Identification of 1ß,2α-epoxytagitinin C as a Notch inhibitor, oxidative stress mechanism and its anti-leukemia activity.

Notch signaling plays crucial roles in cell differentiation and proliferation, but aberrant activation of this signaling results in tumorigenesis and cancer progression. Notch signaling is thus a promising drug target for oncotherapy, and the development of Notch signaling inhibitors is eagerly awaited. Notch inhibitory activity-guided fractionation of a Spilanthes acmella extract led to the identification of five sesquiterpene lactones: tagitinin A (1), 1ß,2α-epoxytagitinin C (2), tagitinin C (3), orizabin (4), and 2α-hydroxytirotundin (5). 1ß,2α-Epoxytagitinin C (2) exhibited Notch signaling inhibition, with an IC50 of 25.6 µM, and was further evaluated for its activity against HPB-ALL, a Notch-activated leukemia cell line. Compound 2 showed potent cytotoxicity against HPB-ALL (IC50 1.7 µM) and arrested the cell cycle at the G2/M phase, but did not induce apoptotic cell death. Notch inhibitory mechanism analysis suggested that compound 2 transcriptionally suppresses Notch1 mRNA. In addition, we found that oxidative stress induction is critical for Notch signaling inhibition and the cytotoxicity of compound 2. This is the first mechanism of small molecule Notch inhibition. Our results demonstrate that 1ß,2α-epoxytagitinin C (2) is a potential anti-leukemia agent and further investigation of this compound is warranted.

Acacienone, a terpenoid-like natural product having an unprecedented C20 framework isolated from Acacia mangium leaves.

A novel C20 natural product, acacienone (1), was isolated from the leaves of Acacia mangium collected in Bangladesh. The structure of compound 1 was elucidated by spectral studies and X-ray crystallographic analysis. Acacienone (1) possesses a terpenoid-related tetracyclic framework containing 20 carbons with biogenetically unusual structural features: (i) vicinal C1-branches at the C-3 and C-4 positions in the A ring, and (ii) a cyclopentenone D ring in an androsterone-like assembly, lacking a methyl group at the C-13 position.

Isolation and evaluation of cardenolides from Lansium domesticum as Notch inhibitors.

Since Notch signaling plays important roles in cell proliferation and differentiation, aberrant activation of this signaling contributes to cancer progression. In neural stem cells, Notch signaling inhibits differentiation by activating HES1 expression. Therefore, Notch signaling inhibitors may be candidates for new anticancer drugs or have applications in neural regenerative medicine. In this study, six naturally occurring Notch inhibitors were isolated from the methanol (MeOH) extract of Lansium domesticum using our novel cell-based assay. Hongherin (2), a cardiac glycoside, demonstrated potent Notch inhibitory activity with an IC50 of 0.62 µM and was found to be cytotoxic in HPB-ALL human T cell acute lymphoblastic leukemia cells. Hongherin (2) also induced the differentiation of C17.2 neural stem cells to neurons, causing a 65% increase in differentiation compared to the control. Mechanistically, hongherin (2) reduced the amount of Notch1 (full length) and mastermind-like protein (MAML). This indicates that hongherin (2) inhibits Notch signaling through a dual mechanism involving the reduction of both Notch1 and MAML protein levels.

Cadinane sesquiterpenoids isolated from Santalum album using a screening program for Wnt signal inhibitory activity.

Upon screening compounds having Wnt signal inhibitory activity through evaluating TCF/ß-catenin transcriptional (TOP) activity, eight cadinane sesquiterpenoids, including three new compounds (1-3), were isolated from wood extracts of Santalum album (Santalaceae). Structures of compounds 1-3 were elucidated by spectral data to have a cadinane skeleton with an aromatic ring. Of the eight compounds isolated, compound 4, identified as mansonone I, was found to be active against TOP, having an IC50 of 1.2 µM.

The Notch Inhibitors Isolated from Nerium indicum.

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.

Total syntheses of schizandriside, saracoside and (±)-isolariciresinol with antioxidant activities.

Lignans are widely distributed in plants and exhibit significant pharmacological effects, including anti-tumor and antioxidative activities. Here, we describe the total synthesis of schizandriside (1), a compound we previously isolated from Saraca asoca by monitoring antioxidative activity using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Starting from a tandem Michael-aldol reaction, the lignan skeleton was synthesized in 6 steps, including a cyclization step. To determine the stereochemistry of 1, we synthesized the natural product (±)-isolariciresinol (18) from alcohol 17. Comparison of the spectral data showed good agreement. Glycosylation was investigated using four different glycosyl donors. Only the Koenigs-Knorr condition using silver trifluoromethanesulfonate with 1,1,3,3-tetramethylurea provided the glycosylated product. Deprotection and purification using reverse-phase high-performance liquid chromatography gave schizandriside (1) and its diastereomer saracoside (2). Synthesized 1, 2 and 18 showed antioxidant activity with IC50 = 34.4, 28.8, 53.0 µM, respectively.

Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of animal cells.

A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from D-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a "co-culture" in which the genus Nocardia was cultured in the presence of an animal cell line.

Bioassay-Guided Isolation of Compounds from Datura stramonium with TRAIL-Resistance Overcoming Activity.

TRAIL is a potent inducer of apoptosis in most cancer cells, but not in normal cells, and therefore has deserved intense interest as a promising agent for cancer therapy. In the search for bioactive natural products for overcoming TRAIL-resistance, we previously reported a number of active compounds. In our screening program on natural resources targeting overcoming TRAIL-resistance, activity-guided fractionation of the MeOH extract of Datura stramonium leaves led to the isolation of three alkaloids--scopolamine (1), trigonelline (2), and tyramine (3). Compounds 1, 2, and 3 exhibited TRAIL-resistance overcoming activity at 50, 150, and 100 µM, respectively in TRAIL-resistant AGS cells.

Constituents from Entada scandens with TRAIL-resistance Overcoming Activity.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent because of its ability to selectively kill tumor cells. But TRAIL-resistance is a major problem of its therapy. A search for compounds for abrogating TRAIL-resistance has, thus, become an important strategy for anticancer drug discovery. In search of bioactive natural products for overcoming TRAIL-resistance, we previously reported some compounds with TRAIL-resistance overcoming activity. Bioassay guided fractionation of Entada scandens led to the isolation of four compounds (1-4). Of the isolates, compounds 1 and 3 showed moderate TRAIL-resistance overcoming activity in TRAIL-resistant human gastric adenocarcinoma (AGS) cells.

Sesquiterpenes with TRAIL-resistance overcoming activity from Xanthium strumarium.

The ability of TRAIL to selectively induce apoptosis in cancer cells while sparing normal cells makes it an attractive target for the development of new cancer therapy. In search of bioactive natural products for overcoming TRAIL-resistance from natural resources, we previously reported a number of active compounds. In our screening program on natural resources targeting overcoming TRAIL-resistance, activity-guided fractionations of the extract of Xanthium strumarium led to the isolation of five sesquiterpene compounds (1-5). 11α,13-dihydroxanthinin (2) and 11α,13-dihydroxanthuminol (3) were first isolated from natural resources and xanthinosin (1), desacetylxanthanol (4), and lasidiol p-methoxybenzoate (5) were known compounds. All compounds (1-5) showed potent TRAIL-resistance overcoming activity at 8, 20, 20, 16, and 16 µM, respectively, in TRAIL-resistant AGS cells. Compounds 1 and 5 enhanced the levels of apoptosis inducing proteins DR4, DR5, p53, CHOP, Bax, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 and also decreased the levels of cell survival protein Bcl-2 in TRAIL-resistant AGS cells in a dose-dependent manner. Compound 1 also enhanced the levels of DR4 and DR5 proteins in a time-dependent manner. Thus, compounds 1 and 5 were found to induce both extrinsic and intrinsic apoptotic cell death. Compound 1 also exhibit TRAIL-resistance overcoming activity in DLD1, DU145, HeLa, and MCF7 cells but did not decrease viability in non-cancer HEK293 cells up to 8 µM.

Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity.

The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 µM, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear ß-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing ß-catenin levels.

Hedgehog/GLI-mediated transcriptional activity inhibitors from Crinum asiaticum.

The inhibition of the hedgehog (Hh) signaling pathway has emerged as an attractive anti-cancer strategy. As part of our continuing search for natural inhibitors of the Hh/GLI1 signaling pathway, we isolated three alkaloids (1-3) from Crinum asiaticum. Compounds 1 and 3 showed potent Hh/GLI1-mediated transcriptional inhibitory activity and exhibited cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells. Our data revealed that compounds 1 and 3 clearly inhibited the Hh signaling pathway by down-regulating the expression of GLI-related proteins (PTCH and BCL2) in DU145 cells.

Phenolic compounds from the bark of Oroxylum indicum activate the Ngn2 promoter.

A reporter gene assay that detects neurogenin 2 (Ngn2) promoter activity was utilized to identify compounds that induce neuronal differentiation. Ngn2 is a basic helix-loop-helix transcription factor that activates transcription of pro-neural genes. Using this assay system and an activity-guided approach, seven phenolic compounds were isolated from the methanol extract of Oroxylum indicum: 1 oroxylin A, 2 chrysin, 3 hispidulin, 4 baicalein, 5 apigenin, 6 baicalin, and 7 isoverbascoside. Compounds 1 and 2 induced an estimated 2.7-fold increase in Ngn2 promoter activity, whereas 3 increased the activity by 2.5-fold. Furthermore, 1 and 2 enhanced neuronal differentiation of C17.2 cells, which are multipotent stem cells.

ß-Sitosterol and flavonoids isolated from Bauhinia malabarica found during screening for Wnt signaling inhibitory activity.

Screening with a cell-based luciferase assay was conducted to identify bioactive natural products which inhibit Wnt signaling activity-guided separation of an MeOH extract of Bauhinia malabarica (Caesalpiniaceae) leaves yielded five compounds, which were identified as ß-sitosterol (1), quercetin (2), 6,8-C-dimethyl kaempferol-3-O-rhamnopyranoside (3), hyperin (4), and 6,8-C-dimethyl kaempferol-3-methyl ether (5). The tested compounds 1, 3, and 5 exhibited Wnt signaling inhibitory activity, with IC50 values of 0.77, 0.74, and 16.6 µM, respectively.

Naturally occurring Ngn2 promoter activators from Butea superba.

Neurogenin2 (Ngn2), an activator-type bHLH transcriptional factor, promotes differentiation of neural stem cells into neurons by transcription of pro-neural genes. To find neural stem cell accelerators from the extract library of natural resources, we used a two-step screening including a Ngn2 promoter reporter gene screening and differentiation assay screening of neural stem cells. A reporter gene assay that can detect Ngn2 promoter activity by luciferase expression was constructed using C3H10T1/2 cells. Using this primary cell-based screening, Butea superba was found to include Ngn2 promoter activators from our tropical plant extract libraries. Bioassay-guided fractionation of this plant extract led to the isolation of 18 natural products, including pterocarpans and isoflavonoids. Dehydromaackiain (1), formononetin (6), ()-variabilin (13), ()-medicarpin (14), rothindin (17) and ononin (18) showed 1.8­2.8 times higher Ngn2 promoter activity at 5 mM compared with control. Of active natural compounds, 30-methoxydaidzein (3) showed promotion of neurite outgrowth of C17.2 in a secondary screen. 30-Methoxydaidzein (3) increased mRNA expression of pro-neural transcriptional factors (Ngn2, Ngn1, NeuroD2), a mature neuron-specific enzyme GAD1 and a pro-neural neurotrophic growth factor neurotrophin 3 (NT3) in C17.2 neural stem cells.

Cycloartane triterpenes and ingol diterpenes isolated from Euphorbia neriifolia in a screening program for death-receptor expression-enhancing activity.

In our screening program for natural products that increase death-receptor 5 expression, seven new cycloartane triterpenes, euphonerins A-G (1-7), and 3-O-acetyl-8-O-tigloylingol (8), a new ingol diterpene, were isolated from the MeOH extract of Euphorbia neriifolia leaves, together with 3,12-di-O-acetyl-8-O-tigloylingol (9), (24R)-cycloartane-3ß,24,25-triol (10), and three known flavonols (11-13). The structures of 1-8 were elucidated by spectroscopic analysis. Among these compounds, 1-11 showed death-receptor 5 expression enhancing activity.

Phorbol esters with wnt signal-augmenting effects isolated from Excoecaria indica.

A screening of natural products using a luciferase assay targeting the Wnt signaling pathway was carried out, and the bioassay-guided fractionation of Excoecaria indica (Euphorbiaceae) collected from Bangladesh afforded three phorbol esters (1 - 3). These compounds exhibited Wnt signal-augmenting effects with 1 causing a 25-fold increase in TCF/beta-catenin (TOP) transcriptional activity at 95 nM.

A new resin glycoside from Ipomoea maxima.

A new resin glycoside (1) was isolated from the aerial part of Ipomoea maxima, together with three known compounds, pescaprein XX (2), stoloniferin X (3), and stoloniferin IX (4). The structure of 1 was elucidated on the basis of 1D NMR spectroscopy, a fragmentation study by APCIMS, and HRESIMS analysis.

Acoschimperoside P, 2'-acetate: a Hedgehog signaling inhibitory constituent from Vallaris glabra.

Complete (1)H and (13)C NMR assignments of acoschimperoside P, 2'-acetate (1) and a new cardiac glycoside (2), isolated from the leaves of Vallaris glabra, are described. Compound 1 was active in the assay for Hedgehog signaling inhibition. In further experiments, this compound showed a strong cytotoxicity against human pancreatic (PANC1) and human prostate (DU145) cancer cells. The expression of GLI-related proteins (PTCH and BCL-2) in a dose-dependent manner was also inhibited by 1.

2-methoxy-1,4-naphthoquinone isolated from Impatiens balsamina in a screening program for activity to inhibit Wnt signaling.

A screening study using a luciferase assay to identify natural products which inhibit Wnt signaling was carried out. The bioassay-guided fractionation of aerial parts of a plant, Impatiens balsamina, led to the isolation of 2-methoxy-1,4-naphthoquinone (1) as an active compound. Compound 1 inhibited the TCF/ß-catenin (TOP) transcriptional activity (IC(50) 2.9 µM), while it decreased the transcriptional activity of FOP (mutated TCF-binding site)-transfected cells at >5 µM.