Pyrrocidine A is a known antimicrobial compound produced by endophytic fungi and has a unique 13-membered macrocyclic alkaloid structure with an α,β-unsaturated carbonyl group. We have previously reported that pyrrocidine A shows potent cytotoxicity against human acute promyelocytic leukemia HL60 cells, and the activity is 70-fold higher than that of pyrrocidine B which is the analog lacking the α,β-unsaturated carbonyl group. Pyrrocidine A induced nuclear condensation, DNA fragmentation and caspase activation in HL60 cells. Since the DNA fragmentation was suppressed by pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-fmk), caspase-mediated apoptosis contributes to pyrrocidine A-induced cytotoxicity. JFCR39 human cancer cells panel indicated that the mechanism of action of pyrrocidine A is different from other clinical anticancer drugs, and this compound broadly inhibited the growth of various cancer cell lines. The apoptosis induction by pyrrocidine A was suppressed by both N-acetyl-l-cysteine (NAC) and glutathione, both of which are thiol-containing antioxidants. Furthermore, pyrrocidine A directly bound to N-acetyl-l-cysteine methyl ester (NACM) through the Michael-type addition at the α,β-unsaturated carbonyl group and was detected by HPLC and liquid chromatography-ESI-tandem MS (LC-ESI-MS/MS) analyses. This indicates that this moiety is crucial for the potent apoptosis-inducing activity of pyrrocidine A.

A new biological activity of falcarindiol isolated from Japanese parsley (Oenanthe javanica) using the mutant yeast YNS17 strain (zds1Δ erg3Δ pdr1Δ pdr3Δ) was discovered as an inhibitor of glycogen synthase kinase-3β (GSK-3β). Falcarindiol inhibited GSK-3β in an ATP noncompetitive manner with a Ki value of 86.9 μM using a human enzyme and luminescent kinase assay platform. Falcarindiol also both suppressed gene expression of glucose-6-phosphatase (G6Pase) in rat hepatoma H4IIE cells and protected mouse neuroblastoma HT22 cells from glutamate-induced oxidative cell death at 10 μM. During an oral glucose tolerance test (OGTT), the blood glucose level was significantly decreased in the rats treated with oral administration of O. javanica extract containing falcarindiol (15 mg/kg). These findings indicate that Japanese parsley could be a useful food ingredient against type-2 diabetes and Alzheimer’s disease.

A bisabolane sesquiterpene endoperoxide compound, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from edible wild plants grown in the northern area of Japan, Cacalia delphiniifolia and Cacalia hastata, using a mutant yeast (cdc2-1 rad9Δ). It showed cytotoxicity at IC50 = 3.4 μM and induced apoptosis against the human promyelocytic leukemia cell line HL60 through a new stable rearrangement product (1) when in the presence of FeSO4. This conversion mechanism is different from another sesquiterpene endoperoxide lactone compound, dihydroartemisinin (DHA), which is an anti-malarial drug. The cytotoxicity of EDBD decreased in the presence of the ferrous ion chelating drug deferoxamine mesylate (DFOM), and this suggested that the structural change of the drug caused by Fe2+ may be responsible for its biological activities. EDBD induced apoptosis via phosphorylation of p38 mitogen-activated protein kinase (MAPK) in HL60 cells, and was detected by Western blot. EDBD resulted in an immediate increase in DCF fluorescence intensity in HL60 cells using DCFH-DA (2′,7′-dichlorofluorescin diacetate) assay. The in vitro reaction of EDBD with FeSO4 also increased DCF fluorescence intensity in a dose dependent manner. These results showed that the biological activity of EDBD involves an unstable carbon-centered radical intermediate. Furthermore, there was no similarity between the JFCR39 fingerprints of EDBD and DHA (correlation coefficient on COMPARE Analysis γ = 0.158). EDBD showed anti-tumor effects against a xenograft of Lox-IMVI cells in vivo.

In the course of our screening program for a new Ca2+-signal transduction inhibitor using the hypersensitive mutant strain of Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ), new eremophilane sesquiterpenoid compounds eremoxylarins A and B were found to restore the growth inhibition caused by the hyperactivated Ca2+-signal. These compounds showed lethal activity against the mpk1Δ strain, specifically, compared to the cnb1D strain, and ion-sensitive activity against the wild-type strain in the presence of LiCl, indicating that their molecular target might be the calcineurin pathway. They inhibited calcineurin directly without immunophilins at IC50 = 2.7 and 1.4μM with competitive inhibition in vitro. The eremophilane sesquiterpenoid structure in eremoxylarins could be a good leading compound for immunosuppressants and anti-allergy drugs.

A novel inhibitor of prolyl endopeptidase, the propeptin analog propeptin-2 (C105H130N24O24), missing two amino acid residues from the propeptin C-terminus was isolated from the fermentation broth of propeptin-producing Microbispora sp. SNA-115 grown using a large inoculum. It shows the same enzyme inhibition activity as propeptin against prolyl endopeptidase (Ki=1.5 M), but its antimicrobial activity against Mycobacterium phlei is more than 100-fold lower.

A new norlabdane compound, named kujigamberol has previously been isolated from Kuji amber (but not from Baltic amber) by activity guided fractionation. However, there has been no study of biological compounds in Dominican amber. Biological activities were examined using the hypersensitive mutant yeast (zds1Δ erg3Δ pdr1Δ pdr3Δ) with respect to Ca2 +-signal transduction, enzymes and rat basophilic leukemia (RBL)-2H3 cells. The structures were elucidated on the basis of spectral analysis including high resolution (HR)-EI-MS, 1D NMR and 2D NMR. Three diterpenoid compounds, 5(10)-halimen-15-oic acid (1), 3-cleroden-15-oic acid (2) and 8-labden-15-oic acid (3), which are different from the bioactive compounds in Kuji and Baltic ambers, were isolated from Dominican amber. They inhibited both calcineurin (CN) (IC50 = 40.0, 21.2 and 34.2 μM) and glycogen synthase kinase-3β (GSK-3β) (IC50 = 48.9, 43.8 and 41.1 μM) which are involved in the growth restored activity against the mutant yeast. The most abundant compound 2 showed inhibitory activity against both degranulation and Ca2 +-influx in RBL-2H3 cells. The compounds having the growth restoring activity against the mutant yeast have potential as anti-allergic compounds.“Yeast Ca2 +-signal transduction inhibitors isolated from Dominican amber prevent the degranulation of RBL-2H3 cells through the inhibition of Ca2 +-influx”, T. Abe et al.Download high-res image (68KB)Download full-size image

Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates and regulates apoptosis, stress response and growth-related pathways. In the course of screening for PP2C activators from natural sources, we isolated abietane-type diterpenes, pisiferdiol and pisiferic acid from Chamaecyparis pisifera. Pisiferdiol having a unique seven-membered ring showed more specific PP2C activation activity (1.3-fold at 100 μM) than pisiferic acid having a normal six-membered ring and oleic acid, which is known to activate PP2C. Pisiferdiol and pisiferic acid showed mixed-type activation with respect to α-casein, and this differed from the non-competitive activation of oleic acid in vitro. In vivo, the cytotoxicity of pisiferdiol toward human promyelocytic leukemia cell line HL60 with an IC50 value of 18.3 μM was 2-fold and 7-fold stronger than those of pisiferic acid and oleic acid, and pisiferdiol induced apoptosis through a caspase 3/7-dependent mechanism involving the dephosphorylation of Bad1, which is a PP2C substrate. We thus conclude that pisiferdiol and pisiferic acid are novel PP2C activators, and the more specific activator, pisiferdiol, may be a useful chemical probe to study PP2C-mediated signaling pathways, and a lead compound for pharmaceutical agents.