Masataka Itoigawa

Cancer chemopreventive agents, 4-phenylcoumarins from Calophyllum inophyllum

In a search for anti-tumor-promoting agents, we carried out a primary screening of ten 4-phenylcoumarins isolated from Calophyllum inophyllum L. (Guttiferae), by examining their possible inhibitory effects on Epstein--Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All of the compounds tested in this study showed inhibitory activity against EBV, without showing any cytotoxicity. Calocoumarin-A (5) showed more potent activity than any of the other compounds tested. Furthermore, calocoumarin-A (5) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. The results of the present investigation indicate that some of these 4-phenylcoumarins might be valuable as potential cancer chemopreventive agents (anti-tumor-promoters).

Cancer Chemopreventive Activity of Terpenoid Coumarins from Ferula Species

Several natural products have been found to have anti-tumor promoting activity. In the present study, we carried out a primary screening of ten terpenoid coumarins isolated from plants of the Ferula species, examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12- O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells. Auraptene (7-geranyloxycoumarin, 1) and umbelliprenin (7-farnesyloxycoumarin, 2) were found to significantly inhibit EBV-EA activation and preserved the high viability of Raji cells, suggesting that they might be valuable anti-tumor-promoting agents (IC (50) 8.3 and 9.1 nM, respectively). Our findings revealed that the presence of a prenyl moiety in the terpenoid coumarins plays an important role in anti-tumor promoting activity as previously reported for xanthones, coumarins, flavonoids and phenylpropanoids.

Cancer chemopreventive activity of naphthoquinones and their analogs from Avicennia plants

As a part of screening studies for cancer chemopreventive agents (anti-tumor promoters), six natural and four synthetic naphthoquinones and five of their analogs were tested for their inhibitory activities against Epstein-Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. Some of the 1,4-naphthoquinones and their analogs were found to show remarkably potent activities, without showing any cytotoxicity. 1,4-Furanonaphthoquinone (5) and its analog (9) isolated from Avicennia plants (Avicenniaceae), having an alcoholic OH group on the dihydrofuran-ring, displayed the most potent activity. Furthermore, avicenol-A (9) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. The result of the present investigation indicated that some of these 1,4-naphthoquinones and their analogs might be valuable as potent cancer chemopreventive agents.

Distinct involvement of NF-κB and p38 mitogen-activated protein kinase pathways in serum deprivation-mediated stimulation of inducible nitric oxide synthase and its inhibition by 4-hydroxynonenal

Cytokine‐induced expression of inducible nitric oxide synthase (iNOS) and concomitant production of nitric oxide (NO) involve activation of mitogen‐activated protein (MAP) kinases and are in most cases mediated by the transcription factor NF‐κB. We investigated the role of p38 MAP kinase activation and IκB phosphorylation in iNOS expression in a novel iNOS‐inducing model in mouse macrophages. Deprivation of serum from the culture medium of RAW 264.7 cells up‐regulated iNOS and NO production, which were inhibited by 4‐hydroxy‐2‐nonenal (HNE), a component of oxidatively modified low‐density lipoprotein (oxLDL). Serum withdrawal induced phosphorylation of Akt, IκB, and p38 MAP kinase. Pretreatment with the potent PI3 kinase inhibitor wortmannin, the NF‐κB inhibitor PDTC or the specific p38 MAP kinase inhibitor SB203580 each partially attenuated the induction of iNOS and NO production, demonstrating that both p38 activation and IκB phosphorylation are required for iNOS expression. SB203580, however, did not prevent the phosphorylation of Akt and IκB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an IκB‐phosphorylation‐independent pathway. HNE, which markedly inhibited iNOS expression and NO production, prevented the serum withdrawal‐triggered IκB phosphorylation but not that of Akt or p38 MAP kinase. A high concentration of HNE stimulated dephosphorylation of IκB but promoted activation of p38 MAP kinase. Taken together, these results suggest that NF‐κB and p38 MAP kinase lie in separate signal pathways for serum deprivation‐stimulated iNOS expression and NO production. HNE selectively suppresses the former pathway, targeting a site downstream of Akt. J. Cell. Biochem. 83: 271–280, 2001.

γ-Lactone Carbazoles from Clausena anisata

The study of chemical constituents of the stems of Clausena anisata collected in Thailand led to the isolation and identification of eight known and two new carbazole alkaloids named furanoclausamines A (1) and B (2). Clausamine E (3) was found to exhibit cytotoxicity against the human leukemia cell line HL-60.

Triphasic inotropic response of guinea-pig papillary muscle to murrayaquinone-A isolated from Rutaceae.

Murrayaquinone-A, a carbazole alkaloid, was found to produce a triphasic inotropic response (first positive, second negative and third positive phases) of guinea-pig papillary muscle that normally paced at a slow rate of 0.2 Hz in Krebs-Henseleit solution at 30 degrees C. Murrayaquinone-A produced a concentration-dependent (10(-6) M-10(-4) M) positive inotropic effect (pD2 value 5.27 evaluated at the first phase). The triphasic pattern of inotropism of murrayaquinone-A was unaffected by reserpine, metoprolol or cimetidine treatment. Murrayaquinone-A increased the initial upstroke and the duration of the slow action potential in partially depolarized muscle (external K+ = 30 mEq). Murrayaquinone-A did not cause any positive inotropy under anoxic conditions and in the presence of 2,4-dinitrophenol and dicumarol. These results indicated that the triphasic inotropic effect of murrayaquinone-A is not mediated through a receptor mechanism but through a novel mechanism involving mitochondrial ATP production, thereby increasing the slow inward calcium current across the cardiac cell membrane via cyclic AMP converted from mitochondrial ATP.

Xanthones as inhibitors of Epstein–Barr virus activation

To search for possible antitumor promoters, we carried out a primary screening of 20 xanthones isolated from plants of the Guttiferae family, using their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Some of these xanthones, namely 1,3,7-trihydroxy-2-(3-methyl-2-butenyl)xanthone (8), dulxanthone-B (10) and latisxanthone-C (15), were observed to significantly inhibit the EBV-EA activation. The investigation indicated that 8, 10 and 15 might be valuable antitumor promoters.

Isolation of the Antioxidant Pyranonigrin-A from Rice Mold Starters Used in the Manufacturing Process of Fermented Foods

Rice mold starters prepared from Aspergillus species are commonly used for the manufacture of koji in the production of oriental fermented foods. Methanol extracts of rice mold starters fermented by the Aspergillus species, A. awamori, A. kawachii, A. oryzae, A. saitoi, and A. sojae, were examined for their antioxidative activity by using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging system. The extracts of A. awamori, A. kawachii, and A. saitoi exhibited higher activity than those of A. oryzae and A. sojae. An antioxidant was isolated from the extract of A. saitoi and identified as pyranonigrin-A by 1H-NMR, 13C-NMR, and FAB-MS analyses. The antioxidative activity of pyranonigrin-A was approximately equivalent to that of ferulic acid, an antioxidant in cereal grain. It was present in rice mold starters prepared by A. awamori, A. kawachii, and A. saitoi, although there was no pyranonigrin-A in the A. oryzae and A. sojae starters. The results suggest that the content of pyranonigrin-A in rice mold starters has a correlation with the antioxidative activity, and that it is induced in rice mold starters at the sporulation stage.

Cancer chemopreventive activity of flavanones on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis.

To search for possible cancer chemopreventive agents from natural sources, we performed primary screening of ten flavanones isolated from plants belonging to Rutaceae and Leguminosae by examining their possible inhibitory effects on Epstein-Barr virus (EBV) early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All of the flavanones tested in this study showed inhibitory activity against EBV, without showing any cytotoxicity. Amorilin (3), which has three prenyl (3-methyl-2-butenyl) side-chains in the molecule, showed the most potent activity. Furthermore, lupinifolin (5) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These results indicate that some of these prenylated flavanones might be valuable as potential cancer chemopreventive agents (anti-tumor promoters).

Anti-tumor-promoting effects of 8-substituted 7-methoxycoumarins on Epstein-Barr virus activation assay.

In a search for anti-tumor-promoting agents, we carried out a primary screening of twenty-nine 8-substituted and four 6-substituted derivatives of 7-methoxycoumarins isolated from plants of the Murraya and/or Citrus species (Rutaceae), examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. This investigation indicated that the prenyl (3-methyl-2-butenyl) or 2-hydroxy-3-methylbutyl (or butenyl) unit as an isoprenoid moiety at C-8 on the 7-methoxycoumarin nucleus plays an important role in the anti-tumor-promoting activity. Some of the 8-substituted 7-methoxycoumarins isolated from Murraya species, murrangatin (7), minumicrolin (10) and chloticol (18), were found to significantly inhibit EBV-EA activation, and preserved the high viability of Raji cells, suggesting that 7, 10 and 18 might be valuable anti-tumor-promoting agents.