Published papers

BACKGROUND AND PURPOSE Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells. EXPERIMENTAL APPROACH P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca2+ channel blocker. KEY RESULTS Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 mu M) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca2+ channel blocker; 100 nM) and a small interfering RNA for the T-type Ca2+ channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. CONCLUSIONS AND IMPLICATIONS Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca2+ channel gene.

The coupling reactions of two diazotized 3-aminothieno[3,4-c]coumarins were investigated. Compounds 1a,b both react with sodium nitrite in concentrated sulphuric acid at 0-5 degrees C to give the diazotized intermediates 2 and 3, the latter resulting from the acid -catalyzed hydrolysis of the lactonic ring of 2. The in situ formed diazonium salts react with aromatic amines (4) to afford a series of arylazothiophenes dyes in the form of their ammonium sulfate salts. With diazotized aniline, besides the normally expected phenylazothiophene 10 from the reaction with compound 1a, the corresponding product of acid hydrolysis 11 was also isolated. In at least one of the cases, the thienyl diazonium salt 2 undergoes a Gomberg-Bachmann arylation reaction with p-nitroaniline to give the 2-arylthiophene 9. The direct hydrolysis of compounds 1a, b by concentrated sulphuric acid and subsequent oxidative dimerization of the primary product of acid hydrolysis led to compound 12.

Separation of somatic cells from germ-line cells is a crucial event for multicellular organisms, but how this step was achieved during evolution remains elusive. In Dictyostelium discoideum and many other dictyostelid species, solitary amoebae gather and form a multicellular fruiting body in which germ-line spores and somatic stalk cells differentiate, whereas in Acytostelium subglobosum, acellular stalks form and all aggregated amoebae become spores. In this study, because most D. discoideum genes known to be required for stalk cell differentiation have homologs in A. subglobosum, we inferred functional variations in these genes and examined conservation of the stalk cell specification cascade of D. discoideum mediated by the polyketide differentiation-inducing factor-1 (DIF-1) in A. subglobosum. Through heterologous expression of A. subglobosum orthologs of DIF-1 biosynthesis genes in D. discoideum, we confirmed that two of the three genes were functional equivalents, while DIF-methyltransferase (As-dmtA) involved at the final step of DIF-1 synthesis was not. In fact, DIF-1 activity was undetectable in A. subglobosum lysates and amoebae of this species were not responsive to DIF-1, suggesting a lack of DIF-1 production in this species. On the other hand, the molecular function of an A. subglobosum ortholog of DIF-1 responsive transcription factor was equivalent with that of D. discoideum and inhibition of polyketide synthesis caused developmental arrest in A. subglobosum, which could not be rescued by DIF-1 addition. These results suggest that non-DIF-1 polyketide cascades involving downstream transcription factors are required for fruiting body development of A. subglobosum.

Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), -2, and -3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5-20 mu M) dose-dependefitly suppressed LPA-induced cell migration with associated IC50 values of 5.5, 4.6, and 4.2 mu M, respectively. On the other hand, the IC50 values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 mu M, respectively, in LM8 cells, and >20, 14.8, and 4.3 mu M, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. (C) 2015 Elsevier Inc. All rights reserved.

Tenuipesine A (1), a novel trichothecane with an unprecedented carbon-migrated skeleton that embodies of a cyclopropane ring, was isolated from cultivated fruiting bodies of Paecilomyces tenuipes (Isaria japonica), a popular e ntomo pathogenic fungi employed in folk medicine and health foods in China, Korea, and Japan. The structure was determined on the basis of two-dimensional NMR data. Its stereochemistry was elucidated by spectroscopic data and the chemical transformation of the coexisting trichothecene, 4beta-acetoxy-12,13-epoxytrichothec-g-ene3cc,15-diol (2).

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

Innate immunity is the front-line of self-defense against microbial infection. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. From a pharmaceutical point of view, innate immunity is an ideal target for the development of immunoregulators. Therefore, we aimed to isolate and characterize a novel mammalian immunoregulator isolated from the thermophilic cellulotic fungus Talaromyces sp. 2'-(R)-hydroxy-C(24) phytoceramide (C(24)(2'OH)Phy) was isolated from Talaromyces sp. using a Drosophila ex vivo culture system. C(24)(2'OH)Phy suppressed the immune deficiency (IMD) pathway-dependent expression of antibacterial peptides in Drosophila, whereas it stimulated the production of chemokines in human cells. Structure activity relationship studies of C(24)(2'OH) Phy analogs revealed that both the 2'-(R)-hydroxylignoceroyl group and phytoceramide backbone are essential for the biologic activity of C(24)(2'OH)Phy. Microarray analysis revealed that C(24)(2'OH)Phy selectively activates the transcription of inflammatory response genes, including chemokines. Furthermore, a reporter gene assay and small interfering RNA analysis demonstrated that C(24)(2'OH)Phy stimulates chemokine production through cAMP response element-binding protein activation in human cells. C(24)(2'OH)Phy may be a lead immunostimulating compound in humans. (C) 2011 Elsevier B.V. All rights reserved.

Brefelamide is an aromatic amide isolated from Dictyostelium cellular slime molds. We found that brefelamide has a potent inhibitory growth effect measured by MTT assay in 1321N1 human astrocytoma cells. The inhibition was associated with reduced phosphorylation of extracellular signal-regulated kinase (ERK). Brefelamide inhibited epidermal growth factor (EGF)-induced phosphorylation of ERK in a concentration-dependent manner. Furthermore, brefelamide diminished EGF-induced phosphorylation of EGF receptor at Tyr(1068), a Grb2 binding site that leads to an activation of the Ras/Raf/ERK system. Brefelamide also reduced the expression level of the EGF receptor. These results suggest that one of the mechanisms of action of brefelamide is assumed to be inhibition of phosphorylation of ERK through a reduction of EGF receptor activity in 1321 NI human astrocytoma cells. (C) 2009 Elsevier B.V. All rights reserved.

An abnormally high serum phosphate level induces calcific aortic stenosis (CAS), which is characterized by ectopic valve calcification and stenosis of the orifice area. Inhibition of ectopic calcification is a critical function of any internal medical therapy for CAS disease. The aim of the present study was to investigate the inhibitory effects of several derivatives of evocarpine, methanolic extracts from the fruits of Evodia rutaecarpa Bentham (Japanese name: Go-Shu-Yu) on the high phosphate-induced calcification of human aortic valve interstitial cells (HAVICs) obtained from patients with CAS. High phosphate (3.2 mM) concentrations significantly increased the calcification of HAVICs after 7 days of culture. This calcification was completely inhibited in the presence of sodium phosphonoformate (PFA), a selective inhibitor of the type III sodium-dependent phosphate cotransporter (PiT-1). PiT-1 contributes to phosphate uptake, resulting in calcification. 1-Methyl-2-undecyl-4(1H)-quinolone (MUQ; 30-300 nM), but not evocarpine or its derivatives dihydroevocarpine and 1-methyl-2-nonyl-4(1H)-quinolone, inhibited the high phosphate-induced HAVICs calcification in a concentration-dependent manner. Although all of the evocarpine derivatives attenuated alkaline phosphatase activity, only MUQ also decreased PiT-1 gene expression with cellular PiT-1 protein diminution. These results suggest that MUQ mitigated high phosphate-induced HAVICs calcification by inhibiting PiT-1 gene expression. (C) 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Since neurotrophic factors are essential for neurons to form neuronal networks and maintain neuronal functions, neurotrophic factor-like substances or inducers of neurotrophic factors can be useful for the treatment of serious neuronal diseases such as Alzheimer's and Parkinson's diseases. In the present study, we examined an effect of 5,19-cyclo-9 beta,10 xi-androstane-3,17-dione (CAD) on neurotrophic factor synthesis in glial cells and scopolamine-induced impairment of learning in mice. 1321N1 human astrocytoma cells promoted secretion of certain neurotrophic factors in response to CAD with no cytotoxicity, which caused dramatic neurite outgrowth in rat pheochromocytoma (PC12) cells. In fact, CAD significantly enhanced nerve growth factor (NGF) secretion and its gene expression in 1321N1 cells, in a time and concentration-dependent manner. Because second messengers such as cAMP, inositol 1,4,5-trisphosphates and Ca2+ induce NGF gene expression, we measured activities of adenylyl cyclase and phospholipase C and intracellular Ca2+ concentration in 1321N1 cells. However, CAD changed neither second messenger levels. CAD enhanced the gene expression of proto-oncogene, c-fos that is one of the components of transcription factor (AP-1). In addition to those above, the in vivo effects of CAD were also examined. Although injection of muscarinic receptor antagonist scopolamine impaired passive avoidance learning in mice, pretreatment with CAD significantly reversed the adverse effect in a dose-dependent manner. Taking these results together, CAD has enormous therapeutic potential for serious neuronal diseases. (C) 2007 Elsevier B.V. All rights reserved.

Innate immunity comprises evolutionarily conserved self-defense mechanisms against microbial infections. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Using Drosophila ex vivo culture systems, we isolated a cyclopentanediol analogue from Aspergillus sp. as an immunosuppressive substance. This compound selectively suppressed activation of the IMD pathway in Drosophila in vivo and the target molecules of the compound lie between the Imd adaptor protein and dTAK1 kinase in the IMD pathway. In human cells, the compound suppressed TNF-alpha, but not IL-1 beta, stimulation-induced activation of NF-kappa B, suggesting that its target molecules are upstream of TAK1 in mammalian innate immunity. (C) 2008 Elsevier Inc. All rights reserved.

Innate immunity is the first line of defence against infectious micro-organisms, and the basic mechanisms of pathogen recognition and response activation are evolutionarily conserved. In mammals, the innate immune response in combination with antigen-specific recognition is required for the activation of adaptive immunity. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Here, for the purpose of pharmaceutical screening, we established an in vitro culture based on the innate immune response of Drosophila. The in vitro system is capable of measuring lipopolysaccharide (LPS)-dependent activation of the immune deficiency (imd) pathway, which is similar to the tumour necrosis factor signalling pathway in mammals. Screening revealed that well-known inhibitors of phospholipase A(2) (PLA(2)), dexamethasone (Dex) and p-bromophenacyl bromide (BPB) inhibit LPS-dependent activation of the imd pathway. The inhibitory effects of Dex and BPB were suppressed by the addition of an excess of three (arachidonic acid, eicosapentaenoic acid and gamma-linolenic acid) of the fatty acids so far tested. Arachidonic acid, however, did not activate the imd pathway when used as the sole agonist. These findings indicate that PLA, participates in LPS-dependent activation of the imd pathway via the generation of arachidonic acid and other mediators, but requires additional signalling from LPS stimulation. Moreover, PLA(2) was activated in response to bacterial infection in Sareophaga. These results suggest a functional link between the PLA(2)-generated fatty acid cascade and the LPS-stimulated imd pathway in insect immunity.

Entomopathogenic fungi infect insects through the cuticle, grow as hyphal bodies or hyphae in the hemocoel, and cause host death by nutritional destruction of tissues, and producint toxic metabolites and pathogenic enzymes. To date, entomopathogenic fungi have not been extensively studied by natural product chemists. We focused on entomopathogenic fungi as new resources for biologically active compounds, and studied the diversity of their secondary metabolites. In this study, we show the isolation and structure elucidation of novel pyrone diterpene-type compounds, metarhizin A-H (1-8) from Metarhizium flavoviride, and their antiproliferative activities on human cancer cell lines. Additionally, we synthesized many metarhizin derivatives, evaluated their antiproliferative activities, and discuss the structural requirements for this activity. A novel pyrone diterpene-type compound, Nf-1 (9), was also isolated from Nectria flammea. Oomycetes from fungus-like mycelium and were calssified as fungi in the past. However, the cell walls of them are c omposed of cellulose rather than chitin, as in the fungi. In the present, the ultrastructure, biochemistry and molecular sequences of them indicate that the oomycetes belong to the Kingdom Chromista, not the Kingdom Fungi. We also focused on oomycetes as new resources for natural product chemistry. A new oxidized lanostane-type compound, St-1 (23), was isolated from the mycelium of oomycete Saprolegnia terrestris. The structure of 23 was determined by spectral means including EIMS, and ^1H and ^<13>C NMR.

EM represents Effective Microorganisms, a microbial consortium consisting mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects on cells of EM fermentation product were reported in the previous WBW25 Meeting in 2017. Here, we report our attempt to identify its active principle and the ongoing preliminary results. The activity was assayed by promoted formation of fruiting body of Dictyostelium discoideum. The EM fermentation product was first subjected to liquid-liquid separation, with the activity being recovered in aqueous phase. Concentrated aqueous fraction was further subjected to column chromatography. No activity was detected in any eluant, while almost all activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, these residues revealed spherical particles of μm range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence of magnesium and, to a certain extent, potassium. In a separate experiment, acid treatment and alkali neutralization of EM fermentation product completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticle to be active principle of EM fermentation product, at least, in regard to fruiting body formation in Dictyostelium discoideum. How these particles exert their effect is currently under extensive investigation.