Natural Compounds with BMI1 Promoter Inhibitory Activity from Mammea siamensis and Andrographis paniculata.

A new coumarin derivative (1) and 30 known compounds were isolated from Mammea siamensis and Andrographis paniculata, guided by B cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) promoter inhibitory activity. Among the isolated compounds, 15 compounds showed BMI1 promoter inhibitory activity, and five compounds were found to be cytotoxic. 14-Deoxy-11,12-dehydroandrographolide (18) was highly cytotoxic to DU145 cells with an IC50 value of 25.4 µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

Two new coumarins and a new xanthone from the leaves of Rhizophora mucronata.

Two new coumarins (1, 2) and a new xanthone (3), together with 14 known compounds-eight coumarins (4, 5, 9, 10, 12-15), three xanthones (11, 16, 17), a benzoic acid (6) and two flavonones (7, 8)-were isolated from the leaves of Rhizophora mucronata. The structures of the compounds were elucidated by spectroscopic (IR, MS, and NMR) analyses. The isolated compounds were tested for cytotoxicity against human cancer cell lines HL-60 and HeLa. Among these compounds, only compound 16 inhibited the growth of both HeLa (IC50 = 4.8 µM) and HL-60 (IC50 = 1.0 µM) cells. Compounds 4, 7, 10, and 12 exhibited moderate activity against HeLa cells (IC50 = 3.8-8.3 µM). Compounds 5, 9, 11, and 17 showed moderate activity against HL-60 cells (IC50 = 2.2-6.3 µM). Higher selectivity against HL-60 cell lines was observed for compounds 5, 9, 11, and 16 with SI values (NIH 3T3/HL-60) of 8.6, 19.2, 9.4, and 10.2, respectively.

Hes1-Binding Compounds Isolated by Target Protein Oriented Natural Products Isolation (TPO-NAPI).

Hairy and enhancer of split 1 (Hes1) is a transcription factor that acts in neural stem cells to inhibit differentiation. We recently developed target protein oriented natural products isolation (TPO-NAPI) using Hes1-immobilized beads to identify activators of neural stem cells. Isomicromonolactam (1), staurosporin (2), and linarin (3) were isolated as Hes1-binding compounds using the TPO-NAPI method. Of these, compound 1 enhanced neural stem cell differentiation. Using truncated Hes1 proteins, the binding region of Hes1 for 1 was estimated to be in the C-terminal half that includes a TLE/Grg binding site. The differentiation-promoting activity of inohanamine (4) is also reported.

Notch Inhibitors from Calotropis gigantea That Induce Neuronal Differentiation of Neural Stem Cells.

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.

Hedgehog inhibitors from Withania somnifera.

The hedgehog (Hh) signaling pathway performs an important role in embryonic development and in cellular proliferation and differentiation. However, aberrant activation of the Hh signaling pathway is associated with tumorigenesis. Hh signal inhibition was evaluated using a cell-based assay system that targets GLI1-mediated transcription. Activity-guided isolation of the Withania somnifera MeOH extract led to the isolation of six compounds: withaferin A (1) and its derivatives (2-6). Compounds 1 and 2 showed strong inhibition of Hh/GLI1-mediated transcriptional activity with IC50 values of 0.5 and 0.6 µM, respectively. Compounds 1, 2, 3, and 6 were cytotoxic toward human pancreatic (PANC-1), prostate (DU145) and breast (MCF7) cancer cells. Furthermore, 1 also inhibited GLI1-DNA complex formation in EMSA.

Coronaridine, an iboga type alkaloid from Tabernaemontana divaricata, inhibits the Wnt signaling pathway by decreasing ß-catenin mRNA expression.

Four alkaloids, voacangine (1), isovoacangine (2), coronaridine (3), and coronaridine hydroxyindolenine (4), were isolated from the MeOH extract of Tabernaemontana divaricata aerial parts by activity-guided fractionation for Wnt signal inhibitory activity. Compounds 1-4 exhibited TCF/ß-catenin inhibitory activities with IC50 values of 11.5, 6.0, 5.8, and 7.3 µM, respectively. Of these, coronaridine (3) decreased ß-catenin levels in SW480 colon cancer cells, while this decrease in ß-catenin was not suppressed by a co-treatment with 3 and MG132, a proteasome inhibitor. These results suggested that the decrease observed in ß-catenin levels by coronaridine (3) did not depend on a proteasomal degradation process. On the other hand, the treatment of SW480 cells with coronaridine (3) caused a decrease in ß-catenin mRNA levels. Thus, coronaridine (3) may inhibit the Wnt signaling pathway by decreasing the mRNA expression of ß-catenin.

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 inhibitors from Artocarpus communis and Hyptis suaveolens.

The hedgehog (Hh) signaling pathway plays crucial roles in cell maintenance and proliferation during embryonic development. Naturally occurring Hh inhibitors were isolated from Artocarpus communis and Hyptis suaveolens using our previously constructed cell-based assay system. Bioactivity guided fractionation led to the isolation of 15 compounds, including seven new compounds (4, 5, 6, 7, and 9-11). The isolated compounds showed cytotoxicity against a cancer cell line (PANC1) in which Hh signaling was abnormally activated. Several compounds (12-14; GLI1 transcriptional inhibition IC50=7.6, 4.7, and 4.0 µM, respectively) inhibited Hh related protein (BCL2) expression. Moreover, compounds 1, 12, and 13 disrupted GLI1 and DNA complex formation.

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.

Scopadulciol, Isolated from Scoparia dulcis, Induces ß-Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells.

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased ß-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of ß-catenin induced by 1. The 1-induced degradation of ß-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of ß-catenin, which resulted in the inhibition of TCF/ß-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

Calotropin: a cardenolide from calotropis gigantea that inhibits Wnt signaling by increasing casein kinase 1α in colon cancer cells.

Wnt signaling plays key roles in embryonic development and various human diseases. Activity-guided testing to isolate Wnt signaling inhibitors from the methanol extract of Calotropis gigantea (Asclepiadaceae) exudutes identified six Wnt inhibitory cardenolides (1-6), of which 1, 3, 5, and 6 exhibited potent TCF/ß-catenin inhibitory activities (IC50 0.7-3.6 nM). Calotropin (1) inhibited Wnt signaling by decreasing both nuclear and cytosolic ß-catenin in a dose-dependent manner, and promoted degradation of ß-catenin by increasing the phosphorylation of ß-catenin at Ser45 through casein kinase 1α (CK1α). Moreover, 1 significantly increased CK1α protein and mRNA levels. The results suggest that 1 inhibits the Wnt signaling pathway by increasing CK1α protein levels. To the best of our knowledge, calotropin is the first small molecule to increase CK1α levels.

Ricinine: a pyridone alkaloid from Ricinus communis that activates the Wnt signaling pathway through casein kinase 1α.

Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/ß-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30µM, respectively. Cells treated with ricinine (1) had higher ß-catenin and lower of p-ß-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1α (CK1α) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1α, had lower ß-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher ß-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1α activity. Furthermore, 1 increased ß-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1α.

Central-stimulating and analgesic activity of the ethanolic extract of Alternanthera sessilis in mice.

BACKGROUND: Alternanthera sessilis is a popular vegetable and used in traditional medicinal practice of Bangladesh and other parts of Asia to relive tiredness, laziness, and sleeps as well as pain and inflammation. However, no report was found on the neuropharmacological and analgesic activity of this plant to-date. Present study was undertaken to evaluate the neuropharmacological and analgesic activity of the ethanol extract of A. sessilis whole plant (ETAS) in mice models. METHODS: Central stimulating activity was investigated by pentobarbitone induced sleeping time, open field, and hole cross tests. Analgesic activity was evaluated by acetic acid induced writhing and hot-plate methods. The tests were performed at 250 and 500 mg/kg body weight dose levels. RESULTS: In sleeping time test, ETAS significantly (p < 0.001) increased the onset of sleep, and decreased the duration of sleep. In open field and hole cross tests, ETAS significantly (p < 0.001) increased the movements of mice which persisted throughout the study period. In writhing test, ETAS showed, significant (p < 0.001) inhibition of writhing reflex. In hot plate test, ETAS significantly (p < 0.001) raised the pain threshold. In HPLC analysis for polyphenols, (+)-catechin, rutin, ellagic acid, and quercetin were detected in ETAS (117.72, 490.74, 3007.26, and 13.85 mg/100 g of dry extract, respectively). CONCLUSION: Present study supported the traditional uses of A. sessilis and indicated that the plant can be a potential source of bioactive molecules.

Physalin H from Solanum nigrum as an Hh signaling inhibitor blocks GLI1-DNA-complex formation.

Hedgehog (Hh) signaling plays an important role in embryonic development, cell maintenance and cell proliferation. Moreover, Hh signaling contributes to the growth of cancer cells. Physalins are highly oxidized natural products with a complex structure. Physalins (1-7) were isolated from Solanum nigrum (Solanaceae) collected in Bangladesh by using our cell-based assay. The isolated physalins included the previously reported Hh inhibitors 5 and 6. Compounds 1 and 4 showed strong inhibition of GLI1 transcriptional activity, and exhibited cytotoxicity against cancer cell lines with an aberrant activation of Hh signaling. Compound 1 inhibited the production of the Hh-related proteins patched (PTCH) and BCL2. Analysis of the structures of different physalins showed that the left part of the physalins was important for Hh inhibitory activity. Interestingly, physalin H (1) disrupted GLI1 binding to its DNA binding domain, while the weak inhibitor physalin G (2) did not show inhibition of GLI1-DNA complex formation.

Eudesmane-type sesquiterpenoid and guaianolides from Kandelia candel in a screening program for compounds to overcome TRAIL resistance.

In a screening program for natural products that can overcome TRAIL resistance, a new eudesmane-type sesquiterpenoid (1), three new guaianolides, mehirugins A-C (2-4), and two known guaianolides (5 and 6) were isolated from a MeOH extract of Kandelia candel leaves. Compounds 1 and 3-6 in combination with TRAIL showed cytotoxic activity in sensitizing TRAIL-resistant human gastric adenocarcinoma cells.

Thannilignan glucoside and 2-(ß-glucopyranosyl)-3-isoxazolin-5-one derivative, two new compounds isolated from Terminalia bellirica.

Two new compounds, thannilignan 9-O-ß-glucoside (1) and 2-(ß-glucopyranosyl)-3-isoxazolin-5-one derivative (2), and seven known compounds were isolated from the methanol extract of Terminalia bellirica leaves, collected in Bangladesh. The structures of the compounds were elucidated using spectroscopic analysis. Among these isolated compounds, corilagin (3) was cytotoxic against human gastric adenocarcinoma cell line AGS at an IC50 of 20.8 µM, and ß-D-glucopyranose 1,3,6-trigallate (4) exhibited the ability to overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance.

New Hedgehog/GLI signaling inhibitors from Excoecaria agallocha.

The inhibition of the Hedgehog (Hh) signaling pathway has emerged as an anti-cancer strategy. Three flavonoid glycosides including 2 new compounds (1-2) were isolated from Excoecaria agallocha as Hedgehog/GLI1-mediated transcriptional inhibitors and exhibited cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells. Our data revealed that compound 1 clearly inhibited the expression of GLI-related proteins (PTCH and BCL-2) and blocked the translocation of GLI1 transcription factors into the nucleus in PANC1. Deleting the Smoothened (Smo) function in PANC1 treated with 1 led to downregulation of the mRNA expression of Ptch. This study describes the first Hh signaling inhibitor which blocks GLI1 movement into the nucleus without interfering with Smo.

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.

Constituents of Amoora cucullata with TRAIL resistance-overcoming activity.

In search of bioactive natural products for overcoming TRAIL resistance from natural resources, we previously reported a number of active compounds. Bioassay-guided fractionation of mangrove, Amoora cucullata, collected from Sundarbans Mangrove Forest, Bangladesh, led to the isolation of four new compounds (1-4), along with seven known compounds (5-11). Of the isolates, compounds 1, 5, 8, and 9 showed TRAIL resistance-overcoming activity, among which 8 showed the most potent activity and enhanced TRAIL-induced apoptosis in TRAIL-resistant human gastric adenocarcinoma (AGS) cells through the activation of caspase-3/7, enhancing the expression of DR4 and DR5 mRNA in AGS cells. Cell death caused by the combined treatment of 8 and TRAIL was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, indicating that 8 sensitizes TRAIL-resistant AGS cells to TRAIL through the induction of DR4 and DR5.

Target protein-oriented isolation of Hes1 dimer inhibitors using protein based methods.

Natural products isolation using protein based methods is an attractive for obtaining bioactive compounds. To discover neural stem cell (NSC) differentiation activators, we isolated eight inhibitors of Hes1 dimer formation from Psidium guajava using the Hes1-Hes1 interaction fluorescent plate assay and one inhibitor from Terminalia chebula using the Hes1-immobilized beads method. Of the isolated compounds, gallic acid (8) and 4-O-(4"-O-galloyl-α-L-rhamnopyranosyl)ellagic acid (11) showed potent Hes1 dimer formation inhibitory activity, with IC50 values of 10.3 and 2.53 µM, respectively. Compound 11 accelerated the differentiation activity of C17.2 NSC cells dose dependently, increasing the number of neurons with a 125% increase (5 µM) compared to the control.