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.

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.

GLI1 Inhibitors Identified by Target Protein Oriented Natural Products Isolation (TPO-NAPI) with Hedgehog Inhibition.

This report describes the development of a target-protein-oriented natural-products-isolation (TPO-NAPI) method for Hedgehog inhibitors and the direct GLI1 inhibitor, 5'- O-methyl-3-hydroxyflemingin A (3), which inhibited hedgehog (Hh) signal transduction and diminished characteristics of cancer stem cells. Eight natural products (including three newly described products) that directly bind to GLI1 were rapidly obtained via the TPO-NAPI method developed using GLI1 protein-immobilized beads. 5'- O-Methyl-3-hydroxyflemingin A (3) inhibited Hh signaling (IC50 7.3 µM), leading to decreasing production of the Hh target proteins BCL2, PTCH1, and BMI1. 5'- O-Methyl-3-hydroxyflemingin A (3) was cytotoxic to Hh-related cancer cells. CD experiments revealed that 5'- O-methyl-3-hydroxyflemingin A (3) directly bound GLI1 ( Kd = 7.7 µM). Moreover, 5'- O-methyl-3-hydroxyflemingin A (3) diminished cancer stem cell characters of Huh7 such as sphere formation and production of the cancer stem cell marker EpCAM. These results suggest that Hh inhibitors can efficiently suppress the activity of cancer stem cells.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Xylogranin B: a potent Wnt signal inhibitory limonoid from Xylocarpus granatum.

Xylogranin B (2) was isolated from Xylocarpus granatum (Meliaceae) leaves, by use of a cell-based luciferase screening system targeting a Wnt signaling pathway. Compound 2 inhibited TCF/ß-catenin transcriptional activity (IC50 48.9 nM) and exhibited strong cytotoxicity against colon cancer cell lines. Compound 2 significantly decreased ß-catenin protein levels in nuclei but not in the cytosol. These results indicated that a decrease in ß-catenin levels in nuclei by 2 resulted in the Wnt signal inhibitory effects of 2.

Hh signaling inhibitors from Vitex negundo; naturally occurring inhibitors of the GLI1-DNA complex.

The hedgehog (Hh) signaling pathway has crucial roles in embryonic development, cell maintenance and proliferation, and is also known to contribute to cancer cell growth. New naturally occurring Hh inhibitors (1, 7 and 9) were isolated from Vitex negundo using our previously constructed cell-based assay. Bioactivity guided isolation provided 9 natural compounds including a new diterpene, nishindanol (9). Compounds 7 and 9 showed cytotoxicity against cancer cell lines in which Hh signaling was aberrantly activated. Vitetrifolin D (7; GLI1 transcriptional inhibition IC50 = 20.2 µM) showed inhibition of Hh related protein (PTCH and BCL2) production. Interestingly, the constructed electrophoresis mobility shift assay revealed that vitetrifolin D (7) disrupted GLI1 binding on its DNA binding domain. epi-Sclareol (8; inactive), possessing a similar structure to 7, did not show inhibition of GLI1­DNA complex formation. This is the first example of naturally occurring inhibitors of GLI1­DNA 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.

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.