Isoko Kuriyama

A novel DNA topoisomerase inhibitor: dehydroebriconic acid, one of the lanostane-type triterpene acids from Poria cocos

Traditional Chinese medicinal plants are a treasure house for screening novel inhibitors of DNA polymerases and DNA topoisomerases from mammals; in the present study, nine lanostanetype triterpene acids were found in sclerotium of Poria cocos. Among the nine compounds, only dehydroebriconic acid could potently inhibit DNA topoisomerase II (topo II) activity (IC50=4.6 μM), while the compound moderately inhibited the activities of DNA polymerases α, β, γ, δ, ɛ, η, iota;, κ and λ only from mammals, to similar extents. Another compound, dehydrotrametenonic acid, also showed moderate inhibitory effects against topo II (IC50=37.5 μM) and weak effects against all the polymerases tested. Both compounds showed no inhibitory effect against topo I, higher plant (cauliflower) DNA polymerase I (α‐like polymerase) or II (βlike polymerase), calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type‐1 reverse transcriptase, prokaryotic DNA polymerases such as the Klenow fragment of E. coli DNA polymerase I, Taq DNA polymerase and T4 DNA polymerase, or DNA metabolic enzymes such as T7 RNA polymerase, T4 polynucleotide kinase and bovine deoxyribonu‐clease I. These findings suggest that dehydroebriconic acid and dehydrotrametenonic acid should be designated as topo II‐preferential inhibitors, although they also moderately inhibited all the mammalian DNA polymerases tested. Both dehydrotrametenonic acid and dehydroebriconic acid could prevent the growth of human gastric cancer cells, and their LD50 values were 63.6 and 38.4 μM, respectively. The cells were halted at the G1 phase in the cell cycle. The relation between the structure of triterpene acids and their inhibitory activities is discussed.

Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase lambda.

We previously reported that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were a selective inhibitor of DNA polymerase λ (pol λ) in vitro. The purpose of this study was to investigate the molecular structural relationship of curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol λ (full‐length, i.e. intact pol λ including the BRCA1 C‐ terminal [BRCT] domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound 13) was the strongest pol λ inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 µm. The compound did not influence the activities of replicative pols such as α, δ, and ɛ. It had no effect on pol β activity either, although the three‐dimensional structure of pol β is thought to be highly similar to that of pol λ. Compound 13 did not inhibit the activity of the C‐terminal catalytic domain of pol λ including the pol β‐like core, in which the BRCT motif was deleted from its N‐terminal region. MALDI‐TOF MS analysis demonstrated that compound 13 bound selectively to the N‐terminal domain of pol λ, but did not bind to the C‐terminal region. Based on these results, the pol λ‐inhibitory mechanism of compound 13 is discussed.

Pinophilins A and B, Inhibitors of Mammalian A-, B-, and Y-Family DNA Polymerases and Human Cancer Cell Proliferation

Pinophilins A (1) and B (2), new hydrogenated azaphilones, and Sch 725680 (3) were isolated from cultures of a fungus (Penicillium pinophilum Hedgcok) derived from a seaweed, and their structures were determined using spectroscopic analyses. These compounds selectively inhibited the activities of mammalian DNA polymerases (pols), A (pol γ), B (pols α, δ, and ε), and Y (pols η, ι, and κ) families, but did not influence the activities of the four X-family pols (pols β, λ, μ, and terminal deoxynucleotidyl transferase). Compound 1 was the strongest inhibitor, with IC₅₀ values of 48.6 to 55.6 μM. Kinetic analysis showed that compound 1 is a noncompetitive inhibitor of both pol α and κ activities with the DNA template-primer substrate, and a competitive inhibitor with the nucleotide substrate. In contrast, compounds 1-3 showed no effect on the activities of plant and prokaryotic pols or any other DNA metabolic enzymes tested. The compounds suppressed cell proliferation and growth in five human cancer cell lines, but had no effect on the viability of normal human cell lines.

Inhibitory effects of a major soy isoflavone, genistein, on human DNA topoisomerase II activity and cancer cell proliferation

The inhibitory activity of 3 soy isoflavones (daidzein, genistein and glycitein) and their glycosides (daidzin, genistin and glycitin) on mammalian DNA polymerases (pols) and topoisomerases (topos) was investigated. Of the compounds tested, only genistein selectively inhibited human topo II activity and had an IC50 value of 37.5 µM. These isoflavones had no effect on the activity of human topo I; mammalian pols α, β, γ and κ; or on any other DNA metabolic enzyme tested. Thermal transition analysis indicated that genistein did not influence the direct binding to double-stranded DNA. Genistein prevented the proliferation of HCT116 human colon carcinoma cells with an LD50 of 94.0 µM and it halted the cell cycle in G2/M phase. These results suggest that decreases in cell proliferation due to genistein may result from the inhibition of cellular topo II and that genistein, a major soy isoflavone, may be an anticancer food component. The relationship between the structures and these bioactivities of soy isoflavones is discussed.

Anti-Tumor Effects of the Glycolipids Fraction from Spinach which Inhibited DNA Polymerase Activity

Abstract: We succeeded in purifying the fraction of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG) containing the major glycolipids from a green vegetable, spinach (Spinacia oleracea L.). This glycolipids fraction inhibited the activities of replicative DNA polymerases (pols) such as α, δ, and ϵ, and mitochondrial pol γ with IC50 values of 44.0–46.2 μg/ml, but had no influence on the activity of repair-related pol β. The fraction also inhibited the proliferation of human cervix carcinoma (HeLa) cells with LD50 values of 57.2 μg/ml. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, the fraction was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that tumor necrosis with hemorrhage was significantly enhanced with the glycolipids fraction in vivo. The spinach glycolipids fraction might be a potent anti-tumor compound, and this fraction may be a healthy food substance with anti-tumor activity.

Lipid components, fatty acid distributions of triacylglycerols and phospholipids in rice brans

Endogenous tocochromanols in extracted lipids from rice brans of the five cultivars were determined by high-performance liquid chromatography, and were investigated in relation to the fatty acid (FA) distribution of triacylglycerols (TAG) and phospholipids (PL). The dominant tocols were α-tocopherol and γ-tocotrienol, followed by α-tocotrienol and with much smaller amounts of γ-tocopherol and δ-tocotrienol. The lipids of these rice brans comprised mainly TAG (80.6-86.0wt.%), free FA (4.2-9.0wt.%), and phospholipids (5.5-6.7wt.%), whilst other components were also detected in minor proportions (0.2-2.1wt.%). The PL components included phosphatidyl choline (31.8-46.8wt.%), phosphatidyl ethanolamine (25.0-38.9wt.%) and phosphatidyl inositol (20.2-23.2wt.%). Comparison of these different cultivars showed, with a few exceptions, no significant differences (P>0.05) in FA distribution. FA distribution of TAG among the five cultivars was evident in the rice brans: unsaturated FA were predominantly concentrated at the sn-2 position and saturated FA primarily occupying the sn-1 or sn-3 position. These results suggest that the tocopherol content, lipid component, and FA distribution in rice brans are not dependent on the cultivation areas during the growing season.

Inhibitory effects of α-mangostin on mammalian DNA polymerase, topoisomerase, and human cancer cell proliferation.

We found that the ethanol extract of mangosteen (Garcinia mangostana L.) fruit rind had a strong inhibitory effect on mammalian DNA polymerase (pol) activity and isolated α-mangostin as a potent pol inhibitor from the extract. In this study, the inhibitory activities against mammalian pols by α-mangostin and its related five compounds, 3-isomangostin, xanthone, 9,10-anthraquinone, 9-anthracenecarboxylic acid, and anthracene, were investigated. α-Mangostin was the most potent inhibitor of the mammalian pol species among the tested compounds, with IC₅₀ values of 14.8-25.6 μM. This compound also inhibited human DNA topoisomerases (topos) I and II activities with IC₅₀ values of 15.0 and 7.5 μM, respectively, but did not inhibit the activities of other DNA metabolic enzymes tested. α-Mangostin also did not directly bind to double-stranded DNA as determined by thermal transition analysis. α-Mangostin was found to suppress human colon HCT116 carcinoma cell proliferation with an LC₅₀ of 18.5 μM, inhibit the activity of cellular topos, halt cell cycle in the G2/M phase, and induce apoptosis. These results suggest that decreased proliferation by α-mangostin may be a result of the inhibition of cellular topos rather than pols, and α-mangostin might be an anticancer chemotherapeutic agent.

Effects of Essential Oils from Herbal Plants and Citrus Fruits on DNA Polymerase Inhibitory, Cancer Cell Growth Inhibitory, Antiallergic, and Antioxidant Activities

In this study, the biological activity of 20 essential oils (EOs) from herbal plants and citrus fruits were investigated in terms of mammalian DNA polymerase (pol) inhibitory activity, cancer cell (human colon carcinoma, HCT116) growth inhibitory activity, antiallergic activity, as anti-β-hexosaminidase release activity in rat basophilic leukemia RBL-2H3 cells treated with calcium ionophore A23187, and antioxidant activity by a lipophilic-oxygen radical absorbance capacity method. These EOs showed patterns of inhibition of pol α, a DNA replicative pol, similar to their cancer cell growth inhibitory activity, and their inhibitory activity on pol λ, a DNA repair/recombination pol, by the EOs showed correlation with anti-β-hexosaminidase release activity. Among these EOs, chamomile (Matricaria chamomilla L.) was the strongest inhibitor of pols α and λ and showed significant effects on both cancer cell growth and mast cell degranulation. On the basis of these results, chamomile EO can be recommended as a potentially useful, bioactive candidate for therapeutic applications.

β-Sitosterol-3-O-β-D-glucopyranoside : A eukaryotic DNA polymerase λ inhibitor

Beta-sitosterol-3-O-beta-D-glucopyranoside (compound 1), a steroidal glycoside isolated from onion (Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta and epsilon, but also showed no effect even on the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of compound 1 such as beta-sitosterol (compound 2) and D-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol lambda inhibition. The inhibitory effect of compound 1 on both intact pol lambda (i.e. residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133-575, del-1 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 microM, respectively. The compound 1-induced inhibition of del-1 pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.

Inhibitory effect of novel somatostatin peptide analogues on human cancer cell growth based on the selective inhibition of DNA polymerase β

The present study was designed to investigate the anticancer activity of novel nine small peptides (compounds 1-9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, compounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1-naphthylamide), 5 (Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1-naphthylamide) containing tyrosine (Tyr) but no carboxyl groups, selectively inhibited the activity of rat pol β, which is a DNA repair-related pol. Compounds 3-6 strongly inhibited the growth of human colon carcinoma HCT116 p53(+/+) cells. The influence of compounds 1-9 on HCT116 p53(-/-) cell growth was similar to that observed for HCT116 p53(+/+) cells. These results suggest that the cancer cell growth suppression induced by these compounds might be related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol β and cancer cell growth among the nine compounds tested. This compound specifically inhibited rat pol β activity, but had no effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sulfonate (MMS) treatment synergistically suppressed HCT116 p53(-/-) cell growth compared with MMS alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results, the influence of compound 4, a specific pol β inhibitor, on the relationship between DNA repair and cancer cell growth is discussed.