Hiroyuki Kumagai

Thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces paxillin in HUVECs and suppresses tumor cell-induced angiogenesis

Recent studies have shown that integrin αvβ3, a receptor for vitronectin, plays an important role in tumor‐induced angiogenesis and tumor growth and that antagonists of αvβ3 inhibit angiogenic processes including endothelial cell adhesion and migration. On the other hand, most inhibitors of integrin αvβ3 are peptide antagonists that include the Arg‐Gly‐Asp (RGD) motif. We therefore reasoned that non‐peptide inhibitors of endothelial cell adhesion to vitronectin might be useful for inhibition of tumor angiogenesis in vivo. We screened for low‐molecular‐weight natural products able to inhibit adhesion of human umbilical vein endothelial cells (HUVECs) to vitronectin, and pyrrothine group compounds including aureothricin, thioaurin and thiolutin were isolated from microbial culture broths. Of these compounds, thiolutin inhibited adhesion of HUVECs to vitronectin the most effectively (IC50, 0.83 μM). In vivo experiments showed that thiolutin significantly suppressed angiogenesis induced by tumor cells (S‐180), a pathological form of neovascularization, in a mouse dorsal air sac assay system. To explore the mechanism of inhibition of HUVEC adhesion to vitronectin by thiolutin, we examined the effect of this agent on intracellular cell adhesion signaling. We found that the amount of paxillin in HUVECs was significantly reduced by thiolutin treatment, while those of other focal adhesion proteins including vinculin and focal adhesion kinase (FAK) were not. Metabolic labeling experiments showed that thiolutin enhanced degradation of paxillin in HUVECs. Protease inhibitors (MG115 and E64‐D) decreased the rate of degradation of the paxillin induced by thiolutin and partially restored thiolutin‐induced inhibition of HUVEC adhesion to vitronectin. Based on these findings, we concluded that thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces the paxillin level in HUVECs and suppresses tumor cell‐induced angiogenesis in vivo.

Ophiobolin A, a Novel Apoptosis-inducing Agent from Fungus Strain f-7438

We isolated ophiobolin A from the f-7438 fungus strain. In the L1210 cell line, ophiobolin A (0-01-1 μM) showed cytotoxicity in a concentration-dependent manner. Morphological observations revealed that ophiobolin A (1 μM) induced shrinkage in cell soma and chromatin condensation, suggesting apoptotic cell death. Moreover, in DNA gel electrophoretic experiments, a typical apoptotic DNA ladder pattern was observed after treatment with this compound. The flow cytometric experiment indicated that ophiobolin A (0.01-1 μM) caused a concentration-dependent apoptosis in close agreement with concentrations that induced cytotoxicity in L1210 cells. The results suggested that ophiobolin A caused the death of L1210 cells through the apoptotic process. Ophiobolin A may become a powerful pharmacological tool for studying the apoptotic mechanism.

4-hydroxy-17-methylincisterol, an inhibitor of DNA polymerase-α activity and the growth of human cancer cells in vitro

An ergosterol derivative, 4-hydroxy-17-methylincisterol (HMI), was found to be an inhibitor of mammalian DNA polymerases in vitro. HMI inhibited the activity of calf thymus DNA polymerase alpha (pol. alpha). Among the polymerases tested, pol. alpha was the most sensitive to inhibition by HMI, and the inhibition was concentration dependent. The inhibitory effect of HMI on pol. alpha was almost the same as that shown by aphidicolin, a well-known potent pol. alpha inhibitor. HMI had relatively less effect on rat DNA pol. beta, human immunodeficiency virus type 1 reverse transcriptase (HIV-RT), and calf thymus terminal deoxynucleotidyl transferase (TdT) in vitro, and did not influence the activities of prokaryotic DNA polymerases such as Klenow Fragment of DNA polymerase I, or the DNA-metabolic enzyme DNase I. HMI was found to be able to prevent the growth of human cancer cell lines originating from patients with leukemia or various solid tumors; its IC50 values ranged from 7.5 to 12 microM. We also synthesized other ergosterol derivatives and tested them, and found that two compounds, 17-methylincisterol and 4-acetyl-17-methylincisterol, have similar inhibitory effects.

Ceramidastin, a novel bacterial ceramidase inhibitor, produced by Penicillium sp. Mer-f17067

Decrease of ceramide in the skin is one of the aggravating factors of atopic dermatitis. The skin is often infected by ceramidase-producing bacteria, such as Pseudomonas aeruginosa. The bacterial ceramidase then degrades ceramide in the skin. To develop anti-atopic dermatitis drugs, we searched for ceramidase inhibitors, which led to the discovery of ceramidastin, a novel inhibitor of bacterial ceramidase, from the culture broth of Penicillium sp. Mer-f17067. Ceramidastin inhibited the bacterial ceramidase with an IC50 value of 6.25 μg ml−1. Here we describe the isolation, physicochemical properties, structure determination and biological activity of ceramidastin.

New potent immunosuppressive eremophilane isolated from Trichothecium roseum.

Abstract -A new sesquiterpene, cuspidatol (1), was isolated from Trichothecium roseum and its structure was elucidated by spectral analyses. This eremophilane type compound displays potent immunosuppressive activity, IC50 11μ/ml in MLR assay.

Generation of new benanomicin analogues by biotransformation using Escherichia coli expressing actinomycete cytochrome P450.

Benanomicins were found as antifungal antibiotics from the culture of an actinomycete with potent antifungal activities in vitro and in vivo. We aimed to generate derivatives superior to benanomicin A by biotransformation using Escherichia coli constructed with bacterial P450 expression system. We found transformation of benanomicin A into two derivatives, 10-hydroxybenanomicin A and 11-O-demethylbenanomicin A by one of the P450-expressed strains which harbored a plasmid carrying a CYP105C1-homologous gene. Unexpectedly, the biotransformed compounds showed weak antifungal activities in vitro compared with those of benanomicin A.

Effect of Cytogenin, a Novel Immunomodulator, on Streptozotocin-induced Diabetes in Mice

The anti-diabetic effect of cytogenin was examined using streptozotocin-induced diabetes in mice. Cytogenin suppressed not only the increase of plasma glucose level but also the body weight reduction in diabetic mice. Histological examination of the pancreas taken from diabetic mice given cytogenin showed that cytogenin decreased the number of macrophages infiltrated into islet of pancreas. Further, cytogenin suppressed the nitric oxide generation by macrophages treated with lipopolysaccharide through decreasing of inducible nitric oxide synthase expression. Cytogenin suppressed interleukin-6 expression by macrophage treated with LPS, suggesting that the anti-diabetic activity of cytogenin might be partly attributed to the suppressive activity against nitric oxide generation.

Suppression of type II collagen-induced arthritis by ICM0301B, a new angiogenesis inhibitor

Suppression of type II collagen-induced arthritis by ICM0301B, a new angiogenesis inhibitor

New Naphthoquinones, f13102A and B, from a Fungus Strain F-13102

Deficiency of Fas-mediated apoptosis is one of the mechanisms involved in the immune evasion by tumors. Thus, it might be a practical approach for cancer treatment that Fas-mediated apoptosis in tumor cells is modified by drugs. In the course of screening, we have isolated two new naphthoquinones, f13102A and B, from the culture broth of fungus strain F-13102. Coumpound f13102A sensitizes Fas-resistant human lung adenocarcinoma A549 cells to apoptosis.