Diglucosyl-glycerolipids from the marine sponge-associated Bacillus pumilus strain AAS3: their production, enzymatic modification and properties.

The marine strain Bacillus pumilus strain AAS3, isolated from the Mediterranean sponge Acanthella acuta, produced a diglucosyl-glycerolipid, 1,2-O-diacyl-3-[beta-glucopyranosyl-(1-6)-beta-glucopyranosyl)]glycerol, with 14-methylhexadecanoic acid and 12-methyltetradecanoic acid as the main fatty acid moieties (GGL11). On a 30 l scale, using artificial seawater supplemented with glucose (20 g/l), yeast extract (10 g/l), and suitable nitrogen/phosphate sources, growth-associated glycoglycerolipid production reached its maximum yield of 90 mg/l after 11 h. Lipase-catalyzed modification of the native substance led to the deacylated parent compound (GG11), which could be reacylated using the same enzyme system to afford a new dipentenoyl-diglucosylglycerol (GGL12) as the major product upon addition of 4-pentenoic acid to the medium. GGL11 decreased the surface tension of water from 72 mN/m to 29 mN/m and the interfacial tension of the water/ n-hexadecane system from 44 to 5 mN/m. Anti-tumor-promoting studies on this class of diglucosyl glycerol products showed that the carbohydrate/glycerol backbone (GG11) has a more potent inhibitory activity than the acylated compounds. The diglucosyl-glycerol GG11 strongly inhibited growth of the tumor cell lines HM02 and Hep G2 (50% inhibition at approximately 1 microg/ml), while the glycerolipids GGL11 and GGL12 were less active or had no effect.

Crown gall -- a plant tumour with biological activities.

Petroleum ether, acetone, 80% MeOH and water extracts of crown gall, a plant tumour, obtained from Eucalyptus globulus tree were screened for cytotoxic, antioxidant, antiinflammatory, embryotoxic, antitumour-promoting and antimicrobial activities. In terms of bioactivity the 80% MeOH extract was most effective followed by the acetone extract. The petroleum ether extract showed weak to moderate cytotoxic activity in dose-dependent manner against PC12 cells, mouse L fibroblasts and 1321N1 glia cells, whereas the hydroalcohol extract had no or a weak cytotoxic effect. The 80% MeOH extract exhibited strong antioxidant activity. Based on the in vitro HET-CAM assay all the extracts were effective against inflammation. The extracts did not show any embryotoxic effect at the concentrations tested. Antitumour-promoting activity (100% inhibition; 100 microg/mL) was observed in the 80% MeOH and acetone extracts. In the antimicrobial screening all extracts displayed predominantly antifungal activity against Candida sp. The extracts also showed various levels of antibacterial activity against E. faecalis, Ps. aeruginosa, Bac. subtilis and Staph. epidermidis. From the results of the investigations it can be concluded that crown gall is a valuable plant tumour tissue having interesting biological activities.

Cancer chemopreventive activity of odorine and odorinol from Aglaia odorata.

In the course of our continuing search for novel cancer chemo-preventive agents from natural sources, we have carried out a primary screening in vitro assay of the compounds isolated from Aglaia odorata. Consequently, aminopyrrolidine-diamides, odorine and odorinol, were obtained as active constituents. These compounds exhibited potent anti-carcinogenic effects in a two-stage carcinogenesis test of mouse skin induced by 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Further, both compounds showed remarkable inhibitory effects in two-stage mouse skin carcinogenesis models induced by nitric oxide (NO) donors such as (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR-1) or peroxynitrite as an initiator and TPA as a promoter. From these results, it was concluded that odorine and odorinol inhibited both the initiation and promotion stages of two-stage skin carcinogenesis.

Cancer chemoprevention by ginseng in mouse liver and other organs.

Oral administration of red ginseng extracts (1% in diet for 40 weeks) resulted in the significant suppression of spontaneous liver tumor formation in C3H/He male mice. Average number of tumors per mouse in control group was 1.06, while that in red ginseng extracts-treated group was 0.33 (p<0.05). Incidence of liver tumor development was also lower in red ginseng extracts-treated group, although the difference from control group was not statistically significant. Anti-carcinogenic activity of white ginseng extracts, besides red ginseng extracts, was also investigated. In the present study, the administration of white ginseng extracts was proven to suppress tumor promoter-induced phenomena in vitro and in vivo. It is of interest that oral administration of the extracts of Ren-Shen-Yang- Rong-Tang, a white ginseng-containing Chinese medicinal prescription, resulted in the suppression of skin tumor promotion by 12-o-tetradecanoylphorbol-13-acetate in 7,12-dimethylbenz[a]anthracene-initiated CD-1 mice. These results suggest the usefulness of ginseng in the field of cancer prevention.

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.

Effects of lycopene and Sho-saiko-to on hepatocarcinogenesis in a rat model of spontaneous liver cancer.

The Long-Evans Cinnamon (LEC) rat is a well-characterized model of spontaneous hepatocarcinogenesis. It has been shown that dietary administration of lycopene or the herbal medicine Sho-saiko-to (TJ-9) has anticarcinogenic activity, although the mechanism by which these products protect against carcinogenesis is not well known. We investigated the outcome of administration of lycopene and TJ-9 on the occurrence of hepatic neoplasia in LEC rats. A diet containing 0.005% lycopene (originally the product of tomato oleoresin containing 13% lycopene) and 1% TJ-9 (crude extracts of 7 herbs: bupleurum root, pinellia tuber, scutellaria root, jujube fruit, ginseng root, glycyrrhiza root, and ginger rhizome) was administered from 6 weeks of age until the rats were sacrificed at 76 weeks of age, at which time most of the nontreated animals were known to have hepatocellular carcinoma (HCC). Development of HCC in treated groups was analyzed histologically by comparison with untreated controls. Glutathione S-transferase placental form (GST-P) was analyzed by an immunohistochemical method. Concentration of copper, iron, and zinc, which appear to play a role in hepatocarcinogenesis in LEC rats, was analyzed. The percent areas of HCC in the liver specimens of control, lycopene, and TJ-9 groups were 17.9 +/- 17.1%, 27.2 +/- 20.8%, and 27.6 +/- 18.4%, respectively. These intergroup differences were not significant. The percent area, number of areas, and mean size of area staining positively for GST-P revealed no significant differences between the groups. The number of GST-P-positive areas within the HCC lesions was greater in the TJ-9 group than in the control or lycopene group (p = 0.024 and p = 0.012, respectively). The study also demonstrated a lower concentration of iron in livers of the lycopene group than the control group (p = 0.019). There were no differences in serum alpha-fetoprotein levels or the cumulative survival rates between the groups. In conclusion, long-term administration of lycopene or TJ-9 did not reduce the risk of hepatocarcinogenesis in LEC rats.

Cancer chemopreventive activity of carotenoids in the fruits of red paprika Capsicum annuum L.

Capsanthin and related carotenoids isolated from the fruits of red paprika Capsicum annuum L. showed potent in vitro anti-tumor-promoting activity with inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Among them, capsanthin diester and capsorbin diester showed strong inhibitory effects. Furthermore, capsanthin , capsanthin 3'-ester and capsanthin 3,3'-diester , major carotenoids in paprika, exhibited potent anti-tumor-promoting activity in an in vivo mouse skin two-stage carcinogenesis assay using 7, 12-dimethylbenz[a]anthracene as an initiator and TPA as a promoter.

Chemopreventive activity of isoquinoline alkaloids from Corydalis plants.

Eighteen isoquinoline alkaloids including protoberberines (1-12), benzophenanthridines (13-16) and an aporphine (17) isolated from plants of Corydalis species (Fumariaceae) were tested for inhibitory effects on Epstein-Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol 13-acetate in Raji cells. In a primary screening test, all of the isoquinoline alkaloids showed inhibitory activity with the IC50 values being in the range of 140-410 mol ratio/32 pmol TPA. The data demonstrate that these isoquinoline alkaloids might be valuable as anti-tumor promoters.

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 agents. New depsidones from Garcinia plants.

In a search for cancer chemopreventive agents from natural sources, chemical constituents of two kinds of Garcinia plants, Garcinia neglecta and Garcinia puat, collected in New Caledonia, were examined. Five new depsidones, garcinisidone-B (2), -C (3), -D (4), -E (5), and -F (6), were isolated, and their structures were determined by spectrometric analyses. Inhibitory effects of these depsidones on EBV-EA activation induced by TPA in Raji cells were also demonstrated.

Isolation and characterization of a human thiamine pyrophosphokinase cDNA.

A human thiamine pyrophosphokinase cDNA clone (hTPK1) was isolated and sequenced. When the intact hTPK1 open reading frame was expressed as a histidine-tag fusion protein in Escherichia coli, marked enzyme activity was detected in the bacterial cells. The hTPK1 mRNA was widely expressed in various human tissues at a very low level, and the mRNA content in cultured fibroblasts was unaffected by the thiamine concentration of the medium. The chromosome localization of the hTPK1 gene was assigned to 7q34.

Lignans as anti-tumor-promoter from the seeds of Hernandia ovigera.

Seven lignans (2-8) isolated from the seeds of Hernandia ovigera L. (Hernandiaceae) were tested for their inhibitory effects on Epstein-Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol 13-acetate in Raji cells. Using a primary screening test, all the lignans showed inhibitory activity with IC50 470-590 mol ratio/32 pmol TPA. The data demonstrated that these lignans might be valuable anti-tumor-promoters.

Abietane diterpenoids from the cones of Larix kaempferi and their inhibitory effects on Epstein-Barr virus activation.

Four known (1-3, 8) and four new abietane diterpenes, 15-hydroxy-8alpha,14alpha,12alpha,13alpha-diepoxyabietan-18-oic acid (4), 7alpha,8alpha,13beta,14beta-diepoxyabietan-18-oic acid (5), 18-nor-abieta-8,11,13-triene-4alpha,7alpha,15-triol (6), and abieta-8,11,13-triene-7alpha,15,18-triol (7) were isolated from the CHCl3 extract of the cones of Larix koempferi. A known compound, 13,14-seco-13,14-dioxoabiet-13-en-18-oic acid (8) was isolated from natural sources for the first time. Their structures were determined by chemical and spectroscopic methods including 1D and 2D NMR techniques. The absolute stereostructure of 5 was determined by X-ray crystallographic analysis. The inhibitory effects of these compounds on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), were examined as a primary screening for antitumor promotors.

Cancer chemopreventive activity of 3,5,6,7,8,3',4'-heptamethoxyflavone from the peel of citrus plants.

Nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone (HPT), isolated from the peel of Citrus plants, were examined for the anti-tumor-initiating activity on two-stage carcinogenesis of mouse skin tumors induced by a nitric oxide donor, (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide, as an initiator and 12-O-tetradecanoylphorbol-13-acetate as a promoter. HPT exhibited the remarkable anti-tumor-initiating effect on mouse skin and it suggested the possibility of HPT being a chemopreventive agent against nitric oxide (NO) carcinogenesis.

Variance of vestibular-evoked myogenic potentials.

OBJECTIVES/HYPOTHESIS: Vestibular-evoked myogenic potential (VEMP) has been thought to originate from sacculus. The variance of this potential and the effectiveness of the adjustments of pInII amplitudes using average muscle tonus of ipsilateral sternocleidomastoid muscle were evaluated. In addition, clinical application of VEMP was examined in patients with acoustic tumors (ATs) and vestibular neurolabyrinthitis (VNL). STUDY DESIGN: Prospective evaluation of the VEMP in 18 normal volunteers and 6 patients. METHODS: Variance and left-right difference of each parameter, including pI latency, nII latency, pInII amplitude, and threshold, was analyzed. Input-output function of pInII amplitude was evaluated. Average muscle tonus was calculated in 20 ears and applied for adjustment of pInII amplitude. Sensitivity of each parameter of VEMP was examined in 3 patients with ATs and 3 patients with VNL. RESULTS: VEMP was present in all 36 ears of 18 control subjects. Thresholds of VEMP for normal subjects were 80 to 95 dB normal hearing level (nHL). The muscle tonus affected pInII amplitude significantly; however, no statistically significant improvement was observed in test-retest investigation after adjustment using muscle tonus. The threshold of the affected side was elevated compared with the non-affected side in all patients with ATs, whereas 2 of 3 patients showed normal pInII-ratio. One patient with VNL presented normal VEMP, whereas 2 patients presented no VEMP to the highest stimulus intensity. CONCLUSIONS: Interaural difference of thresholds might be the most useful parameters. Adjustment using average muscle tonus is not necessary when the subject is able to get sufficient muscle tonus.

Bioactive triterpenoids from the stem bark of Picea glehni.

Two new triterpenoids, 3 alpha-methoxyserrat-14-en-21 beta-yl formate (1), and 24-methylcycloartenone (2), were isolated from the stem bark of Picea glehni (Fr. Schm.) Masters together with three known triterpenoids, 3 alpha-methoxyserrat-14-en-21 beta-ol, 3 beta-methoxyserrat-14-en-21 beta-ol, and piceanonol A. Compounds 1, 2, and a synthetic sample, 3 alpha-methoxyserrat-13-en-21 beta-yl formate showed potent inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).

Cancer chemopreventive agents, labdane diterpenoids from the stem bark of Thuja standishii (Gord.) Carr.

Seven labdane-type diterpenoids from the stem bark of Thuja standishii (Gord.) Carr. (Cupressaceae) and their analogues showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among these compounds, 15,16-bisnor-13-oxolabda-8(17), 11E-dien-19-oic acid was revealed to have the strongest inhibitory effect on the EBV-EA activation, being stronger than that of beta-carotene which has been intensively studied in cancer prevention using animal models. 15,16-bisnor-13-Oxolabda-8(17), 11E-dien-19-oic acid was also found to exhibit the excellent anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.

Constituents of Boronia pinnata.

A novel quinolone, pinolinone (1); seven new phenylpropanoids, boropinols A (2), B (3), C (4), boropinals A (5), B (6), C (7), and boropinic acid (8); and a new lignan, boropinan (9), were isolated from the roots of Boronia pinnata, and their structures were elucidated by NMR and MS analyses. In a search for novel cancer chemopreventive agents (antitumor-promoters), we screened 10 compounds isolated from the plant for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. Boropinic acid (8) and 4'-hydroxy-3'-prenylcinnamaldehyde were observed to significantly inhibit the EBV-EA activation.

Inhibitory effect of citrus nobiletin on phorbol ester-induced skin inflammation, oxidative stress, and tumor promotion in mice.

The intake of citrus fruits has been suggested as a way to prevent the development of some types of human cancer. Nitric oxide (NO) is closely associated with the processes of epithelial carcinogenesis. We attempted a search for NO generation inhibitors in Citrus unshiu. The active constituent was traced by an activity-guiding separation. NO and superoxide (O2-) generation was induced by a combination of lipopolysaccharide and IFN-gamma in mouse macrophage RAW 264.7 cells, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocyte HL-60, respectively. Expression of inducible NO synthase and cyclooxygenase 2 proteins were detected by Western blotting. The in vivo anti-inflammatory and antitumor promoting activities were evaluated by topical TPA application to ICR mouse skin with measurement of edema formation, epidermal thickness, leukocyte infiltration, hydrogen peroxide production, and the rate of proliferating cell nuclear antigen-stained cells. As a result, nobiletin, a polymethoxyflavonoid, was identified as an inhibitor of both NO and O2- generation. Nobiletin significantly inhibited two distinct stages of skin inflammation induced by double TPA application [first stage priming (leukocyte infiltration) and second stage activation (oxidative insult by leukocytes)] by decreasing the inflammatory parameters. It also suppressed the expression of cyclooxygenase-2 and inducible NO synthase proteins and prostaglandin E2 release. Nobiletin inhibited dimethylbenz[a]anthracene (0.19 micromol)/TPA (1.6 nmol)-induced skin tumor formation at doses of 160 and 320 nmol by reducing the number of tumors per mouse by 61.2% (P < 0.001) and 75.7% (P < 0.001), respectively. The present study suggests that nobiletin is a functionally novel and possible chemopreventive agent in inflammation-associated tumorigenesis.

Cypellocarpins A-C, phenol glycosides esterified with oleuropeic acid, from Eucalyptus cypellocarpa.

Three new phenol glycosides acylated with (+)-oleuropeic acid, cypellocarpins A (1), B (2), and C (3), along with seven known compounds, were isolated from the dried leaves of Eucalyptus cypellocarpa. Structures of the new compounds were determined on the basis of spectroscopic methods, including 2D NMR experiments and chemical degradation. These new compounds and a known related glucoside (7) showed potent in vitro antitumor-promoting activity in a short-term bioassay evaluating the inhibitory effect on Epstein-Barr virus early antigen activation induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA). These compounds also suppressed an in vivo two-stage carcinogenesis induced with nitric oxide and TPA on mouse skin.