論文

B-cell-specific Moloney murine leukemia virus region 1 (BMI1) is a central component of polycomb repressive complex 1 (PRC1), which maintains epigenetic repression of genes expression via chromatin condensation. BMI1 overexpression downregulates the expression of tumor suppressor genes, such as p16Ink4a and PTEN. BMI1 expression is upregulated in cancer stem cells (CSCs). Therefore, inhibitors of BMI1 expression have potential as therapeutic agents for cancer. This study aimed to identify BMI1 promoter inhibitors from actinomycetes. Using a recently constructed BMI1 promoter assay, we isolated three known compounds, elaiophylin (1), 2-methylelaiophylin (2), and nocardamin (3), from Streptomyces sp. IFM-11958 that inhibited BMI1 promoter activity with IC50 values of 30 nM, 447 nM, 22 µM, respectively. Elaiophylin (1) was the most potent. Western blot and PCR analyses revealed that elaiophylin (1) inhibited BMI1 expression at the mRNA level in human prostate cancer cells (DU145). Elaiophylin (1) also inhibited the sphere-forming activity of human hepatocellular carcinoma cells (Huh7), indicating that elaiophylin (1) suppresses the self-renewal capacity of CSCs. Elaiophylin (1) is the first BMI1 promoter inhibitor isolated from actinomycete metabolites.

Abstract: 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. Graphic abstract: [Figure not available: see fulltext.].

Because research on the genus Nocardia has not progressed as compared to the extensively studied genus Streptomyces, Nocardia is considered as a useful undeveloped resource for exploring natural products. In this study, a new γ-butyrolactone derivative, named inohanalactone (1), was isolated from Nocardia inohanensis IFM0092T. Inohanalactone possesses an aliphatic side chain containing 8 carbons, a vicinal diol, and a cis double bond. Inohanalactone was produced by N. inohanensis IFM0092T in the modified Czapek-Dox medium, whereas it was not produced in other media such as Nutrient broth, Waksman, and Yeast-Malt-Glucose media.

A new aminocyclitol derivative, designated nabscessin C (1), was isolated from Nocardia abscessus IFM 10029T. Nabcessin C is an isomer of nabscessins A (2) and B (3) with different positioning of the acyl group. Absolute configuration of nabscessin A was determined by conversion into the 2-deoxy-scyllo-inosamine pentaacetyl derivative (4) by hydrolysis and acetylation of 2. The biosynthetic pathway of nabscessins is proposed based on gene expression analysis.

Notch signaling inhibitors with the potential of immune suppressor production by pathogenic bacteria for easy host infection were searched from extracts of Nocardia sp. Nocobactin NA-a (compound 1) and nocobactin NA-b (compound 2), which have been suggested as pathogenesis factors, were isolated from N. farcinica IFM 11523 isolated from the sputum of a Japanese patient with chronic bronchitis. Compounds 1 and 2 showed Notch inhibitory activities with IC50 values of 12.4 and 17.6 μM, respectively. Compound 1 and 2 decreased of Notch1 protein, Notch intracellular domain, and hairy and enhancer of split 1, which is a Notch signaling target protein. In addition, compounds 1 and 2 showed cytotoxicity against mouse macrophage-like cell line RAW264.7 with IC50 values of 18.9 and 21.1 μM, respectively. These results suggested that the Notch inhibitors production by pathogenic bacteria may increase pathogen infectivity.

We isolated two peptidolipin NA derivatives, L-Val (6) peptidolipin NA and peptidolipin NA, named as compounds 1 and 2, respectively. These compounds were produced in the co-culture extracts of Nocardia arthritidis IFM10035T and the mouse macrophage cell line J774.1, but not in the single culture of N. arthritidis. Among the two compounds along with nocarjamide (3) isolated from the co-culture of N. tenerifensis and J774.1, compounds 1 and 3 showed higher cytotoxicity against J774.1, with IC50 values of 116 μM and 25 μM, respectively. In addition, compound 1 showed a TRAIL-resistance-overcoming activity in the human gastric adenocarcinoma AGS cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) potently induces apoptosis in most cancer cells, but not in normal cells, and, thus, has attracted intense interest as a promising antitumor agent. Activity-guided fractionation of the culture broth of the actinomycete CKK1172 strain led to the isolation of the antibiotic resistomycin (1) using human gastric adenocarcinoma AGS cells. Compound 1 exhibited TRAIL resistance-overcoming activity at 2 μM with 38% more inhibition than a treatment with 1 alone (without TRAIL) in TRAIL-resistant AGS cells.

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 25.4µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

The Hes1 dimer inhibitor, agalloside (2), which can accelerate the differentiation of neural stem cells is described. Six natural products, including one new natural product, which bind to Hes1 were rapidly isolated by a developed "target protein oriented natural products isolation" (TPO-NAPI) method using Hes1-immobilized beads. Of the six compounds, 2 inhibited Hes1 dimer formation at both the protein-and cellular level. Neural stem cells treated with 2 differentiated to neurons with longer neurites than cells treated with varproic acid or retinoic acid. Moreover, 2 exhibited specificity for neurons. This promotion of differentiation was supported by an increase in the mRNA expression of the proneural genes, Mash1 and Ngn2, which were inhibited by Hes1.

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.

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 GLI1-DNA complex formation. This is the first example of naturally occurring inhibitors of GLI1-DNA complex formation. © 2013 The Royal Society of Chemistry.

A high-throughput screening system for analyzing small molecule-induced coactivator (CoA) recruitment by the vitamin D receptor (VDR) has been developed. The vitamin D-induced protein - protein interactions between VDR and fluorophore (Cy3 or Cy5)-labeled TIF2 or SRC-1 were successfully detected by using a new HCHO fixing method of the protein complex on microplates. The results obtained from this screening of our synthetic vitamin D analogues suggest that the CoA-recruiting activities play an important role in determining the biological activity of various vitamin D analogues and explain the discrepancies between the VDR binding affinity and their biological activity.

B-Cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) is a core component of the polycomb repressive complex 1 (PRC1). Abnormal expression of BMI1 is associated with a number of human malignances and cancer stem cells (CSCs), which cause chemotherapy resistance. Therefore, small molecules that inhibit BMI1 expression are potential candidates for cancer therapy. In this study, a cell-based reporter gene assay was developed that allowed BMI1 promoter activity to be measured in 293T human embryonic kidney cells based on luciferase expression levels. Using this screening assay, the methanol-soluble extracts of Beaumontia murtonii and Eugenia operculata were selected as leads. Bioassay-guided fractionation of the extracts led to the isolation of three known cardenolides (13) and one new compound (4) from B. murtonii and two known triterpenoids (5 and 6) and one new compound (7) from E. operculata. These seven compounds inhibited BMI1 promoter activity (IC50 range 0.09323.0 mu M), and the most active compound, wallichoside (1), was further evaluated. Western blot analysis revealed that wallichoside (1) decreases BMI1 protein levels in HCT116 human colon carcinoma cells, and flow cytometry analysis showed that it significantly reduced levels of the CSC biomarker epithelial cell adhesion molecule. Wallichoside (1) also inhibited sphere formation of Huh7 human hepatocellular carcinoma cells, indicating that it diminished the self-renewal capability of CSCs.

Three new acridine alkaloids, inubosins A (1), B (2), and C (3), were isolated from an extract of a culture of Streptomyces sp. IFM 11440 using bioassay-guided fractionation. Neurogenin2 (Ngn2) is an activator-type basic helix-loop-helix transcription factor that promotes neural stem cell differentiation. Using cell-based Ngn2 promoter activity-guided screening, Streptomyces sp. IFM 11440 was found to induce Ngn2 promoter activity. The structures of 1-3 were established using spectroscopic methods, including 1D- and 2D-NMR measurements. Inubosin B (2) showed potent Ngn2 promoter activity. Moreover, inubosin B (2) increased mRNA expression of genes related to neural stem cell differentiation.

The hedgehog (Hh) signaling pathway is an important regulator of embryonic development, tissue patterning, cellular proliferation and differentiation. However, aberrant activation of the pathway is associated with tumorigenesis. In this study, the Hh signal inhibitory activity of plant extracts was measured with a cell-based assay system that targets GLI1-mediated transcription. Activity guided analysis of MeOH extracts of christmas grass (Themeda arguens) led to the isolation of four inhibitory compounds: aciculatin (1), 7-de-O-methylaciculatin (2), 8-C-beta-D-boivinopyranosylapigenin (3) and aciculatinone (4). Compound 1 strongly inhibited Hh/GLI1-mediated transcriptional activity with an IC50 value of 1.8. mu M, and disrupted the formation of the GLI1-DNA complex by EMSA. 1 and 3 were cytotoxic for human prostate (DU-145) and breast (MCF-7) cancer cells. These compounds may serve as new tools for the molecular dissection of Hh pathway activation.

In the screening program for natural bioactive compounds that enhance the expression of death receptor 5 (DR5), activity-guided fractionation of the Piper chaba (Piperaceae) root MeOH extract led to the isolation of four alkamides (1-4). Compounds 1, 3, and 4 enhanced DR5 promoter activity while pellitorine (3) exhibited TRAIL-resistance-overcoming activity.

An unusual alkaloid with a 2-azaquinone-phenylhydrazone structure, katorazone (1), and other metabolites were isolated from the ethyl acetate extract of Streptomyces sp. IFM 11299. The chemical structure of katorazone (1) was elucidated by 1D and 2D NMR analyses together with HR-ESI mass spectrometry. Katorazone (1) showed a synergistic effect in combination with TRAIL and decreased the viability of human gastric adenocarcinoma (AGS) cells. (C) 2012 Elsevier Ltd. All rights reserved.

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D-3[25(OH)-19-nor-D-3] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D-3 [1 alpha, 25(OH)(2)D-3], suggesting that 25(OH)-19-nor-D-3 might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D-3 [1 alpha,25(OH)(2)-19-nor-D-3] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D-3 and 25-hydroxyvitamin D-3 [25(OH) D3] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D3 and 1 alpha, 25(OH)(2)D-3, we found that k(cat)/ Km for 1 alpha-hydroxylation of 25(OH)-19-nor-D-3 was less than 0.1% of that for 25(OH) D-3, and the kcat/ Km value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)219- nor-D-3 and 1 alpha, 25( OH)(2)D-3. The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D-3 compared with 1 alpha,25(OH)(2)D-3. We then analyzed the metabolites of 25(OH)D-3 and 25(OH)-19-nor-D-3 in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha, 25(OH)(2)D-3 was detected in cells incubated with 25(OH)D-3, whereas no 1 alpha,25(OH)(2)-19-nor-D-3 was detected in cells incubated with 25(OH)-19-nor-D-3. Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D-3 is converted to 1 alpha,25(OH)2-19-nor-D-3 by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D-3 by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D-3 has potential as an attractive agent for prostate cancer therapy.

The Wnt signal regulates various biological processes, and its aberrant activation is associated with the development of diseases. Thus, inhibiting the Wnt signal provides a promising strategy to treat these diseases. Our cell-based luciferase assay system, which targets the Wnt signal (TOP assay), revealed that Azadirachta excelsa inhibited the Wnt signal. The activity-guided isolation of the MeOH fruit extract of A. excelsa provided one new (1) and seven known (2-8) limonoids. Their structures were elucidated based on their spectroscopic data, and their NMR data were compared with those in the literature. Compounds 3-6 potently inhibited the Wnt signal with IC50 values of 127 nM, 300 nM, 252 nM, and 121 nM, respectively. Compound 4 exhibited selective cytotoxicity against AGS and HCT116. Western blot analysis showed that 4 did not affect the level or localization of beta-catenin, but downregulated the level of c-myc. Our results suggested that 4 may have inhibited the Wnt signal by affecting the components downstream of beta-catenin. (C) 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

According to the prevailing paradigm, 1 alpha-hydroxylation of 25-hydroxyvitamin D-3 (25(OH)D-3) and its analogs is a prerequisite step for their biological effects. We previously reported that 25-hydroxy-19-nor-vitamin D-3 (25(OH)-19-nor-D-3) had anti-proliferative activity in a cell line, PZ-HPV-7, which was derived from human non-cancerous prostate tissue, and suggested that 25(OH)-19-nor-D-3 acted after 1 alpha-hydroxylation by vitamin D 1 alpha-hydroxylase (CYP27B1). However, metabolic studies of 25(OH)-19-nor-D-3 using recombinant CYP27B1 revealed that 25(OH)-19-nor-D-3 was rarely subjected to 1 alpha-hydroxylation. Therefore, in this report, we attempted to clarify the mechanism of 25(OH)-19-nor-D-3 action in intact cells using PZ-HPV-7 prostate cells. After incubating the cells with 25(OH)-19-nor-D-3, eight metabolites of 24-hydroxylase (CYP24A1) were detected, whereas no products of CYP27B1 including 1 alpha,25-dihydroxy-19-nor-vitamin D-3 (1 alpha,25(OH)(2)-19-nor-D-3) were found. Furthermore, the time-dependent nuclear translocation of vitamin D receptor (VDR) and the subsequent transactivation of cyp24A1 gene in the presence of 25(OH)-19-nor-D-3 were almost identical as those induced by 1 alpha,25(OH)(2)-19-nor-D-3. These results strongly suggest that 25(OH)-19-nor-D-3 directly binds to VDR as a ligand and transports VDR into the nucleus to induce transcription of cyp24A1 gene. In addition, knock down of cyp27B1 gene did not affect the anti-proliferative activity of 25(OH)-19-nor-D-3, whereas knock down of VDR attenuated the inhibitory effect. Thus, our results clearly demonstrate that the anti-proliferative activity of 25(OH)-19-nor-D-3 is VDR dependent but 1 alpha-hydroxylation independent, suggesting that 25(OH)D-3 analogs such as 25(OH)-19-nor-D-3 could be attractive candidates for anticancer therapy. Journal of Molecular Endocrinology (2011) 47, 209-218