Koichi Koshimizu

Inhibitory effects of ursolic and oleanolic ancid on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

Ursolic acid (UA) and oleanolic acid (OA), which had been isolated from Glechoma hederacea as inhibitors of Epstein-Barr virus (EBV) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), were tested against inhibitory effect on tumor promotion by TPA in vivo. They inhibited effectively the tumor promotion in mouse skin and the activities were comparable to that of a known inhibitor of tumor promotion, retinoic acid (RA). Interestingly, UA was more effective on a single application before initial TPA-treatment than on a continuous application before each TPA-treatment, while OA and RA were ineffective in the same treatment. These data suggest that the role of UA for inhibitory action on tumor promotion differs slightly from those of RA and OA.

Search for possible antitumor promoters by inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation; Ursolic acid and oleanolic acid from an anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L.

From an anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L., two triterpene carboxylic acids, ursolic acid (UA) and oleanolic acid (OA) have been isolated as inhibitors of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus (EBV) activation in Raji cells. Both acids significantly inhibited the activation at a 1000-fold molar ratio to TPA, and also teleocidin B-4. The dose responses of the acids were very similar to those of the antitumor promoters, retinoic acid (RA) and glycyrrhetinic acid (GA). However, a characteristic property that UA and OA possess, far higher cell viability to the Raji cells than RA to the Raji cells, has been pointed out. Furthermore, enhancement of the inhibitory activity was found in 3-keto derivatives of UA and OA, while either loss of oxygen functionality at C-3 position of UA or oxidation at C-3 of GA led to reduction of the activity. Binding assay suggested that the inhibitory activity should be exhibited by some event caused after binding of TPA to the receptor in the cells.

Auraptene, a Citrus Coumarin, Inhibits 12-O-Tetradecanoylphorbol-13-acetate-induced Tumor Promotion in ICR Mouse Skin, Possibly through Suppression of Superoxide Generation in Leukocytes

Coumarin‐related compounds, auraptene and umbelliferone, have been isolated from the cold‐pressed oil of natsumikan (Citrus natsudaidai HAYATA), and tested as inhibitors of tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced Epstein‐Barr virus activation in Raji cells. The 50% inhibitory concentration (IC50) of auraptene (18μM)was almost equal to that of genistein. Umbelliferone, which lacks a geranyloxyl group present in auraptene, was less active (IC50=450 μM). In a two‐stage carcinogenesis experiment with 7, 12‐dimethylbenz[α]anthracene (topical application at 0.19 μmol) and TPA (topical application at 1.6 nmol) in ICR mouse skin, topical application of auraptene (at 160 nmol) significantly reduced tumor incidence and the numbers of tumors per mouse by 27% (P < 0.01) and 23% (P < 0.05), respectively. Auraptene at a concentration of 50 μM markedly suppressed superoxide (O2−) generation induced by 100 nM TPA in differentiated human promyelocytic HL‐60 cells. Having no O2− ‐scavenging potential, auraptene may inhibit the multicomponent NADPH oxidase system. Inhibition of intracellular hydroperoxide formation in differentiated HL‐60 cells by auraptene was also confirmed by flow‐cytometric analysis using 2′,7′‐dichlorofluorescein diacetate as a fluorescence probe. Quantitative analyses using high‐performance liquid chromatography showed the occurrence of auraptene not only in both the peels and sarcocarps of natsumikan, but also in those of hassaku orange (C. hassaku) and grapefruit (C. paradisi,) and even in their bottled fresh juice form. These results indicate that auraptene is a chemopreventer of skin tumorigenesis, and implies that suppression of leukocyte activation might be the mechanism through which it inhibits tumor promotion.

Effects of selected food factors with chemopreventive properties on combined lipopolysaccharide- and interferon-γ-induced IκB degradation in RAW264.7 macrophages

Degradation of IkappaB (IκB) is a key step for nuclear factor-kappaB (NF-κB)-induced transcription of certain proinflammatory genes, including inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. We selected seven chemopreventive agents and examined their effects on combined lipopolysaccharide- and interferon-γ-induced IκB degradation in RAW264.7 murine macrophages. IκB degradation was notably suppressed by 1′-acetoxychavicol acetate (ACA), zerumbone (ZER), and benzylisothiocyanate (BITC), however, not by auraptene (AUR), while the suppressive potencies of nobiletin (NOB), genistein (GEN), and resveratrol (RES) were low, but significant. These results suggest that ACA, ZER, and BITC suppress iNOS/COX-2 gene expression mainly by attenuating IκB degradation, while other chemopreventive agents use alternative pathway(s) to suppress the expression of proinflammatory genes.

Chemoprevention of azoxymethane-induced rat aberrant crypt foci by dietary zerumbone isolated from Zingiber zerumbet

The modifying effects of dietary feeding of zerumbone isolated from Zingiber zerumbet on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Expression of cyclooxygenase (COX)-2 in colonic mucosa exposed to AOM and/or zerumbone was also assayed. In addition, we assessed the effects of zerumbone on cell proliferation activity of crypts by counting silver-stained nucleolar organizer regions protein (AgNORs) in colonic cryptal cell nuclei. To induce ACF rats were given three weekly subcutaneous injections of AOM (15 mg/kg body weight). They were also fed the experimental diet containing 0.01% or 0.05% zerumbone for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced 84+/-13 ACF/rat at the end of the study (week 5). Dietary administration of zerumbone caused reduction in the frequency of ACF: 72+/-17 (14% reduction) at a dose of 0.01% and 45+/-18 (46% reduction, p<0.001) at a dose of 0.05%. Feeding of zerumbone significantly reduced expression of COX-2 and prostaglandins in colonic mucosa. Zerumbone feeding significantly lowered the number of AgNORs in colonic crypt cell nuclei. These findings might suggest possible chemopreventive ability of zerumbone, through suppression of COX-2 expression, cell proliferating activity of colonic mucosa, and induction of phase II detoxification enzymes in the development of carcinogen-induced ACF.

FA15, a hydrophobic derivative of ferulic acid, suppresses inflammatory responses and skin tumor promotion: comparison with ferulic acid

In our previous study, FA15 (2-methyl-1-butyl ferulic acid) was chemically synthesized as a novel ferulic acid (FA) analog, and found to notably suppress phorbol ester-induced Epstein-Barr virus activation and superoxide anion generation in vitro. In this report, we demonstrated that FA15, in contrast to FA, markedly suppressed the combined lipopolysaccharide and interferon-gamma-induced protein expressions of inducible nitric oxide synthase and cyclooxygenase-2, and also inhibited the release of tumor necrosis factor-alpha accompanied by suppression of I-kappa B degradation in RAW264.7, a murine macrophage cell line. In ICR mouse skin, topical application of FA15 significantly attenuated phorbol ester-triggered hydrogen peroxide production and edema formation as well as papilloma development while that of FA did not. Our results suggest that FA15, derived from natural sources, is a novel chemopreventive agent, both structurally and functionally.

1,1-Dimethylallylcoumarins potently supress both lipopolysaccharide- and interferon-γ-induced nitric oxide generation in mouse macrophage RAW 264.7 cells

We investigated the suppressive effects of 16 coumarin-related compounds on both lipopolysaccharide (LPS)- and interferon (IFN)-gamma-induced nitric oxide (NO) generation in a mouse macrophage cell line, RAW 264.7. Notably, coumarins possessing prenyl unit(s) were found to be highly active, a tendency consistent with our previous study. Among the coumarins tested, 1,1-dimethylallylcoumarins showed the highest inhibitory activity. Western blotting analysis revealed that they inhibited NO generation by suppressing inducible NO synthase (iNOS) protein expression. Our ongoing studies suggest that coumarins are prominent natural compounds that attenuate excessive and prolonged NO generation at inflammatory sites.

Bitter steroid glucosides, vernoniosides A1, A2, and A3, and related b1 from a possible medicinal plant, Vernonia amygdalina, used by wild chimpanzees

Abstract From Vernonia amygdalina, a possible medicinal plant used by wild chimpanzees, three bitter steroid glucosides, vernoniosides A1, A2, and A3 and a nonbitter vernonioside B1, were isolated. The oxygenation patterns of the aglycone parts were new, especially the pattern of the car☐yl group at C21. The oxygen functionalities at C16 were important for the bitter taste.

Toward the chemical ecology of medicinal plant use in chimpanzees: The case ofVernonia amygdalina, a plant used by wild chimpanzees possibly for parasite-related diseases

The bitter and related constituents have been isolated fromVernonia amygdalina (Compositae), a plant ingested by wild chimpanzees possibly suffering from parasite-related diseases in the Mahale Mountains National Park, Tanzania. Isolated from the plant were four known sesquiterpene lactones, seven new steroid glucosides, and two aglycones of the glucosides. The sesquiterpene lactones showed significant in vitro antischistosomal, plasmodicidal, and leishmanicidal activities. Antischistosomal activity was also found for the major steroid glucoside, vernonioside B1. A trend in the glucosides to show significant antischistosomal, plasmodicidal, and amebicidal activities when the sugar moiety was removed, was observed. Vernodalin, judged as the most significant constituent for antiparasitic activities in vitro, was tested for in vivo antischistosomal effect. It was, however, highly toxic to the cercaria-infected mouse. Chimpanzees have been only rarely observed to ingest anything but the pith of the young stem. The occurrence of vernonioside B1 and its aglycone vernoniol B1, the major constituents among the steroid-related constituents, were detected at significant levels in the pith. However, vernodalin was abundant only in the leaves and bark. Thus, chimpanzees at Mahale were hypothesized to control parasite-related diseases by ingesting the young pith of this tree containing steroid-related constituents.

Use of Vernonia amygdalina by wild chimpanzee: possible roles of its bitter and related constituents.

Bitter principles and related constituents have been isolated from Vernonia amygdalina (Compositae), a plant ingested by wild chimpanzees sometimes suffering from parasite-related diseases in the Mahale Mountains National Park, Tanzania. These isolated constituents were the known sesquiterpene lactones (vernodalin, vernolide, hydroxyvernolide), and new stigmastane-type steroid glucosides (vernonioside A1-A4: for bitter tasting constituents and vernonioside B1-B3; for nonbitter related constituents). Antiparasitic activity tests of these constituents together with quantitative analyses of the major active constituents, vernodalin and vernonioside B1, supported the hypothesis that Mahale chimpanzees control parasite-related diseases by ingesting the pith of this plant, found to contain several steroid-related constituents. While the major active steroid-related constituents (vernonioside B1 and its primary aglycone, vernoniol B1) do not taste bitter themselves, it was hypothesized that the highly bitter constituents including vernodalin may play an important role as signals to the ingester guiding their choice of the appropriate plant, plant part, and possibly also as signals which help to control the amount of intake.