Yukihiro Asami

Protuboxepins A and B and Protubonines A and B from the Marine-Derived Fungus Aspergillus sp. SF-5044

Two new oxepin-containing (1 and 2) and two diketopiperazine-type alkaloids (3 and 4) have been isolated from an EtOAc extract of the marine-derived fungus Aspergillus sp. SF-5044. The structures of these metabolites were determined through analysis of NMR and MS data, along with Marfeys method. Compound 1 showed weak growth inhibitory activity against a small panel of cell lines.

Aflaquinolones A–G: Secondary Metabolites from Marine and Fungicolous Isolates of Aspergillus Spp.

Seven new compounds (aflaquinolones A-G; 1-7) containing dihydroquinolin-2-one and terpenoid units have been isolated from two different fungal sources. Two of these metabolites (1 and 2) were obtained from a Hawaiian fungicolous isolate of Aspergillus sp. (section Flavipedes; MYC-2048 = NRRL 58570), while the others were obtained from a marine Aspergillus isolate (SF-5044) collected in Korea. The structures of these compounds were determined mainly by analysis of NMR and MS data. Relative and absolute configurations were assigned on the basis of NOESY data and (1)H NMR J-values, comparison of calculated and experimental ECD spectra, and analysis of a Moshers ester derivative of 2. Several known compounds, including alantrypinone, aspochalasins I and J, methyl 3,4,5-trimethoxy-2((2-((3-pyridinylcarbonyl)amino)benzoyl)amino)benzoate, and trans-dehydrocurvularin were also encountered in the extract of the Hawaiian isolate.

Verrucarin A sensitizes TRAIL-induced apoptosis via the upregulation of DR5 in an eIF2α/CHOP-dependent manner.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. However, resistance to TRAIL in some cancers remains a current problem in recent. The protein-folding compartment of the endoplasmic reticulum (ER) is particularly sensitive to disturbances, which, if severe, may trigger apoptosis. Therefore, we examined whether verrucarin A (VA) sensitize TRAIL-induced apoptosis in cancer cells by induction of ER stress. We first found that VA induces a major molecule of ER stress, CCAAT/enhancer binding protein homologous protein (CHOP)-dependent DR5 induction and subsequently increases TRAIL-induced cleavage of caspases and PARP in TRAIL-resistant Hep3B cells. Importantly, the transient knockdown using siRNA for CHOP abrogated VA-induced DR5 expression and attenuated TRAIL-induced apoptosis. Treatment with VA also increased the levels of phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which is a common cellular response of ER stress. Furthermore, salubrinal, a specific eIF2α phosphorylation-inducing agent, increased CHOP and DR5 expression in the presence of VA. In contrast, transfection of mutant-eIF2α significantly reversed VA-induced apoptosis with downregulation of CHOP-dependent DR5 expression. Therefore, VA-induced eIF2α phosphorylation seemed to be important for CHOP and DR5 upregulation and TRAIL-induced apoptosis. In addition, generation of reactive oxygen species (ROS) is an effector molecular in sensitization of VA-induced ER stress. We concluded that VA triggers TRAIL-induced apoptosis by eIF2α/CHOP-dependent DR5 induction via ROS generation.

Protuboxepin A, a marine fungal metabolite, inducing metaphase arrest and chromosomal misalignment in tumor cells

Previously we reported the identification of a new oxepin-containing diketopiperazine-type marine fungal metabolite, named protuboxepin A which showed antiproliferative activity in several cancer cell lines. In this study we elucidated the mechanism by which protuboxepin A induces cancer cell growth inhibition. Here we report that protuboxepin A induced round-up morphology, M phase arrest, and an increase in the subG(1) population in tumor cells in a dose dependent manner. Our investigations revealed that protuboxepin A directly binds to α,β-tubulin and stabilizes tubulin polymerization thus disrupting microtubule dynamics. This disruption leads to chromosome misalignment and metaphase arrest which induces apoptosis in cancer. Overall, we identified protuboxepin A as a microtubule-stabilizing agent which has a distinctly different chemical structure from previously reported microtubule inhibitors. These results indicate that protuboxepin A has a potential of being a new and effective anti-cancer drug.

Combined treatment with verrucarin A and tumor necrosis factor-α sensitizes apoptosis by overexpression of nuclear factor-kappaB-mediated Fas.

Verrucarin A (VA) is a member of the family of macrocyclic trichothecenes, which exhibit anti-cancer and immune-modulating activities. However, VA has not yet been demonstrated to be involved in the sensitization of tumor necrosis factor-alpha (TNF-α)-mediated apoptosis. In the present study, we found that VA triggers TNF-α-induced apoptosis in human breast cancer MDA-MB-231 and MCF-7 cells. In particular, activation of caspas-3 and caspase-8 as well as release of cytochrome c were significantly enhanced in response to the combined treatment with VA and TNF-α (VA/TNF-α) and the pan-caspase inhibitor z-VAD-fmk completely reversed the apoptosis, suggesting that caspases are the main effector molecules in VA/TNF-α-induced apoptosis via the intrinsic and extrinsic pathway. Moreover, we confirmed that enhanced Fas expression plays a critical role, because the Fas-blocking antibody partially inhibited VA/TNF-α-induced apoptosis. VA also increased specific DNA-binding activity of nuclear factor-kappaB (NF-κB) via nuclear translocation of p50 and p65. In addition, pretreatment with the NF-κB inhibitor MG132 blocked VA/TNF-α-induced apoptosis by suppression of NF-κB-dependent Fas expression. These results indicated that VA enhances TNF-α-induced apoptosis via NF-κB-dependent Fas overexpression.

Verrucarin A enhances TRAIL-induced apoptosis via NF-κB-mediated Fas overexpression

We investigated whether verrucarin A (VA) sensitizes HepG2 hepatoma cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. We found that VA alone induces little apoptosis, but when combined with TRAIL (VA/TRAIL), it triggered significant apoptosis, causing little or no toxicity in normal mouse splenocytes. VA/TRAIL-induced cell death is involved in the loss of mitochondrial transmembrane potential and the consequent activation of caspases. Because nuclear factor (NF)-κB inhibition has been known as a critical target in TRAIL-mediated apoptosis, we also investigated the role of NF-κB in VA/TRAIL treatment. We found that VA upregulated the DNA binding activity of NF-κB, but that the antioxidants glutathione and N-acetyl-l-cysteine, as well as NF-κB inhibitor MG132, and mutant-IκB (m-IκB) transfection, significantly downregulated VA/TRAIL-induced cell death by inhibiting caspase-3 and NF-κB activities. Transfection of mutant-eIF2α also resulted in a decrease in VA/TRAIL-induced cell death by inhibiting of caspase-3, but not NF-κB activity. Although VA/TRAIL treatment led to an increase of DR5 expression, transfection of m-IκB had no influence on the DR5 expressional level. Finally, we showed that NF-κB-mediated Fas expression is critical to VA/TRAIL-induced apoptosis. Taken together, these results indicate that VA/TRAIL sensitizes HepG2 cells to apoptosis via NF-κB-mediated overexpression of Fas.

Xestospongin C induces monocytic differentiation of HL60 cells through activation of the ERK pathway

Xestospongin C (XC), which is a group of macrocyclic bis-1-oxaquinolizidines, is a potent inhibitor of sarcoendoplasmic reticulum calcium transport ATPase and IP3 receptor. Nevertheless, very less information is available regarding whether XC induces AML differentiation. We investigated the potential role of XC in the differentiation of human leukemia HL60 cells and mechanisms underlying XC actin. XC treatment inhibited proliferation by inducing G1-phase cell cycle arrest in the HL60 cells. In addition, XC induced differentiation of HL60 cells into the CD14(+) monocytic lineage, which was indicated by morphological changes, nitroblue tetrazolium reduction assay, and expressions of CD11b and CD14 surface antigens. Our results also showed that XC promotes phagocytic activity and granularity in HL60 cells, suggesting that the cells are functionally activated. Furthermore, XC enhanced tumor necrosis factor (TNF)-α-mediated cytotoxic effect by increasing the numbers of TNF receptors. Moreover, we showed that XC activates extracellular signal-regulated kinase (ERK) pathway in the differentiation stages. Inhibition of ERK activation using PD98059 significantly decreased NBT+HL60 cells induced by XC treatment. Taken together, the results show that XC promotes monocytic differentiation of HL60 cells via ERK pathway activation, suggesting that XC could be a candidate for use as a differentiation-inducing agent for AML treatment.