3,6-Epidioxy-1,10-bisaboladiene and sulfasalazine synergistically induce ferroptosis-like cell death in human breast cancer cell lines.

3,6-Epidioxy-1,10-bisaboladiene (EDBD) is an endoperoxide compound isolated from edible wild plants that induces iron-dependent ferroptosis-like cell death in HL-60 cells by decreasing the expression of GPX4 and glutathione. In contrast, sulfasalazine (SSZ), a clinically used anti-inflammatory drug, induces ferroptosis through the system xc-. In this study, we investigated the synergistic effects of these 2 compounds on 3 human breast cancer cell lines (HBC-5, MCF-7, and MDA-MB-231). EDBD-induced cell death was relieved by the lipid peroxidation inhibitor ferrostatin-1 and the iron chelator deferoxamine mesylate (DFOM), indicating that EDBD induced ferroptosis-like cell death. Moreover, cotreatment with EDBD and SSZ synergistically induced cell death in all 3 cell lines. Because the cytotoxicity of the cotreatment was inhibited by DFOM and ferrostatin-1, the combination of EDBD and SSZ synergistically induced ferroptosis. Collectively, EDBD enhanced the effects of SSZ as a clinical anti-inflammatory and anticancer drug candidate.

Cytotoxicity and induction of G1 phase cell cycle arrest in human acute promyelocytic leukemia HL60 cells by indole alkaloids isolated from Dialium corbisieri seeds.

The phytochemical investigation of Dialium corbisieri seeds led to the isolation of five monoterpenoid indole alkaloids along with a phytoserotonin, 1-6 and among the known compounds, the spectroscopic data of (5S)-methoxy-akuammiline (1) was reported for the first time. The structures were elucidated based on nuclear magnetic resonance spectroscopic techniques such as ultraviolet, infrared, high-resolution electrospray ionization time-of-flight mass spectrometry, and electron-capture dissociation spectrum calculations. The isolated compounds were evaluated for their cytotoxicity and cell progression in the human acute promyelocytic leukemia HL60 cell line.

Synthesis and structure-activity relationship of kujigamberol B, a dinorlabdane diterpenoid isolated from an ancient Kuji amber.

The versatile methodology was developed for synthesizing kujigamberol B, a dinorlabdane diterpenoid isolated from the methanol extract of Kuji amber. A highly efficient intramolecular cyclization is followed by a Sonogashira-coupling reaction during the total synthesis. The synthesized compounds were evaluated for the growth-restoring activity against the mutant yeast (zds1Δ erg3Δ pdr1Δ pdr3Δ) and for the degranulation of RBL-2H3 cells. We found that in both activities, primary alcohol and secondary alcohol analogs are as active as kujigamberol B.

Golden berry leaf extract containing withanolides suppresses TNF-α and IL-17 induced IL-6 expression in HeLa Cells.

Inflammation, characterized by the overexpression of IL-6 in various tissues, has been reported as a symptom of coronavirus disease 2019. In this study, we established an experimental system for overexpression of IL-6 in HeLa cells stimulated by TNF-α and IL-17, along with identification of anti-inflammatory materials and components from local agricultural, forestry, and fishery resources. We constructed a library of extracts from natural sources, of which 111 samples were evaluated for their anti-inflammatory activities. The MeOH extract of Golden Berry (Physalis peruviana L) leaf was found to exhibit strong anti-inflammatory properties (IC50 = 4.97 µg/mL). Preparative chromatography identified two active constituents, 4ß-hydroxywithanolide E (4ß-HWE) (IC50 = 183 nM) and withanolide E (WE) (IC50 = 65.1 nM). Withanolides are known anti-inflammatory ingredients of Withania somnifera, an Ayurvedic herbal medicine. P. peruviana leaves containing 4ß-HWE and WE should be considered as useful natural resources for anti-inflammatory products.

Anti-melanogenic effect of furanoeremophilanes identified from edible wild plants belonging to the genus Cacalia.

Cacalia delphiniifolia and Cacalia hastata are edible wild plants in Japan. We found that these plants have anti-melanogenic activity in B16F10 mouse melanoma cells. Three furanoeremophilanes, cacalol (from C. delphiniifolia), dehydrocacalohastin, and cacalohastin (from C. hastata), were identified as the main active components. The genus Cacalia may be a good source of beneficial materials with anti-melanogenic effects.

KIFC1 regulates ZWINT to promote tumor progression and spheroid formation in colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. Kinesin Family Member C1 (KIFC1) has been proposed as a promising therapeutic target due to its pivotal role in centrosome clustering to mediate cancer cell progression. This study aimed to analyze the expression and biological function of KIFC1 in CRC. Immunohistochemically, 67 (52%) of 129 CRC cases were positive for KIFC1 and statistically associated with poorer overall survival. KIFC1 small interfering RNA (siRNA)-transfected cells demonstrated lower cell proliferation as compared to the negative control cells. A specific KIFC1 inhibitor, kolavenic acid analog (KAA) drastically inhibited CRC cell proliferation. Microarray analysis revealed that KAA-treated CRC cells presented reduced ZW10 interacting kinetochore protein (ZWINT) expression as compared to control cells. Immunohistochemical analysis demonstrated that 61 (47%) of 129 CRC cases were positive for ZWINT and ZWINT expression was significantly correlated with KIFC1 expression. ZWINT-positive cases exhibited significantly worse overall survival. KIFC1 siRNA-transfected cells showed reduced ZWINT expression while ZWINT siRNA-transfected cells decreased cell proliferation. Both KIFC1 and ZWINT knockdown cells attenuated spheroid formation ability. This study provides new insights into KIFC1 regulating ZWINT in CRC progression and its potential as a therapeutic target.

Omphaloprenol A: a new bioactive polyisoprenepolyol isolated from the mycelium of poisonous mushroom Omphalotus japonicus.

Mushrooms of the Omphalotus genus are known to be rich in secondary metabolites. In the quest for new bioactive compounds, we analyzed the compounds isolated from the mycelium of the poisonous mushroom Omphalotus japonicus. As a result, a new polyisoprenepolyol, which was named omphaloprenol A, was identified, along with known substances such as hypsiziprenol A10 and A11, illudin S, and ergosterol. The chemical structure of omphaloprenol A was elucidated by nuclear magnetic resonance and infrared spectroscopies and mass spectrometry, and its bioactivity was investigated. Omphaloprenol A showed growth promoting activity against the root of lettuce seeds and cytotoxicity against HL60 cells. To the best of our knowledge, this is the first report on the isolation of a polyisoprenepolyol compound from Omphalotaceae mushrooms.

A yeast-based screening system identified bakkenolide B contained in Petasites japonicus as an inhibitor of interleukin-2 production in a human T cell line.

Ca2+ signaling is related to various diseases such as allergies, diabetes, and cancer. We explored Ca2+ signaling inhibitors in natural resources using a yeast-based screening method and found bakkenolide B from the flower buds of edible wild plant, Petasites japonicus, using the YNS17 strain (zds1Δ erg3Δ pdr1/3Δ). Bakkenolide B exhibited growth-restoring activity against the YNS17 strain and induced Li+ sensitivity of wild-type yeast cells, suggesting that it inhibits the calcineurin pathway. Additionally, bakkenolide B inhibited interleukin-2 production at gene and protein levels in Jurkat cells, a human T cell line, but not the in vitro phosphatase activity of human recombinant calcineurin, an upstream regulator of interleukin-2 production. Furthermore, bakkenolide A showed weak activity in YNS17 and Jurkat cells compared with bakkenolide B. These findings revealed new biological effects and the structure-activity relationships of bakkenolides contained in P. japonicus as inhibitors of interleukin-2 production in human T cells.

Kolavenic acid analog restores growth in HSET-overproducing fission yeast cells and multipolar mitosis in MDA-MB-231 human cells.

Although cancer cells often harbor supernumerary centrosomes, they form pseudo-bipolar spindles via centrosome clustering, instead of lethal multipolar spindles, and thus avoid cell death. Kinesin-14 HSET/KIFC1 is a crucial protein involved in centrosome clustering. Accordingly, a compound that targets HSET could potentially inhibit cancer cell proliferation in a targeted manner. Here, we report three natural compounds derived from Solidago altissima that restored the growth of fission yeast cells exhibiting lethal HSET overproduction (positive screening), namely solidagonic acid (SA) (1), kolavenic acid analog (KAA: a stereo isomer at C-9 and C-10 of 6ß-tigloyloxykolavenic acid) (2), and kolavenic acid (KA) (3). All three compounds suppressed fission yeast cell death and enabled reversion of the mitotic spindles from a monopolar to bipolar morphology. Compound 2, which exerted the strongest activity against HSET-overproducing yeast cells, also inhibited centrosome clustering in MDA-MB-231 human breast adenocarcinoma cells, which contained large numbers of supernumerary centrosomes. These natural compounds may be useful as bioprobes in studies of HSET function. Moreover, compound 2 is a prime contender in the development of novel agents for cancer treatment.

Inhibition of melanin production and promotion of collagen production by the extract of Kuji amber.

Kuji amber is fossilized tree resin of the Late Cretaceous in Japan. In this study, new biological activities of ethanol extract of Kuji amber (EtOH ext.) and supercritical carbon dioxide fluid extract of Kuji amber (scCO2 ext.) were examined. Both EtOH ext. and scCO2 ext. inhibited melanin production in B16 mouse melanoma cells and promoted collagen production in human skin fibroblast SF-TY cells. The scCO2 ext. had more potent activity than that of EtOH ext. and may depend on the efficiency of the extraction. The main new biologically active compound in Kuji amber, kujigamberol had no activities against melanin production, however, it promoted collagen production at low concentrations. A biologically active compound having a different structure, spirolactone norditerpenoid, showed both the inhibition activity against melanin production and the promotion activity of collagen production in a dose dependent manner. EtOH ext. and scCO2 ext., which include both kujigamberol and spirolactone norditerpenoid, have not only anti-allergy activity, but also inhibit melanin production and promote collagen production.

Constituents of the Fruiting Body of Poisonous Mushroom Omphalotus japonicus.

Six new sesquiterpenes, tsukiyols A-C, neoilludin C, and 4-O-methylneoilludins A and B, were isolated from the fruiting body of Omphalotus japonicus (Kawam.) Kirchm. & O. K. Mill. Additionally, six known compounds, illudin S, neoilludins A-B, 5-hydroxydichomitol, ergosterolperoxide, and 3ß,5α,9α-trihydroxyergosta-7,22-diene-6-one, were also obtained. Their chemical structures were determined with MS, IR, and NMR spectra and the absolute configurations of neoilludins A-C, 4-O-methylneoilludins A, and B were determined with electronic circular dichroism (ECD). Illudin S and 3ß,5α,9α-trihydroxyergosta-7,22-diene-6-one showed cytotoxicity against human acute promyelocytic leukemia HL60 cells. Illudin S, 4-O-methylneoilludin A, B, and tsukiyol C showed growth-restoring activity against mutant yeast via Ca2+-signal transduction.

A novel sex difference in Drosophila contact chemosensory neurons unveiled using single cell labeling.

Among the sensory modalities involved in controlling mating behavior in Drosophila melanogaster, contact sex pheromones play a primary role. The key receptor neurons for contact sex pheromones are located on the forelegs, which are activated in males upon touching the female abdomen during tapping events in courtship actions. A fruitless (fru)-positive (fru [+]) male-pheromone sensing cell (M-cell) and a fru [+] female-pheromone sensing cell (F-cell) are paired in a sensory bristle on the legs, and some fru [+] chemoreceptor axons project across the midline in the thoracic neuromere in males but not in females. However, the receptor cells that form sexually dimorphic axon terminals in the thoracic ganglia remain unknown. By generating labeled single-cell clones, we show that only a specific subset of fru [+] chemosensory neurons have axons that cross the midline in males. We further demonstrate that there exist two male-specific bristles, each harboring two chemosensory neurons; neither of which exhibits midline crossing, a masculine characteristic. This study reveals hitherto unrecognized sex differences in chemosensory neurons, imposing us to reinvestigate the pheromone input pathways that impinge on the central courtship circuit.

Identification of neomacrophorins isolated from Trichoderma sp. 1212-03 as proteasome inhibitors.

Neomacrophorins I-III (1-3) and X have previously been isolated from Trichoderma sp. 1212-03. Their mode of action against cancer cells and the mechanism of biosynthesis of the characteristic [4.4.3] propellane framework in neomacrophorin X have not been reported. The isolation and characterization of neomacrophorins IV (4), V (5), and VI (6) is reported. Epoxyquinones 1, 4, and 6 potently induced apoptotic cell death in human acute promyelocytic leukemia HL60 cells, while epoxysemiquinols 2, 3, and 5 showed weak activity. This indicates that the epoxyquinone moiety is crucial for apoptosis-inducing activities of neomacrophorins. We also found that neomacrophorins inhibit proteasome in vitro, and 1, 4, and 6 induced significant accumulation of ubiquitinated proteins in HL60 cells. These activities were completely suppressed by a nucleophile, N-acetyl-l-cysteine (NAC). The analysis of reaction mechanisms using LC-MS suggested that C2' and C7' of neomacrophorins could be Michael acceptors in the reaction with NAC methyl ester (NACM). These findings indicated that the electrophilic properties of neomacrophorins are responsible for both their potent biological effects and the biosynthesis of unique [4.4.3] propellane framework in neomacrophorin X.

A novel Ca2+-signal transduction inhibitor, kujigamberol C, isolated from Kuji amber.

A novel labdane type diterpenoid, 15-nor-8-labden-13-ol, named kujigamberol C, was isolated from Kuji amber using a modified isolation method to increase the yield of biologically active compounds. The structure was determined using HREIMS, 1D and 2D NMR. Kujigamberol C showed growth-restoring activity against mutant yeast via Ca2+-signal transduction.

Kujigamberoic acid A, a carboxylic acid derivative of kujigamberol, has potent inhibitory activity against the degranulation of RBL-2H3 cells.

The aldehyde and carboxylic acid derivatives of kujigamberol were synthesized using pyridinium dichromate (PDC). The carboxylic acid derivative exhibited lower cytotoxicity and inhibited the degranulation of rat basophilic leukemia-2H3 (RBL-2H3) cells stimulated by thapsigargin more than kujigamberol. The carboxylic acid derivative was detected and isolated from the methanol extract of Kuji amber (MEKA) by the modified isolation procedure. Thus, it has been named as kujigamberoic acid A.

Identification and Total Synthesis of Two Previously Unreported Odd-Chain Bis-Methylene-Interrupted Fatty Acids with a Terminal Olefin that Activate Protein Phosphatase, Mg2+/Mn2+-Dependent 1A (PPM1A) in Ovaries of the Limpet Cellana toreuma.

Diverse non-methylene-interrupted (NMI) fatty acids (FAs) with odd-chain lengths have been recognized in triacylglycerols and polar lipids from the ovaries of the limpet Cellana toreuma, however their biological properties remain unclear. In this study, two previously unreported odd-chain NMI FAs, (12Z)-12,16-heptadecadienoic (1) and (14Z)-14,18-nonadecadienoic (2) acids, from the ovary lipids of C. toreuma were identified by a combination of equivalent chain length (ECL) values of their methyl esters and capillary gas chromatography-mass spectrometry (GC-MS) of their 3-pyridylcarbinol derivatives. On the basis of the experimental results, both 1 and 2 were synthesized to prove their structural assignments and to test their biological activity. The ECL values and electron impact-mass (EI-MS) spectra of naturally occurring 1 and 2 were in agreement with those of the synthesized 1 and 2. In an in vitro assay, both 1 and 2 activated protein phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) up to 100 µM in a dose-dependent manner.

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors.

Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.

Synthesis of polyozellin, a prolyl oligopeptidase inhibitor, and its structural revision.

Polyozellin is a p-terphenyl compound which was isolated from Polyozellus multiplex, and exhibits an inhibitory activity against prolyl oligopeptidase (POP). Its structure was assigned as 1 having a p-terphenyl skeleton including a p-substituted dibenzofuran moiety by spectroscopic analyses and chemical means. This paper describes the total syntheses of the proposed structure 1 for polyozellin and its o-isomer 2, revising the structure of polyozellin to the latter. These syntheses involved a double Suzuki-Miyaura coupling using chlorophenylboronic acid as a common key building block, and Cu mediated Ullmann cyclization as key steps. The inhibitory activities of synthetic compounds against POP and cancer cells were also evaluated.

Ca2+-Signal Transduction Inhibitors, Kujiol A and Kujigamberol B, Isolated from Kuji Amber Using a Mutant Yeast.

A podocarpatriene and a labdatriene derivative, named kujiol A [13-methyl-8,11,13-podocarpatrien-19-ol (1)] and kujigamberol B [15,20-dinor-5,7,9-labdatrien-13-ol (2)], respectively, were isolated from Kuji amber through detection with the aid of their growth-restoring activity against a mutant yeast strain ( zds1Δ erg3Δ pdr1Δ pdr3Δ), which is known to be hypersensitive with respect to Ca2+-signal transduction. The structures were elucidated by spectroscopic data analysis. Compounds 1 and 2 are rare organic compounds from Late Cretaceous amber, and the mutant yeast used seems useful for elucidating a variety of new compounds from Kuji amber specimens, produced before the K-Pg boundary.

Cyclohelminthol X, a Hexa-Substituted Spirocyclopropane from Helminthosporium velutinum yone96: Structural Elucidation, Electronic Circular Dichroism Analysis, and Biological Properties.

Helminthosporium velutinum yone96 produces cyclohelminthol X (1), a unique hexa-substituted spirocyclopropane. Although its molecular formula and NMR spectral data resemble those of AD0157, being isolated from marine fungus Paraconiothyrium sp. HL-78-gCHSP3-B005, our detailed analyses disclosed a totally different structure. Chemical shift calculations and electronic circular dichroism spectral calculations were quite helpful to establish the structure, when those were performed based on density functional theory. The carbon framework of cyclohelminthols I-IV is found at the C1-C8 propenylcyclopentene substructure of 1. Thus, 1 is assumed to be biosynthesized by cyclopropanation between an oxidized form of cyclohelminthol IV and a succinic anhydride derivative 4. Cytotoxicity for two cancer cell lines and proteasome inhibition efficiency are measured.