Kimihiro Matsunaga

Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells

In nerve growth factor-treated PC12 cells, 12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-3,6,8,11(2H,12bH)-tetrone (halenaquinone) caused cytotoxicity in a concentration-dependent manner (EC(50) value; 10 microM). Gel electrophoretic DNA analysis of PC12 cells treated with halenaquinone (10 microM) and 11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-[1S-(1 alpha,6b alpha,9a beta,11 alpha,11b beta)]-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione (wortmannin) (3 microM) showed a typical apoptotic DNA ladder. In the flow cytometric analysis, halenaquinone caused apoptosis in a concentration- and time-dependent manner (EC(50) value; 10 microM), whereas 2,3-dihydro-12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-6,8,11(12bH)-trione (xestoquinone) with the methylene group at the C-3 position failed to cause apoptosis, suggesting that the carbonyl group at the C-3 position in halenaquinone is important for exerting apoptotic effects in PC12 cells. Phosphatidylinositol 3-kinase was inhibited by halenaquinone (IC(50) value; 3 microM) as well as wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Halenaquinone inhibited phosphatidylinositol 3-kinase activity at lower concentrations than those at which it induced apoptosis in PC12 cells. These results suggest that halenaquinone causes the death of PC12 cells through an apoptotic process and that the mechanism of halenaquinone-induced apoptosis may be partially explained by the inhibition of phosphatidylinositol 3-kinase activity.

Goniodomin A, an antifungal polyether macrolide, exhibits antiangiogenic activities via inhibition of actin reorganization in endothelial cells

Goniodomin A (GDA) is an antifungal polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax. Previous studies revealed that GDA profoundly affected cytoskeletal reorganization. We examined the effect of GDA on the angiogenic properties of vascular endothelial cells. GDA itself did not affect proliferation of, migration of, and tube formation in type I collagen gels by, bovine aortic endothelial cells (BAECs). Proliferation of BAECs stimulated by bFGF was not affected by GDA at concentrations of up to 10 nM. However, at similar concentrations, GDA significantly inhibited bFGF‐induced migration and tube formation in type I collagen gels by BAECs. Actin reorganization is required for cell migration. GDA caused the perinuclear aggregation of filamentous actin and inhibited stress fiber formation in bFGF‐ or VEGF‐stimulated BAECs and lysophosphatidic acid‐stimulated HeLa cells. However, GDA did not affect stress fiber structures already formed through Gβγ expression or in constitutively active RhoA mutant HeLa cells. Finally, GDA inhibited forming of vasucular system in a chorioallantoic membrane. Our results indicated that GDA suppressed angiogenic properties of ECs at least in part through the inhibition of actin reorganization and inhibited angiogenesis in vivo. J. Cell. Physiol. 190: 109–116, 2002.

Picrosides I and II, selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.

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.

Nardosinone, the first enhancer of neurite outgrowth-promoting activity of staurosporine and dibutyryl cyclic AMP in PC12D cells.

Nardosinone was isolated as an enhancer of nerve growth factor (NGF) from Nardostachys chinensis [Neurosci. Lett. 273 (1999) 53]. Nardosinone (0.1-100 microM) enhanced dibutyryl cyclic AMP (dbcAMP, 0.3 mM)- and staurosporine (10 nM)-induced neurite outgrowth from PC12D cells in a concentration-dependent manner. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, partially blocked enhancements of dbcAMP (0.3 mM)- or staurosporine (10 nM)-induced neurite outgrowth by nardosinone. Nardosinone alone had no effect on the phosphorylation of MAP kinase. The dbcAMP-induced increase in phosphorylation of MAP kinase was not affected by nardosinone. Staurosporine almost unaffected the phosphorylation of MAP kinase, and nardosinone potentiated the staurosporine-induced neurite outgrowth without stimulation of the phosphorylation of MAP kinase. Since it is known that MAP kinase signaling is required for neurite outgrowth in PC12D cells, these results suggest that nardosinone enhances staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying both the MAP kinase-dependent and -independent signaling pathways of dbcAMP and staurosporine. It is also suggested that nardosinone enhances a downstream step of MAP kinase in the MAP kinase-dependent signaling pathway. Nardosinone is the first enhancer of the neuritogenic action of dbcAMP and staurosporine and may become a useful pharmacological tool for studying the mechanism of action of not only NGF but also both the neuritogenic substances.

Structure–activity relationship studies with (±)-nantenine derivatives for α1-adrenoceptor antagonist activity

A series of (+/-)-nantenine derivatives of the natural aporphine alkaloids was synthesized and examined for a blocking action on alpha1-adrenoceptors in rat aorta and A10-cells. The potency of these derivatives was compared with that of an aporphine-related compounds (+)-boldine, an alpha1-adrenoceptor antagonist. Among nine (+/-)-nantenine derivatives having different substituents at N-6, C-1, or C-4 of the aporphine skeleton, (+/-)-domesticine had the most powerful alpha1-adrenoceptor-blocking action. The order of pA2 values was (+/-)-domesticine (8.06+/-0.06)>(+/-)-nordomesticine (7.34+/-0.03)>(+/-)-nantenine (7.03+/-0.03)>(+)-boldine (6.91+/-0.02)>other derivatives. Study of the structure-activity relationships showed that the replacement of a methoxy moiety at C-1 position of (plus minus)-nantenine with a hydroxyl group increased affinity for the receptor. In contrast, replacement of a methyl group with a hydrogen atom or an ethyl group at N-6 position in the (+/-)-nantenine structure decreased affinity for the receptor. These results suggest that a hydroxyl group at the C-1 position and a methyl group at the N-6 position in the (+/-)-nantenine structure are essential for the enhancement of affinity for the alpha1-adrenoceptor.

Potent apoptotic effects of saponins from Liliaceae plants in L1210 cells.

We isolated eight saponins, a hexacyclic lanosterol tetraglycoside (1), a 27‐norlanosterol tetraglycoside (2) and six spirostanol oligoglycosides (3–8), from the plants of the family Liliaceae. In murine leukaemic L1210 cells, saponins 5 and 7 at a concentration of 1 μM showed potent cytotoxic activity and the activities were in the following decreasing order: 5, 7, 1, 3, 2, 8,4, 6. At a concentration of 10 μM, not only 5 and 7 but also 3 and 8 markedly caused cell death. The flow cytometric analysis indicated that 7 and 8 caused a concentration‐ and time‐ dependent apoptosis of L1210 cells (EC50 value = ∼5 μM). The morphological observation using a light microscope revealed that both 7 and 8 induced shrinkage in cell soma and chromatin condensation, suggesting apoptotic cell death. Moreover, in agarose gel electrophoretic analysis, a typical apoptotic DNA ladder pattern was observed after treatment with both 7 and 8. These results suggest that 7 and 8 caused the death of L1210 cells through the apoptotic process. These compounds may become powerful pharmacological tools for studying the molecular mechanism of apoptosis.

Potentiation of nerve growth factor‐induced elongation of neurites by gelsemiol and 9‐hydroxysemperoside aglucone in PC12D cells

In PC12D cells, nerve growth factor (NGF) increased the proportion of neurite‐bearing cells and made neurites longer. A methanol extract of Verbena littoralis H. B. K. collected in Paraguay only slightly potentiated the proportion of PC12D cells with neurites but markedly increased the length of neurites in the presence of NGF (2 ng mL−1). The methanol extract was partitioned between ethyl acetate and water followed by further extraction of water fraction with n‐butanol. The potentiating activity of NGF‐action was observed in the ethyl acetate and n‐butanol fractions. The n‐butanol fraction was separated by silica gel chromatography, monitoring the NGF‐potentiating activity to give gelsemiol and 9‐hydroxysemperoside aglucone (9‐OHSA). Neither compound (30–300 μM) exhibited neurite‐inducing activity alone. Gelsemiol (100–300 μM) markedly enhanced an increase in the proportion of neurite‐bearing cells and an extension of the neurite length in the presence of NGF (2 ng mL−1). Interestingly, in the presence of NGF (2 ng mL−1), 9‐OHSA (100–300 μM) enhanced the elongation of neurites without affecting the increase in the proportion of cells with neurites. These results suggested that gelsemiol and 9‐OHSA were major active components of V. littoralis in the NGF‐potentiating action. It was possible that the mechanism of neurite elongation by NGF was different from that of the increase in the proportion of neurite‐bearing cells, and that 9‐OHSA selectively affected the neurite elongation mechanism.

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction.

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.

Modulation of actomyosin ATPase by goniodomin A differs in types of cardiac myosin

Goniodomin A causes the conformational change of actin to modify actomyosin ATPase activity [Furukawa, K.-I., Sakai, K., Watanabe, S., Maruyama, K., Murakami, M., Yamaguchi, K., Ohizumi, Y., 1993. Goniodomin A induces modulation of actomyosin ATPase activity mediated through conformational change of actin. J. Biol. Chem. 268, 26026-26031]. Goniodomin A inhibited the ATPase activities of atrial myofibrils, myosin B and reconstituted actomyosin in a concentration-dependent manner. Interestingly, these ATPase activities of ventricular muscle were enhanced by goniodomin A (3 x 10(-8)-3 x 10(-7) M), but were decreased when the concentration was further raised. The stimulatory effect of goniodomin A was significantly inhibited by troponin tropomyosin complex. These results suggest that goniodomin A affects actin to modify cardiac actomyosin ATPase activity, and that this modulation differs in types of cardiac myosin.