論文

ホスファチジルイノシトール　キナーゼの阻害剤をスクリーニングした結果、トヨカマイシンを単離し、更にその間連化合物の阻害活性についても報告した。(4ページ)

ホスファチジルイノシトール代謝回転阻害物質として、イノスタマイシンを発見し、構造解析について報告した。(5ページ)

チロシンキナーゼ阻害剤アーブスタチンの阻害様式を検討し、報告した。(3ページ)

合成されたアーブスタチンの誘導体のチロシンキナーゼ阻害活性と構造活性相関を報告した。(2ページ)

Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade. (C) 2000 Academic Press.

The stimulation of human tumor cells overexpressing epidermal growth factor receptor (EGFR) with EGF enhances tumor development and malignancy. Therefore, compounds that modulate the EGF-mediated signal inducing apoptosis in EGFR-overexpressing cells would represent a new class of antitumor drug and might be useful in the treatment of a subset of human tumors. In the course of screening for compounds that induce apoptosis in EGFR-overexpressing human epidermal carcinoma A431 cells from secondary metabolites of microorganisms, we found that vacuolar-type H+-ATPase (V-ATPase) inhibitors, such as concanamycin B and destruxin E, induced apoptosis only when the cells were stimulated with EGF. The EGF-dependent apoptosis by V-ATPase inhibitors was not observed in other types of human tumor cells which do not overexpress EGFR. The apoptosis in A431 cells was inhibited by anti-FasL antibody which neutralized the cytotoxic effect of FasL, indicating that the Fas/FasL system was involved. The expression of cell surface FasL was upregulated by stimulation with EGF and increased further by V-ATPase inhibitors. Moreover, EGF inhibited cytotoxic Fas antibody-induced apoptosis, whereas V-ATPase inhibitors disrupted the protective effect of EGF on apoptosis in A431 cells. Taken together, these results suggested that V-ATPase inhibitors induced EGF-dependent apoptosis in A431 cells, possibly through both the enhancement of EGF-induced cell surface expression of FasL and the disruption of an EGF-induced survival signal. © 2002 Elsevier Science (USA).

We have isolated an unusual nucleoside, aristeromycin, from the culture filtrate of Actinomycetes as a compound that induces normal morphology in v-ablts-NIH3T3 cells. Aristeromycin also induced erythroid differentiation in abl-expressing human chronic myelogenous leukaemia K562 cells. It did not affect the amount of Abl or the Abl-associated tyrosine kinase activity in either v-ablts-NIH3T3 or K562 cells. As a potent inhibitor of S-adenosylhomocysteine hydrolase, aristeromycin inhibited methylation of phosphatidylethanolamine to form phosphatidylcholine in K562 cells. Among aristeromycin analogues, the activity to inhibit S-adenosylhomocysteine hydrolase was paralleled with the induction of erythroid differentiation. Thus, aristeromycin inhibits abl functions indirectly, possibly by inhibiting biological methylations. © 1995 Academic Press, Inc.

In our previous studies (S. Simizu, et al., 1996, Cancer Res. 56, 4978- 4982), we reported that apoptosis of human small cell lung carcinoma (SCLC) cells induced by protein tyrosine kinase inhibitors, such as erbstatin and herbimycin A, was mediated by H2O2 via a newly synthesized protein(s). In the present study, we demonstrated that induction of apoptosis by erbstatin resulted in activation of caspase-3(like) proteases, which are interleukin- 1β-converting enzyme family proteases (caspases) and that inhibition of these protease activities reduced the extent of cell death and H2O2 generation. We also demonstrated that expression of apoptotic protein Bax was induced by erbstatin. Erbstatin-induced Bax expression was inhibited by the inhibitor of caspase-3(-like) proteases. These results indicate that generation of intracellular H2O2 and Bax expression in tyrosine kinase inhibitor-induced apoptosis were modulated by the activation of caspase-3-(- like) proteases in SCLC cells.

Erbstatin and methyl 2,5-dihydroxycinnamate, related tyrosine kinase inhibitors, induced a morphological change in temperature-sensitive Rous sarcoma virus-transformed rat kidney (RSV15·NRK) that brought the cells close to the morphology of their normal counterpart. Erbstatin did not change the morphology of normal or Kirsten sarcoma virus-transformed rat kidney cells. Erbstatin also inhibited morphological transformation of RSV15·NRK cells induced by a shifting in temperature. Actin stress fibres were observed only in normal cells and not in transformed cells. Erbstatin induced stress fibre organization in transformed cells. Erbstatin and methyl 2.5-dihydroxycinnamate increased fibronectin gene expression in RSV-transformed cells. Thus, tyrosine kinase inhibitors induced normal phenotypes specifically in v-sre-expressing cells. © 1991.

Psi-tectorigenin, an isoflavonoid, was isolated from a culture filtrate of actinomycetes as an inhibitor of epidermal growth factor-induced phosphatidylinositol turnover in cultured A431 cells. It inhibited phosphatidylinositol turnover with an IC50 of about 1 μg/ml; thus, its inhibitory activity was 6-times stronger than that of genistein or orobol. When added to cultured A431 cells psi-tectorigenin inhibited phosphatidylinositol turnover without inhibiting epidermal growth factor receptor tyrosine protein kinase. Thus, psi-tectorigenin is a specific inhibitor of phosphatidylinositol turnover and may be a useful tool for the functional analysis of phosphatidylinositol turnover. © 1988.

Erbstatin, a tyrosine kinase inhibitor, inhibited epidermal growth factor (EGF)-induced inositol phosphate production in cultured A431 cells. However, it did not inhibit ATP-induced inositol phosphate production. Cytosolic but not membrane-associated phospholipase C was iactivated by EGF, and erbstatin inhibited enhancement of the phospholipiase C activity in EGF-treated cells. Thus, tyrosine kinase of A431 cells is suggested to be functionally involved in phospholipase C activation. © 1990 Academic Press, Inc.

We isolated fluvirucin B2from the culture broth of Streptomyces as an inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC). It inhibited PI-PLC of A431 cell cytosol with an IC50 of 1.6μg/ml. Fluvirucin B2also inhibited PI-PLC in cultured A431 cells, whereas it did not inhibit phosphatidylinositol synthesis and macromolecular synthesis markedly. It also inhibited epidermal growth factor-induced rapid rounding of A431 cells, in which PI turnover is involved. © 1995, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION. All rights reserved.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered as a possible therapeutic agent for cancer treatment. This is because of its selective cytotoxicity against various cancer cells without a detrimental effect on normal cells. However, recent studies have reported that the potential application of TRAIL in cancer therapy is limited, as many cancer cells have been found to be resistant to TRAIL. Therefore, small molecule compounds that potentiate the cytotoxicity of TRAIL would be strategic candidates for therapeutic applications in combination with TRAIL. Here we found that a combined treatment of inostamycin and TRAIL synergistically induced caspase-dependent apoptosis in HCT116 cells. Inostamycin upregulated DR5, and a knockdown of DR5 suppressed the apoptosis that was synergistically induced by co-treatment with inostamycin and TRAIL. Moreover, inostamycin increased the expression of DR5 on the cell surface. Therefore, inostamycin-increased cell surface expression of DR5 may have contributed to the enhancement of TRAIL-induced apoptosis. Our study suggests that combined treatment with inostamycin and TRAIL may offer a strategy to overcome TRAIL resistance in tumor cells. © 2012 Japan Antibiotics Research Association All rights reserved.

Inostamycin is an inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase. It significantly reduced epidermal growth factor (EGF)-induced in vitro invasion of the tongue carcinoma cell line, HSC-4, through reconstituted basement membrane Matrigel®. Since phosphatidylinositol (PI) 4,5-biphosphate is important for signal transduction through protein kinase C and actin reorganisation, we further examined the effect of inostamycin on production of two matrix metalloproteinases (MMPs), MMP-2 and -9, and on cell motility. Zymographic analysis showed that inostamycin suppressed pro-MMP-2 and pro-MMP-9 levels at a dose-dependent fashion, while MMP-2 activity was not significantly affected. By reverse transcription-polymerase chain reaction, it was found that inostamycin diminished steady state levels of MMP-2 and -9 but not membrane type 1-MMP mRNA expressions. Inostamycin partially blocked both EGF- and phorbol 12-myristate 13-acetate-stimulated pro-MMP- 9 production. A cytoplasmic calcium chelator (BAPTA-AM) dramatically elevated pro- MMP-9 and slightly elevated pro-MMP-2 secretions. EGF-stimulated motility of HSC-4 cells was suppressed by inostamycin treatment along with reduction of actin cytoskeletal reorganisation, filopodia formation and cdc42 expression. These results suggested that inostamycin would be useful for an anti-invasive agent in tongue cancer.

Predictions of interactions between target proteins and potential leads are of great benefit in the drug discovery process. We present a comprehensively applicable statistical prediction method for interactions between any proteins and chemical compounds, which requires only protein sequence data and chemical structure data and utilizes the statistical learning method of support vector machines. In order to realize reasonable comprehensive predictions which can involve many false positives, we propose two approaches for reduction of false positives: (i) efficient use of multiple statistical prediction models in the framework of two-layer SVM and (ii) reasonable design of the negative data to construct statistical prediction models. In two-layer SVM, outputs produced by the first-layer SVM models, which are constructed with different negative samples and reflect different aspects of classifications, are utilized as inputs to the second-layer SVM. In order to design negative data which produce fewer false positive predictions, we iteratively construct SVM models or classification boundaries from positive and tentative negative samples and select additional negative sample candidates according to pre-determined rules. Moreover, in order to fully utilize the advantages of statistical learning methods, we propose a strategy to effectively feedback experimental results to computational predictions with consideration of biological effects of interest. We show the usefulness of our approach in predicting potential ligands binding to human androgen receptors from more than 19 million chemical compounds and verifying these predictions by in vitro binding. Moreover, we utilize this experimental validation as feedback to enhance subsequent computational predictions, and experimentally validate these predictions again. This efficient procedure of the iteration of the in silico prediction and in vitro or in vivo experimental verifications with the sufficient feedback enabled us to identify novel ligand candidates which were distant from known ligands in the chemical space. © 2009 Nagamine et al.

Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ξ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ξ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ξ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Tyrosine kinase inhibitor, erbstatin, induced morphological apoptosis and DNA fragmentation in human small cell lung carcinoma (SCLC) cells. Erbstatin-induced apoptosis was inhibited by antioxidants, whereas erb- statin-inhibited tyrosine phosphorylation was not affected by them. Erbstatin was shown by means of flow cytometry to induce hydrogen peroxide generation. Furthermore, hydrogen peroxide induced morphological apoptosis and DNA fragmentation in the SCLC cells. We also demonstrated that erbstatin-induced hydrogen peroxide production and DNA fragmentation were partially suppressed by inhibition of protein synthesis. Thus, erbstatin-induced apoptosis would be due to hydrogen peroxide generation via newly synthesized protein.

Leptomycin B (LMB) is a Streptomyces metabolite that inhibits the chromosomal region maintenance (CRM)1-dependent nuclear export of proteins. It also induces apoptosis in several types of cancer cells, by a yet undefined mechanism. We used a functional proteomics approach to delineate the pathways and mediators involved in LMB-induced apoptosis in HeLa cells. Using two-dimensional gel electrophoresis, we searched for proteins accumulated in the nuclei of HeLa cells upon LMB treatment. Among such proteins we found prohibitin and Heat shock protein (Hsp)27, identified by peptide mass fingerprinting and mass spectrometry. Immunocytochemistry was carried out to confirm the LMB-induced nuclear accumulation of these two proteins in HeLa cells. Furthermore, we found that the cytoplasmic expression of Hsp27, but not prohibitin, partially inhibited LMB-induced apoptosis, indicating that nuclear sequestration of Hsp27 was, at least in part, involved in the apoptosis. © Japan Antibiotics Research Association.

Leptomycin B (LMB) is a Streptomyces metabolite that causes the specific inhibition of the nuclear export of proteins containing a nuclear export signal (NES). LMB was reported to inhibit cell cycle progression in fission yeast and mammalian cells, however, the mechanism underlying LMB-induced cell cycle arrest is still obscure. In this study, we found that in serum-starved NIH3T3 cells, LMB inhibited serum-induced cyclin D1 expression at the level of transcription. However, this inhibition was reversed by inhibitors of protein phosphatase 2A (PP2A). Furthermore, we found that PP2A accumulated in the nucleus upon treatment with LMB. The finding prompted us to identify the functional NES in PP2A catalytic subunit α. These results indicated that LMB inhibited the chromosomal region maintenance 1 (CRM1)-dependent nuclear export of PP2A, resulting in sustained dephosphorylation in the nucleus. Although phosphorylation of c-Jun at Ser-63 is required for activator protein 1 (AP-1)-dependent expression of cyclin D1, it decreased in LMB-treated cells compared to untreated cells. Moreover, the inhibitors of PP2A restored the levels of c-Jun phosphorylated at Ser-63. We propose that inhibition of cyclin D1 expression by LMB is mediated by the LMB-induced nuclear accumulation of PP2A, leading to sustained dephosphorylation of c-Jun at Ser-63, which leads to inactivation of the transcription of the AP-1-responsive cyclin D1 gene. © 2007 Nature Publishing Group All rights reserved.