H2O− MeOH, 40:60−0:1, then washed with 0.1% TFA in MeOH) to afford fractions 4A to 4J. A combined mixture of active fr. 4F (86.9 mg) and fr. 4G (91.8 mg) was recrystallized from MeOH−H2O (55:45) to give compound 1 as crystals (24.5 mg). The rest of fr. 4F and fr. 4G (41.7 mg) were subjected to ODS HPLC (YMC-Pack ODS-AM; MeOH− H2O, 65:35, flow rate 1.5 mL/min) to give compound 6 (0.9 mg, tR 31.6 min), compound 4 (2.5 mg, tR 28.8 min), compound 2 (6.4 mg, tR 36.8 min), compound 5 (3.3 mg, tR 40.4 min), compound 7 (2.2 mg, tR 45.4 min), compound 1 (7.9 mg, tR 53.6 min), and compound 3 (0.9 mg, tR 58.0 min). Part of fr. 4H (45.0 mg) was subjected to ODS HPLC (YMC-Pack ODS-AM; MeOH−H2O, 65:35, flow rate 1.5 mL/ min) to give compound 4 (0.7 mg, tR 22.6 min), compound 1 (4.7 mg, tR 30.2 min), and compound 8 (0.4 mg, tR 42.8 min). The part of fr. 4H (49.2 mg) was subjected to ODS HPLC (YMC-Pack ODS-AM; MeOH−H2O, 65:35, flow rate 1.5 mL/min) again to give compound 8 (0.7 mg, tR 42.8 min). Notch Signaling-Mediated Transcriptional Activity Assay. Assay cells (LS174T Notch cells; 2 × 104 cells per well) were seeded in a 96-well white plate. After incubation for 12 h at 37 °C, doxycycline (50 ng/mL) was added to each well to express exogenous Notch1(1704−2531) protein, and cells were incubated at 37 °C for 12 h. Then, the medium was changed to DOX-free medium containing each sample at 37 °C for 12 h. Luciferase activity was measured with a microplate luminometer (Thermo Fisher Scientific Inc.) using the Bright-Glo luciferase assay system (Promega Co., Madison, WI, USA) according to the manufacturer’s protocol. The cell viability of the sample-treated cells was determined by a fluorometric microculture cytotoxicity assay (FMCA). Assay cells were seeded in a 96-well black plate at 2 × 104 cells per well and incubated at 37 °C for 24 h. Samples were added at the same time as the luciferase assay, and the cells were incubated at 37 °C for 12 h. Cell viability was measured by FMCA using a Fluoroskan Ascent (Thermo Fisher Scientific Inc.). The ratio of sample-treated cells to nontreated cells was calculated as cell viability. Cell Culture. HPB-ALL cells were obtained from RIKEN BRC. Assay cells (LS174T Notch cells) and HEK293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Wako, Osaka, Japan) with fetal bovine serum (10% FBS; BioWest, Nuaillé, France; SigmaAldrich Co. LLC., St. Louis, MO, USA), penicillin (100 unit/mL), and streptomycin (100 μg/mL) (Sigma-Aldrich Co.). HPB-ALL cells were cultured in RPMI-1640 (Wako, Osaka, Japan) with FBS (10%), penicillin (100 unit/mL), and streptomycin (100 μg/mL). Cultures were maintained in a humidified incubator at 37 °C in 5% CO2/95% air. Cytotoxicity Test. HEK293 cells were seeded onto 96-well black plates in 100 μL of DMEM medium containing 10% FBS, penicillin (100 unit/mL), and streptomycin (100 μg/mL) at 1 × 104 cells per well. After incubation at 37 °C for 24 h, the medium was then changed to fresh medium containing different concentrations of compound. After incubation for 72 h, the medium was removed, and cell proliferation was determined by FMCA using a fluorescence plate reader (Thermo Fisher Scientific Inc.). HPB-ALL cells were seeded onto 96-well black plates in 100 μL of RPMI-1640 medium containing 10% FBS, penicillin (100 unit/mL), and streptomycin (100 μg/mL) at 1 × 104 cells per well in the presence of different concentrations of compounds. Cells were incubated at 37 °C for 72 h. After incubation, AlamarBlue (00-025, DAL1025; Thermo Fisher Scientific Inc.) was added at 10 μL per well. After incubation for 4 h, fluorescence was measured using a fluorescence plate reader (Thermo Fisher Scientific Inc.) and cell viability was determined. Western Blotting Analysis. Assay cells (LS174T Notch cells; 1 × 106 cells per dish) were seeded onto a 6 cm dish in 5 mL of DMEM containing 10% FBS, penicillin (100 unit/mL), and streptomycin (100 μg/mL) and incubated for 12 h at 37 °C. Then 50 ng/mL of doxycycline was added into each dish and incubated for another 12 h. Medium was removed and fresh medium with each compound was added. After 12 h of incubation at 37 °C, cells were washed with 500 μL of PBS twice and then collected by scraping the whole parts.

Protein lysate was prepared with 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 a 1% proteasome inhibiotor cocktail (Nacalai Tesque, Tokyo, Japan). Protein lysates were subjected to a 7.5%, 10%, 12.5%, and 15% SDS-PAGE electrophoresis, then transferred electrophoretically to a PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking with TBST (10 mM Tris-HCl pH 7.4, 100 mM NaCl and 0.1% Tween 20) containing 5% skimmed milk for 1 h, the membrane was incubated with primary antibodies anti-β-actin (A2228; dilution 1:4000, Sigma-Aldrich, Co.), anticleaved Notch1 (Val1744) (2421, 4147; dilution 1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-FLAG (F7425; dilution 1:1000, Sigma-Aldrich, Co.), antiHES1 (sc-13844; dilution 1:1000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-HES5 (ab25374; dilution 1:250, ab194111; dilution 1:1000, Abcam plc., Cambridge, UK) at 4 °C for 12 h. Then, the membrane was washed with TBST and incubated with secondary antibodies, either anti-rabbit IgG (111-035-144; dilution 1:4000, Jackson Immuno Research Inc., West Grove, PA, USA), anti-rabbit IgG (7074; dilution 1:4000, Cell Signaling Technology, Inc.), anti-goat IgG (705-035-003; dilution 1:10 000, Jackson Immuno Research Inc.), or anti-mouse IgG (NA931; dilution 1:4000, GE Healthcare Japan, Tokyo, Japan) at room temperature for 1 h. After washing with TBST, immunocomplexed bands were detected using an ECL Advance Western (RPN2135; GE Healthcare Japan, Tokyo, Japan), ECL Select Western (RPN2235; GE Healthcare Japan) or Immobilon Western (WBKLS0100; Merck Millipore Co., Darmstadt, Germany) detection system. HPB-ALL cells were seeded onto a 6 cm dish in 5 mL of RPMI1640 medium containing 10% FBS, penicillin (100 unit/mL), and streptomycin (100 μg/mL) at 5 × 105 cells per dish (1 × 106 cells per dish, when treated with MG132). Media also contained different concentrations of compound. Cells were incubated at 37 °C for 72 h (12 h, when treated with MG132). After incubation, cells were centrifuged, collected, and then washed with 500 μL of PBS twice. To prepare cell lysate and confirm protein expression levels, the same method as described above was used.

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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.7b01031. Information regarding the plasmids, isolation charts, 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 a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), Strategic Priority Research Promotion Program, Chiba University, “Phytochemical Plant Molecular Sciences”, Takeda Foundation, the Naito Foundation, Astellas Foundation for Research on Metabolic Disorders, the Uehara Memorial 1239



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Foundation, and a Workshop on Chirality at Chiba University (WCCU). This work was inspired by the international and interdisciplinary environments of the JSPS Core-to-Core Program “Asian Chemical Biology Initiative” and JSPS A3 Foresight Program.

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The Notch Inhibitors Isolated from

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