Tetsuro Ikekawa

Antitumor activity of daphnane-type diterpene gnidimacrin isolated from Stellera chamaejasme L.

The daphnane‐type diterpene gnidimacrin, isolated from the root of the Chinese plant, Stellera chamaejasme L., was found to strongly inhibit cell growth of human leukemias, stomach cancers and non‐small cell lung cancers in vitro at concentrations of 10−9 to 10−10 M. On the other hand, even at 10−8 to 10−5 M, the small cell lung cancer cell line H69 and the hepatoma cell line HLE were refractory to gnidimacrin. The agent showed significant antitumor activity against murine leukemias and solid tumors in an in vivo system. In K562, a sensitive human leukemia cell line, gnidimacrin induced blebbing of the cell surface, which was completely inhibited by staurosporine at concentrations above 10−8 M, and arrested the cell cycle transiently to G2 and finally the G1 phase at growth‐inhibitory concentrations. It inhibited phorbol‐12,13‐dibutyrate (PDBu) binding to K562 cells and directly stimulated protein kinase C (PKC) activity in the cells in a dose‐dependent manner (3–100 nM). Although activation of PKC isolated from refractory H69 cells was observed only with 100 nM gnidimacrin, the degree of activation was lower than that produced by 3 nM in K562 cells. Our results suggest that gnidimacrin acts as a PKC activator for tumor cells and that this mechanism may be responsible for its antitumor activity.

Entada saponin-III, a saponin isolated from the bark of Entada phaseoloides

Abstract The structure of Entada saponin (ES)-III, one of the main saponins of Entada phaseoloides bark, was established to be 3- O -[β- d -xylopyranosyl (1 → 2)-α- l -arabinopyranosyl (1 → 6)] [β- l -glucopyranosyl (1 → 4)]-2-acetamido-2-deoxy-β- l -glucopyranosyl-28- O -[β- l -apiofuranosyl (1 → 3)-β- d -xylopyranosyl (1 → 2)] [(2- O -acetoxyl)-β- d -glucopyranosyl-(1 → 4)] (6 − O ( R ) (−)2,6-dimethyl-2- trans -2,7-octadienoyl)-β- d -glucopyranosyl echinocystic acid.

Mechanism of antitumor action of PKC activator, gnidimacrin.

Daphnane‐type diterpene gnidimacrin isolated from the Chinese plant Stellera chamaejasme L. is an antitumor agent that activates protein kinase C (PKC). The mechanism of antitumor action of gnidimacrin and the possible involvement of PKC were examined using sensitive K562 and refractory HLE cells. Gnidimacrin did bind to K562 cells 3 times more than to HLE cells. Immunoblot analyses revealed pronounced PKCβII expression in gnidimacrin sensitive cell lines including K562 cells, while refractory HLE cells strongly expressed PKCα, but not PKCβII. In a 24‐hr exposure of K562 cells to gnidimacrin, G1 phase arrest and inhibition of cdk2 kinase activity was found at growth‐inhibitory concentration (0.0005 μg/ml). Complete inhibition of cdk2 activity and maximum G1 phase arrest were observed at 0.005 μg/ml, however, these biological effects were reduced at 0.05 μg/ml (260 times the 50% inhibitory concentration). Cellular PKC after a 24‐hr exposure was examined by immunoblot analysis and specific binding of [3H]phorbol‐12,13‐dibutyrate as a ligand of PKC. Expression and the amount of functional PKC of K562 cells were not changed at 0.002 μg/ml, but down‐regulated to less than 1/10th of the control at 0.05 μg/ml. The reduction of biological effects at 0.05 μg/ml is most likely due to PKC down‐regulation. Our results suggest that PKC (particularly βII) is one of the major determinants of the ability of cells to respond to gnidimacrin and that the antitumor action might be associated with cell‐cycle regulation through suppression of cdk2 activity. Int. J. Cancer 77:243–250, 1998.© 1998 Wiley‐Liss, Inc.

Isolation, purification, and structure of components from acidic polysaccharides of pleurotus ostreatus (Fr.) Quél.

Isolation of an antitumor component from polysaccharide fraction A5 of some Basidiomyces was achieved by column chromatography on Sephadex G-200. A detection method based on the specific rotatory characteristics of the polysaccharide was applied to estimate components in effluent fractions from the chromatography, and it was confirmed that a series of eluates having similar specific rotation was made up of homogeneous polysaccharide. Three components (H51, H52, and H53) were isolated, in chromatographically pure state, from fraction A5. Component H51 consisted of a skeleton of beta-(1 leads to 3)-linked glucose residues, probably having branches of galactose and mannose residues, and also containing acidic sugars. Component H53 had a main structure similarly consisting of beta-(1 leads to 3)-linked glucose residues and a larger proportion of acidic sugar than H51. Component H52 was a heteropolysaccharide made up of alpha-linked galactose and mannose residues. Components H51 and H53 had a higher and a lower molecular weight, respectively, than H52. The only antitumor-active component was H51.

Triptinins A and B, two leukotriene D4 antagonistic 19(4→3)-abeo-abietanes from Tripterygium wilfordii

Abstract Bioassay-directed separation of an ethanolic extract of the roots of Tripterygium wilfordii furnished two new 19(4→3)- abeo -abietanes, triptinin A and B, along with two known diterpenes, triptoquinone A and 11-hydroxy-14-methoxy-19(4→3)- abeo -abieta-3, 8, 11, 13-tetraen-19-oic acid, as leukotriene D 4 antagonist constituents. The structures of these diterpenes were elucidated by spectroscopic methods.

Involvement of PKC βII in anti-proliferating action of a new antitumor compound gnidimacrin

Daphnane‐type diterpene gnidimacrin (NSC 252940) shows significant antitumor activity against murine tumors and human tumor cell lines. This compound binds to and directly activates protein kinase C (PKC), arresting the cell cycle at the G1 phase through inhibition of cdk2 activity in human K562 leukemia cells. In our study, we examined whether cellular PKC is involved in the antiproliferating effect of gnidimacrin. In a 24‐hr exposure of K562 cells to high concentrations of bryostatin 1 (0.11–3.3 μM), both expression of PKC α and PKC βII was downregulated, and thereafter these cells became resistant to gnidimacrin in response to the degree of PKC downregulation. In addition, PKC α and PKC βII genes were transfected to gnidimacrin‐resistant human hepatoma HLE cells that demonstrated positive expression of PKC α and negative expression of PKC βII. PKC βII gene‐transfected cells became sensitive to gnidimacrin in relation to the degree of PKC βII expression. The most sensitive clone to show 0.001 μg/mL (1.2 nM) as IC50 in a continuous 4‐day exposure was obtained. While PKC α gene‐transfected cells exhibited an increase in PKC α expression and became sensitive to gnidimacrin, sensitivity was one‐hundredth of that in PKC βII gene‐transfected cells. These results suggest that PKC, in particular PKC βII, is necessary in the antitumor effect of gnidimacrin.

Studies on the structure and stereochemistry of cytotoxic furanonaphthoquinones from Tabebuia impetiginosa: 5- and 8-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-diones

Chemical investigation of the bark of Tabebuia impetiginosa(Bignoniaceae) afforded cytotosic furanonaphthoquinones, including 5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4.9 dione and its 8-hydroxy isomer. The position of the phenolic group in these two compounds was established by X-ray crystallography. The furanonaphthoquinones were found to be mixtures of both enantiomers in diffferent proportions, i.e., (for the 5-hydroxyisomer)R:S1:23 and (for the 8-hydroxy isomer)R:S 3:1. The synthesis of the racemic naphthofurans, their optical resolution into enantiomerically pure forms, and the determination of their absolute stereochemistry are described. The structure of kigelinone was also established to be that of the 5-hydroxy isomer, not the 8-hydroxy one, through this study.

Antitumor action of the PKC activator gnidimacrin through cdk2 inhibition

Daphnane‐type diterpene gnidimacrin (NSC252940), isolated from a Chinese plant, exhibited antitumor activity against murine leukemias and solid tumors. At concentrations of 10–9 to 10–10 M, this agent strongly inhibited the growth of human tumor cell lines. In sensitive human leukemia K562 cells, gnidimacrin is a PKC activator that arrests the cell cycle in the G1 phase by inhibiting cdk2 activity. A 4 hr exposure of K562 cells to gnidimacrin induced the CDK inhibitor p21WAF1/Cip1, but this effect was transient and did not correlate temporally with the onset of G1 arrest. Expression of cdc25A, a phosphatase that activates cdk2, was reduced during 24‐hr exposure to gnidimacrin. Moreover, the suppression corresponded in a concentration‐ and time‐dependent manner to both the inhibition of cdk2 activity and the mobility shift observed when cdk2 was electrophoresed on SDS‐PAGE, indicating that the phosphorylation state of cdk2 must change. Cyclin E, the other regulator of cdk2 activity, was not influenced by gnidimacrin. These results suggest that gnidimacrin exerts antitumor activity through suppression of cdc25A and inhibition of cdk2 activity.