Eiichiro Ichiishi

Chemoprevention of DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot.

Our previous studies identified the extract of Beta vulgaris (beetroot), commercially also known as betanin, as a potent cancer chemopreventive agent in both in vitro Epstein-Barr early antigen activation assay and in an in vivo two-stage mouse lung and skin carcinogenesis. To explore this issue further, we have now investigated its cancer chemopreventive potentials in three different chemical carcinogen initiation-promotion experimental tumor models in mice. Following tumor initiation with 390 nmol of 7,12-dimethylbenz(a)anthracene (DMBA) in 100 microl of acetone, the mouse skin tumor promotion with 3430 J/m(2) of ultraviolet light-B (UV-B) as well as splenomegaly was significantly inhibited by oral administration of 0.0025% betanin. At the same dose, betanin also afforded significant protection in the mouse skin cancer model following the topical application of 390 nmol of (+/-)-(E)-4-methyl-2-[(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) in 100 microl of acetone and promoted by topical administration of 1.7 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA). In the two-stage model of hepatocarcinogenesis in mice with N-nitrosodiethylamine (DEN, 30 mg/kg) as the initiator and phenobarbital as the promoter, oral administration of 0.0025% betanin also showed a very significant inhibition of both the incidence and multiplicity of the liver tumors. These findings along with our initial reports suggest that betanin which is a regularly consumed natural product colorant is an effective cancer chemopreventive agent in mice. The most interesting observation is that the cancer chemopreventive effect was exhibited at a very low dose used in the study and thus indicating that beetroot warrants more attention for possible human applications in the control of malignancy.

Constituents of Compositae plants III. Anti-tumor promoting effects and cytotoxic activity against human cancer cell lines of triterpene diols and triols from edible chrysanthemum flowers.

Fifteen pentacyclic triterpene diols and triols, consisting of: six taraxastanes, faradiol (1), heliantriol B0 (2), heliantriol C (3), 22alpha-methoxyfaradiol (4), arnidiol (5), and faradiol alpha-epoxide (6); five oleananes, maniladiol (7), erythrodiol (8), longispinogenin (9), coflodiol (10), and heliantriol A(1) (11); two ursanes, brein (12) and uvaol (13); and two lupanes, calenduladiol (14) and heliantriol B2 (15), isolated from the non-saponifiable lipid fraction of the edible flower extract of chrysanthemum (Chrysanthemum morifolium) were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, in Raji cells as a primary screening test for anti-tumor-promoters. All of the compounds tested showed inhibitory effects against EBV-EA activation with potencies either comparable with or stronger than that of glycyrrhetic acid, a known natural anti-tumor-promoter. Evaluation of the cytotoxic activity of six compounds, 1-3 and 5-7, against human cancer cell lines revealed that compound 5 possesses a wide range of cytotoxicity, with GI50 values (concentration that yields 50% growth) of mostly less than 6 microM.

Cancer prevention by carotenoids

Various natural carotenoids have been proven to have anticarcinogenic activity. Epidemiological investigations have shown that cancer risk is inversely related to the consumption of green and yellow vegetables and fruits. As b-carotene is present in abundance in these vegetables and fruits, it has been investigated extensively as a possible cancer preventive agent. However, various carotenoids which coexist with b-carotene in vegetables and fruits also have anticarcinogenic activity, and some of these, such as a-carotene, lutein and lycopene, show a higher potency than b-carotene in suppressing experimental carcinogenesis. Thus, we have carried out more extensive studies on cancer preventive activities of natural carotenoids in foods. For example, we found that b-cryptoxanthin showed antitumor initiating activity, as well as antitumor promoting activity. It is of interest that not only carotenoids distributed in vegetables and fruits, but also animal carotenoids, such as astaxanthin, are promising as cancer preventive agents. In the present study, the cancer preventive potential of phytoene was also con®rmed. The establishment of NIH3T3 cells that produce phytoene by introducing the crtB gene provides evidence that resistance against transformation, imposed by transfection of activated H-ras oncogene, was acquired by phytoene production. Analysis of the action mechanism of these natural carotenoids is now in progress, and some interesting results have already been obtained; for example, various carotenoids were suggested to stimulate the expression of RB gene, an antioncogene.

Recent Trends in Functional Food Science and the Industry in Japan

International recognition of functional foods has resulted in the recent global development of this field, which originated in Japan. The national policy on functional foods, in terms of “foods for specified health use”, also has been developing and has motivated the food industry to produce a variety of new food items. In Japan as well as in many other countries, academic and industrial scientists have been working in collaboration for the analysis and practical applications of functional food science. Emphasis has been placed on the study of antioxidant and anticarcinogenic food factors as well as pre- and probiotics. This review pinpoints recent trends in the science and industry in this field.

Cancer chemopreventive activity of majonoside-R2 from Vietnamese ginseng, Panax vietnamensis.

In the course of our continuing search for novel cancer chemopreventive agents from natural sources, several kinds of Panax plants were screened. Consequently, the ocotillol-type saponin, majonoside-R2 (MR2), was obtained from the rhizome and root of Panax vietnamensis (Vietnamese ginseng) as an active constituent. MR2 exhibited potent anti-tumor-promoting activity on two-stage carcinogenesis test of mouse hepatic tumor using N-nitrosodiethylamine (DEN) as an initiator and phenobarbital (PB) as a promoter. Further, MR2 exhibited the remarkable inhibitory effect on two-stage carcinogenesis test of mouse skin induced by nitric oxide (NO) donor/12-O-tetradecanoylphorbol-13-acetate (TPA) or peroxynitrite/TPA.

Inhibitory effect of flavonoid derivatives on Epstein–Barr virus activation and two-stage carcinogenesis of skin tumors

To search for possible anti-tumor promoters, ten flavonoid derivatives (1-10) synthesized from morin and quercetin were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation by a short-term in vitro assay. Of these compounds, pentaallyl ethers (9, 10) showed significant inhibitory effects on EBV-EA activation induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate. Further, quercetin pentaallyl ether (10) exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Anti-tumor promoting effects of multiflorane-type triterpenoids and cytotoxic activity of karounidiol against human cancer cell lines

Forty-nine multiflorane-type triterpenoids consisting of 11 compounds isolated from the seeds of Trichosanthes kirilowii (Cucurbitaceae) and 38 of their derivatives have been evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in Raji cells as a primary screening test for anti-tumor promoters. All of the compounds tested showed an inhibitory effect against EBV-EA activation, and among which 43 were revealed to possess remarkable activity with potencies either comparable to or stronger than that of glycyrrhetic acid, a known natural anti-tumor promoter. Their structure-activity relationship is discussed. Evaluation of the cytotoxic activity of karounidiol (27) against human cancer cell lines exhibited cytotoxicity especially against a human renal cancer.

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).

MIAX: A new paradigm for modeling biomacromolecular interactions and complex formation in condensed phases

A new paradigm is proposed for modeling biomacromolecular interactions and complex formation in solution (protein–protein interactions so far in this report) that constitutes the scaffold of the automatic system MIAX (acronym for Macromolecular Interaction Assessment X). It combines in a rational way a series of computational methodologies, the goal being the prediction of the most native‐like protein complex that may be formed when two isolated (unbound) protein monomers interact in a liquid environment. The overall strategy consists of first inferring putative precomplex structures by identification of binding sites or epitopes on the proteins surfaces and a simultaneous rigid‐body docking process using geometric instances alone. Precomplex configurations are defined here as all those decoys the interfaces of which comply substantially with the inferred binding sites and whose free energy values are lower. Retaining all those precomplex configurations with low energies leads to a reasonable number of decoys for which a flexible treatment is amenable. A novel algorithm is introduced here for automatically inferring binding sites in proteins given their 3‐D structure. The procedure combines an unsupervised learning algorithm based on the self‐organizing map or Kohonen network with a 2‐D Fourier spectral analysis. To model interaction, the potential function proposed here plays a central role in the system and is constituted by empirical terms expressing well‐characterized factors influencing biomacromolecular interaction processes, essentially electrostatic, van der Waals, and hydrophobic. Each of these procedures is validated by comparing results with observed instances. Finally, the more demanding process of flexible docking is performed in MIAX embedding the potential function in a simulated annealing optimization procedure. Whereas search of the entire configuration hyperspace is a major factor precluding hitherto systems from efficiently modeling macromolecular interaction modes and complex structures, the paradigm presented here may constitute a step forward in the field because it is shown that a rational treatment of the information available from the 3‐D structure of the interacting monomers combined with conveniently selected computational techniques can assist to elude search of regions of low probability in configuration space and indeed lead to a highly efficient system oriented to solve this intriguing and fundamental biologic problem. Proteins 2002;48:696–732.

Structure–activity relationship in potentially anti-tumor promoting benzalacetone derivatives, as assayed by the Epstein–Barr virus early antigen activation

The in vitro anti-tumor promoting activities of antimutagenic benzalacetone (4-phenyl-3-buten-2-one), its monosubstituted derivatives and related compounds, cinnamaldehydes and cinnamic acids, were evaluated by determining the inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. In this short-term assay, benzalacetone, which is the basic structure of dehydrozingerone (one-half analog of curcumin) inhibited the EBV-EA activation; the IC(50) value, the molar ratio of benzalacetone to TPA needed for inhibiting 50% of positive cells activated with 32 pmol TPA, was 129. IC(50) values of 2- and 4-methoxybenzalacetones were about one-half of that of benzalacetone and the methoxy compounds were more effective than hydroxybenzalacetones. IC(50) values of chloro- and trifluoromethyl-benzalacetones were higher than that of benzalacetone, indicating that these compounds are weaker inhibitors. In addition, the position of a substituent on the benzene ring affected the inhibitory effect. In benzalacetone derivatives substituted by a hydroxy-, methoxy-, chloro- or trifluoromethyl group, the 2-substituted derivatives exhibited the strongest inhibitory effect, followed by the 3- and the 4-substituents. Cinnamic acid derivatives also decreased the inhibitory effects in the same order. In the side chain of benzalacetone, the terminal group adjacent to the carbon-carbon double bond also affected the inhibitory effect. The conversions of the methylketone to aldehyde and carboxyl groups, i.e., cinnamaldehyde and cinnamic acid, increased the inhibitory effect: the IC(50) values were about one-third of that of benzalacetone. beta-Methyl styrene, which in the side chain has no carbonyl group adjacent to the double bond, inhibited the EBV-EA activation at the concentration of about one-third of that of benzalacetone, indicating that the carbonyl group negatively affects the inhibitory effect. This agreed with the previous observation between isoeugenol and dehydrozingerone, 4-hydroxy-3-methoxy derivatives of beta-methyl styrene and benzalacetone, respectively. The mechanism of the EBV-EA activation inhibition was discussed by being compared with the inhibition of mutagenesis for which the unsaturated bonded-carbonyl system is necessary.