Previously, we observed that purified monogalactosyl diacylglycerol (MGDG), a major glycoglycerolipid from spinach, selectively inhibits the activities of mammalian replicative DNA polymerases (α, δ and ε). However, the function of MGDG following ingestion is not well-known. In the present study, spinach MGDG suppressed the proliferation of Colon26 mouse colon cancer cells with an LD50 of 24 μg/ml in vitro. γ-cyclodextrin (CD)-MGDG complex was prepared and administered orally following Colon26 mouse tumor adhesion for 26 days. It was observed that 20 mg/kg equivalent (eq.) of the CD-MGDG complex reduced tumor volume by ∼60% compared with that of the vehicle-treated controls. In immunohistochemical analysis, the CD-MGDG complex group showed a decreased number of proliferating cell nuclear antigen (PCNA)-positive cells and reduction of mitosis in the tumor cells compared with the control group. In addition, the CD-MGDG complex increased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive apoptotic cells and inhibited CD31-positive tumor blood vessel growth significantly. These results suggest that MGDG has the potential for cancer prevention and health promotion.

Penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG) has been shown by us and others to inhibit the in vivo growth of human prostate cancer (PCa) xenografts in athymic nude mice and mouse lung cancer allograft in syngenic mice without evident adverse effect on their body weight. We observed a rapid inhibition of DNA synthesis in S-phase cells in PGG-exposed cancer cells and in PGG-treated isolated nuclei. The purpose of the present study was to test the hypothesis that PGG inhibits DNA replicative synthesis through a direct inhibition of one or more DNA polymerases (pols). Using purified pols, we show that PGG exhibited a selective inhibition against the activities of B-family replicative pols (α, δ and ε) and Y-family (η, ι and κ) of bypass synthesis pols, and the inhibitory effect of PGG on pol α was the strongest with IC50 value of 13 nM. PGG also inhibited pol β, but the potency was an order of magnitude less than against pol α. PGG inhibition of pol α and κ activity was non-competitive with respect to the DNA template-primer and the dNTP substrate; whereas it inhibited pol β competitively. Docking simulation on pol β, which is the only mammalian pol with solved crystal structure, suggests several favorable interactions with the catalytic pocket/binding site for the incoming dNTP. These results support PGG as a novel inhibitor of select families of mammalian pols by distinct mechanisms, and suggest that the potent pol inhibition may contribute to its anti-cancer efficacy.

We investigated the inhibitory effect of three glycyrrhizin derivatives, such as Glycyrrhizin (compound 1), dipotassium glycyrrhizate (compound 2) and glycyrrhetinic acid (compound 3), on the activity of mammalian pols. Among these derivatives, compound 3 was the strongest inhibitor of mammalian pols α, β, κ, and λ, which belong to the B, A, Y, and X families of pols, respectively, whereas compounds 1 and 2 showed moderate inhibition. Among the these derivatives tested, compound 3 displayed strongest suppression of the production of tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in a cell-culture system using mouse macrophages RAW264.7 and peritoneal macrophages derived from mice. Moreover, compound 3 was found to inhibit the action of nuclear factor-κB (NF-κB) in engineered human embryonic kidney (HEK) 293 cells. In addition, compound 3 caused greater reduction of 12-O-tetradecanoylphorbol-13-acetate-(TPA-) induced acute inflammation in mouse ear than compounds 1 and 2. In conclusion, this study has identified compound 3, which is the aglycone of compounds 1 and 2, as a promising anti-inflammatory candidate based on mammalian pol inhibition.

The lipids extracted from rice brans were classified by thin-layer chromatography into eight fractions, and their fatty acid (FA) compositions were investigated among five different Japanese cultivars. The lipids of these rice brans comprised mainly triacylglycerols (TAG; 84.9-86.0 wt%), free FA (4.2-4.6 wt%), and phospholipids (PL; 6.5-6.7 wt%), whilst other components were also detected in minor proportions (0.2-2.1 wt%). The PL components included phosphatidyl choline (43.3-46.8 wt%) phosphatidyl ethanolamine (25.0-27.3 wt%) and phosphatidyl inositol (20.2-23.2 wt%). Comparison of the different cultivars showed, with a few exceptions, no substantial difference (P > 0.05) in FA distribution. FA distribution of TAG among the five cultivars was characterized as: unsaturated FA predominantly concentrated at the sn-2 position and saturated FA primarily occupying the sn-1 or sn-3 position in these lipids. These results suggest that the rice bran lipids may be well incorporated into our daily diet to improve nutritional value of the Japanese diet.

Previously, we reported that vitamin K3 (VK3), but not VK1 or VK2 (=MK-4), inhibits the activity of human DNA polymerase γ (pol γ). In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS). Moreover, MK-2 was found to inhibit the action of nuclear factor (NF)-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.

Regiospecific distributions of fatty acids (FA) of triacylglycerols (TAG) and phospholipids (PL) isolated from five cultivars of adzuki beans (Vigna angularis) were investigated. The lipids comprised mainly PL (72.2-73.4 wt-%) and TAG (20.6-21.9 wt-%), whilst other components were detected in minor proportions (0.1-3.4 wt-%). The principal profiles of the FA distribution in the TAG and PL were evident in the beans among the five cultivars: unsaturated FA were predominantly distributed in the sn-2 position, whilst saturated FA primarily occupied the sn-1 or the sn-3 position in the these lipids. The results would be useful information to both producers and consumers for manufacturing traditional adzuki confectionaries such as wagashi in Japan.

Acetogenins from the Annonaceous plant are a fatty acid-derived natural product. Chemically synthesized natural acetogenins, such as mucocin (compound 1), jimenezin (compound 2), muconin (compound 4), pyranicin (compound 5) and pyragonicin (compound 6) were investigated. Concomitantly, 19-epi jimenezin (compound 3), 10-epi pyragonicin (compound 7) and a γ-lactone (compound 8), which is estimated to be a biosynthetic precursor of acetogenins, were synthesized and investigated. Compounds 5 and 6 strongly inhibited, and compound 7 moderately inhibited the activities of mammalian DNA polymerases (pols), such as replicative pol α and repair/recombination-related pol β and λ, and also inhibited human DNA topoisomerase (topos) I and II activities. On the other hand, compounds 1–4 and 8 did not influence the activities of any pols and topos. Compound 5 was the strongest inhibitor of the pols and topos tested, and the IC50 values were 5.0–9.6 μM, respectively. These compounds also suppressed human cancer cell growth with almost the same tendency as the inhibition of pols and topos. Compound 5 was the strongest suppressor of the proliferation of the promyelocytic leukemia cell line, HL-60, in human cancer cell lines tested with an LD50 value of 9.4 μM, and arrested the cells at G1 phases, indicating that it blocks DNA replication by inhibiting the activity of pols rather than topos. This compound also induced cell apoptosis. The relationship between the three-dimensional molecular structure of acetogenins and these inhibitory activities is discussed. The results suggested that compound 5 is a lead compound of potentially useful cancer chemotherapy agents.

We isolated a pol inhibitor from the cultured mycelia extract of a fungal strain isolated from natural salt from a sea salt pan in Australia, which was identified as 3-O-methylfunicone by spectroscopic analyses. This compound selectively inhibited the activities of mammalian Y-family DNA polymerases (pols) (i.e., pols η, ι and κ). Among these pols, human pol κ activity was most strongly inhibited, with an IC50 value of 12.5 μM. On the other hand, the compound barely influenced the activities of the other families of mammalian pols, such as A-family (i.e., pol γ), B-family (i.e., pols α, δ and ɛ) or X-family (i.e., pols β, λ and terminal deoxynucleotidyl transferase), and showed no effect on the activities of fish pol δ, plant pols, prokaryotic pols and other DNA metabolic enzymes, such as calf primase of pol α, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase or bovine deoxyribonuclease I. This compound also suppressed the growth of two cultured human cancer cell lines, HCT116 (colon carcinoma cells) and HeLa (cervix carcinoma cells), and UV-treated HeLa cells exhibited lower clonogenic survival in the presence of inhibitor.