Mammalian Pols Research Articles

Screening of mammalian DNA polymerase and topoisomerase inhibitors from Garcinia mangostana L. and analysis of human cancer cell proliferation and apoptosis.

We purified and identified eight xanthones from mangosteen (Garcinia mangostana L.) and investigated whether these compounds inhibited the activities of mammalian DNA polymerases (Pols) and human DNA topoisomerases (Topos). β-Mangostin was the strongest inhibitor of both mammalian Pols and human Topos among the isolated xanthones, with 50% inhibitory concentration (IC50) values of 6.4-39.6 and 8.5-10 µM, respectively. Thermal transition analysis indicated that β-mangostin did not directly bind to double-stranded DNA, suggesting that this compound directly bound the enzyme protein rather than the DNA substrate. β-Mangostin showed the strongest suppression of human cervical cancer HeLa cell proliferation among the eight compounds tested, with a 50% lethal dose (LD50) of 27.2 µM. This compound halted cell cycle in S phase at 12-h treatment and induced apoptosis. These results suggest that decreased proliferation by β-mangostin may be a result of the inhibition of cellular Pols rather than Topos, and β-mangostin might be an anticancer chemotherapeutic agent.

Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites.

Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias toward deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol β did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide.

Inhibition of Mammalian DNA Polymerases and the Suppression of Inflammatory and Allergic Responses by Tyrosol from Used Activated Charcoal Waste Generated during Sake Production

The components adsorbed onto activated charcoal following the fermentation process of the Japanese rice wine "sake" have been studied with the aim of identifying suitable applications for this industrial food waste product. The absorbed materials were effectively extracted from the charcoal, and inhibited the activity of several mammalian DNA polymerases (pols). Subsequent purification of the extract afforded tyrosol [4-(2-hydroxyethyl)phenol] as the active component, which selectively inhibited the activity of 11 mammalian pols with IC50 values in the range of 34.3-46.1 μM. In contrast, this compound did not influence the activities of plant or prokaryotic pols or any of the other DNA metabolic enzymes tested. Tyrosol suppressed both anti-inflammatory and antiallergic effects in vivo, including 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, and immunoglobulin E-induced passive cutaneous anaphylactic reaction in mice. These results suggested that this byproduct formed during the sake-brewing process could be used as an anti-inflammatory and/or antiallergic agent.

Screening of Mammalian DNA Polymerase Inhibitors from Rosemary Leaves and Analysis of the Anti-inflammatory and Antiallergic Effects of the Isolated Compounds

In this study, on screening mammalian DNA polymerase (pol) inhibitors from the extracts of 20 edible plants, we found that leaves of rosemary (Rosmarinus officinalis L.) showed the strongest pol inhibition; furthermore, we isolated four components, carnosic acid, carnosol, ursolic acid, and rosmarinic acid, from an extract of the leaves. Carnosic acid inhibited the activities of all 11 mammalian pols tested. In descending order of their inhibitory effect on pol λ, which is a DNA repair/recombination pol, the four compounds ranked as follows: carnosic acid > ursolic acid > carnosol > rosmarinic acid. The inhibition of pol λ by these compounds was significantly correlated with both in vivo anti-inflammatory and antiallergic effects, including on 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, and immunoglobulin E-induced passive cutaneous anaphylactic reaction in mice. These results indicate that rosemary and its constituent, carnosic acid, are potential therapeutic food candidates for inflammatory and allergic diseases.

Formosusin A, a novel specific inhibitor of mammalian DNA polymerase β from the fungus Paecilomyces formosus

Variotin (1) and three novel compounds, formosusin A (2), B (3), and C (4), were isolated from the cultures of the fungus Paecilomyces formosus, and their structures were determined by spectroscopic analyses. Compound 2 is (6Z,8E,10E)-variotin, a new cis-olefin analog of compound 1. Compound 2 selectively inhibited the activity of mammalian DNA polymerase β (pol β) in vitro, with an IC50 of 35.6μM. By contrast, compounds 1, 3, and 4 did not influence the activity of pol β. These four compounds showed no effect on the activities of other 10 mammalian pols (i.e., pols α, γ, δ, ε, η, ι, κ, λ, and μ, and terminal deoxynucleotidyl transferase). These compounds also did not inhibit the activities of fish, insect, plant, and prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 2 could be a selective inhibitor of mammalian pol β. The compound 2-induced inhibition of rat pol β activity was competitive and non-competitive with respect to the DNA template–primer substrate and the dNTP substrate, respectively. On the basis of these results, the relationship between the three-dimensional structure and pol β inhibitory mechanism of compound 2 is discussed.

A Novel Radiosensitizer of the Functional Food Component, Monogalactosyl Diacylglycerol (MGDG) From Spinach, Enhanced the Cytotoxic Effects for the Pancreatic Cancer In Vitro and In Vivo

Monogalactosyl diacylglycerol (MGDG), a major chloroplast membrane galactolipid extracted from spinach, has been reported as a potent inhibitor of DNA replication and repair. The purpose of the current study is to test whether the MGDG might increase the cytotoxic effects of gemcitabine (GEM) or radiation therapy (IR) in vitro and in vivo by using human pancreas cancer cell lines. Several human cancer cell lines including pancreas cancer cell lines were used in the study. In vitro, DNA polymerase (pol) assay was performed, and cytotoxic effects and cell viability of MGDG and/or GEM and/or IR were evaluated by MTT assay and comet assay. Moreover, detection of apoptosis was assessed by a flow cytometer using the APO-Direct kit. In vivo, BALB/cAJcl-nu/nu mice were subjected to the experiments. Equal numbers of MIAPaCa2 cells were injected subcutaneously and assessed in size, according to 4-groups, such as PBS (sham), MGDG alone, IR alone and a combination of the same doses of MGDG and IR. In vitro, the inhibitory effects of MGDG and/or GEM on mammalian pols were observed clearly. The combination of phosphorylated GEM with MGDG significantly enhanced the inhibition of pols α and γ activities. The cell growth suppressive effects of combined GEM and MGDG treatments were determined using various timed treatments in which three human pancreatic cancer cell lines were treated with mixtures of (a) GEM and MGDG, (b) GEM → MGDG or (c) MGDG → GEM. The effects of schedules (a) and (b) were almost the same strength as MGDG alone, whereas schedule (c) showed synergistic effects on overall cytotoxicity. The synergistic effect of schedule (c) on BxPC-3, MIAPaCa2 and PANC-1 cells was 6.60, 28.7 and 13.0-fold stronger, respectively, than MGDG treatment alone. Moreover, MGDG and IR showed induction of apoptosis in MIAPaCa2 cells (control: 2.6%, MGDG: 5.2% and IR: 8.8%). Interestingly, the combination between MGDG and IR significantly increased the proportion of apoptotic cells (21.5%). In vivo studies, tumor growth inhibition was observed in the xenograft treated with either IR or MGDG alone, and the inhibitory effect was significantly increased by the combined treatment. MGDG showed cytotoxic effects and enhanced the effects of GEM or IR in vitro and in vivo. The combination, as MGDG + GEM + IR, could be a future strategy against pancreas cancer.

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.

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.

Inhibition of repair-related DNA polymerases by vitamin Ks, their related quinone derivatives and associated inflammatory activity

Vitamin Ks (VKs) are fat-soluble quinone compounds known to have various bioactivities. This review describes the inflammatory effects of VKs and their related quinone derivatives based on DNA polymerase (pol) inhibition. VK3, but not VK1 or VK2 (=MK-4), inhibited the activity of human pol γ, which is the DNA replicative pol in mitochondria. Of the intermediate compounds between VK2 and VK3 (namely MK-3, MK-2 and MK-1), 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. Among the VK3 based quinone derivatives, such as 1,4-naphthoquinone (NQ), 2-dimethyl-1,4-naphthoquinone (1,2-dimethyl-NQ), 1,4-benzoquinone (BQ), 9,10-anthraquinone (AQ) and 5,12-naphthacenequinone (NCQ), NQ was the strongest inhibitor of mammalian pols α and λ, in particular, DNA repair-related pol λ. Among the all compounds tested, NQ displayed the strongest suppression of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS) in a cell culture system using RAW264.7 mouse macrophages. NQ also suppressed the expression of pol λ protein in these cells, after LPS-treated RAW264.7 cells were stimulated to induce pol λ expression. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of NQ into mice suppressed TNF-α production in peritoneal macrophages and serum. In an in vivo colitis mouse model induced using dextran sulfate sodium (DSS), NQ markedly suppressed DSS-evoked colitis. The promising anti-inflammatory candidates based on the inhibition of DNA repair-related pols, such as pol λ, by VKs quinone derivatives, such as NQ, are 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.

Inhibitory effects of catechin derivatives on mammalian DNA polymerase and topoisomerase activities and mouse one-cell zygote development

In this study, the inhibitory activities against DNA polymerases (pols) and DNA topoisomerases (topos) by eight major green tea catechin derivatives (flavan-3-ols) were investigated. Some catechins inhibited mammalian pols (α and β) and human topos (I and II), with (-)-epigallocatechin gallate (EGCg) the strongest inhibitor of both enzyme types, showing IC(50) values of 3.8-21.5 and 2.0-20.0μM, respectively. EGCg did not affect the activities of plant (cauliflower) pol α or prokaryotic pols and showed no effect on the activities of other DNA metabolic enzymes tested. Next, a method was established for assay of mouse one-cell zygote development inhibition, the catechin derivatives screened for bioactivity, and the inhibition was assessed and their effects ranked as: EGCg>GCg>Cg>> others. In the mouse one-cell zygote assay, EGCg at 50μM increased abnormal cells and 75μM of EGCg-induced apoptosis. The observed ranking of catechin derivative inhibition effects against mouse one-cell zygote development in vivo was similar to their ranking by topo inhibition in vitro rather than by pol inhibition; therefore, topo inhibition might have been effecting zygote development inhibition. These results suggested that catechin derivatives indeed reached the nuclear DNA where topo inhibition can occur, thus causing the observed cellular effects. From these findings, this zygote development inhibition assay will be useful as an anti-pregnant agent screening.

Inhibitory effects of vitamin K3 derivatives on DNA polymerase and inflammatory activity

Previously, we reported that vitamin K₃ (menadione, 2-methyl-1,4-naphthoquinone) (compound 2) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we investigated the inhibitory effects (IEs) of vitamin K3 and its derivatives, such as 1,4-naphthoquinone (compound 1) and 1,2-dimethyl-1,4-naphthoquinone (compound 3), on the activity of mammalian pols. Among compounds 1-3 (10 µM for each), compound 1 was the strongest inhibitor of mammalian pols α and λ, which belong to the B and X pol families, respectively, whereas compound 2 was the strongest inhibitor of human pol γ, a family A pol. However, these compounds did not affect the activity of human pol κ, a family Y pol. As we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these vitamin K₃ derivatives are able to inhibit inflammatory responses. Among the three compounds tested, compound 1 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ears. In addition, in a cell culture system using RAW264.7 mouse macrophages, compound 1 displayed the strongest suppression of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS). In an in vivo mouse model of LPS-evoked acute inflammation, the intraperitoneal injection of compound 1 into mice suppressed TNF-α production in their peritoneal macrophages and serum. In an in vivo colitis mouse model induced using dextran sulfate sodium (DSS), the vitamin K₃ derivatives markedly suppressed DSS-evoked colitis. In conclusion, this study has identified several vitamin K₃ derivatives, such as compound 1, that are promising anti-inflammatory candidates.

Inhibitory effects of glycyrrhetinic Acid on DNA polymerase and inflammatory activities.

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.

A conserved arginine residue in the terminal protein domain of hepatitis B virus polymerase is critical for RNA pre-genome encapsidation

Hepadnaviruses, including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV), replicate their DNA genome through reverse transcription. Although hepadnaviral polymerase (Pol) is distantly related to retroviral reverse transcriptases, some of its features are distinct. In particular, in addition to the reverse transcriptase and RNase H domains, which are commonly encoded by retroviral reverse transcriptases, the N-terminally extended terminal protein (TP) domain confers unique features such as protein-priming capability. Importantly, the TP domain is also essential for encapsidation of the viral RNA pre-genome. To gain further insight into the TP domain, this study used clustered charged residue-to-alanine mutagenesis of HBV Pol. Of the 20 charged residues examined, only one arginine (R105) was critical for RNA encapsidation. This result contrasts with previous findings for DHBV Pol regarding the critical residue of the TP domain required for RNA binding. Firstly, R128 of DHBV Pol, which corresponds to R105 of HBV Pol, was reportedly tolerable to alanine substitution for RNA binding. Secondly, the C-terminal arginine residue of the DHBV Pol TP domain (R183) was shown to be critical for RNA binding, whereas alanine substitution of the corresponding arginine residue of the HBV Pol TP domain (R160) remained able to support RNA encapsidation. Together, these data highlight the divergence between avian and mammalian hepadnaviral Pols with respect to an arginine residue critical for RNA encapsidation.

Effects of Intermediates between Vitamins K2 and K3 on Mammalian DNA Polymerase Inhibition and Anti-Inflammatory Activity

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.

Effects of Quinone Derivatives, such as 1,4-Naphthoquinone, on DNA Polymerase Inhibition and Anti-Inflammatory Action

Previously, we reported that vitamin K(3), which consists of a quinone component, inhibits the activity of human DNA polymerase γ (pol γ). In this study, we investigated the inhibitory effects of 4 quinone derivatives (1,4-benzoquinone (BQ), 1,4-naphthoquinone (NQ), 9,10-anthraquinone (AQ) and 5,12-naphthacenequinone (NCQ)) on the activity of mammalian pols. BQ and NQ potently inhibited the activity of all the pol species: pols α, β, γ, δ, ε and λ, and NQ was a stronger pol inhibitor than BQ. Because we previously found a positive relationship between pol l inhibition and anti-inflammatory action, we examined whether these quinone derivatives could inhibit inflammatory responses. BQ and NQ caused a marked reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear, although AQ and NCQ did not. In a cell culture system using mouse macrophages, NQ displayed the strongest suppression in the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS) among the quinone derivatives tested. Moreover, NQ was found to inhibit the action of nuclear factor (NF)-κ. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of BQ and NQ to mice led to suppression of TNF-α production in serum. These anti-inflammatory responses of NQ were more potent than those of BQ. In conclusion, this study has identified several quinone derivatives, such as NQ, that are promising anti-inflammatory candidates.

Purified Canola Lutein Selectively Inhibits Specific Isoforms of Mammalian DNA Polymerases and Reduces Inflammatory Response

In the screening of DNA polymerase (pol) inhibitor, we isolated lutein, a carotenoid, from the crude (unrefined) pressed oil of canola (low erucic acid rapeseed, Brassica napus L.). Commercially prepared carotenoids such as lutein (1), zeaxanthin (2), beta-cryptoxanthin (3), astaxanthin (4), canthaxanthin (5), beta-carotene (6), lycopene (7), capsanthin (8), fucoxanthin (9) and fucoxanthinol (10), were investigated for the inhibitory activities of pols. Compounds 1, 2 and 8 exhibited strong inhibition of the activities of mammalian pols beta and lambda, which are DNA repair- and/or recombination-related pols. On the other hand, all carotenoids tested had no influence on the activity of a mammalian pol alpha, which is a DNA replicative pol. Lutein (1) was the strongest pol inhibitor of mammalian pols beta and lambda in the prepared ten carotenoids tested, but did not influence of the activities of mammalian pols alpha, gamma, delta and epsilon. The tendency for pols beta and lambda inhibition by these carotenoids showed a positive correlation with the suppression of TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation. These results suggest that cold pressed unrefined canola/rapeseed oil, or other oils with high levels of lutein and other carotenoids, may be useful for their anti-inflammatory properties.

Anti-cancer gallotannin penta- O-galloyl-beta- d-glucose is a nanomolar inhibitor of select mammalian DNA polymerases

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 ɛ) and Y-family (η, ι and κ) of bypass synthesis pols, and the inhibitory effect of PGG on pol α was the strongest with IC 50 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.

Novel Terpenoids, Trichoderonic Acids A and B Isolated fromTrichoderma virens, Are Selective Inhibitors of Family X DNA Polymerases

Trichoderonic acids A (1) and B (2), novel terpenoids, and (+)-heptelidic acid (3) isolated from cultures of a fungus, Trichoderma virens, and their structures were identified by spectroscopic analysis. These compounds selectively and competitively inhibited the activities of mammalian DNA polymerases beta, lambda (pols beta, lambda), and terminal deoxynucleotidyl transferase (TdT) in family X of pols, and compound 2 was a stronger inhibitor than compound 1 or 3. On the other hand, compounds 1-3 did not influence the activities of the other families (A-, B-, and the Y-families) of the mammalian pols tested, and showed no effect on the activities of plant pol alpha, fish pol delta, prokaryotic pol, or the other DNA metabolic enzymes tested. Compound 2 suppressed the growth of two human cancer cell lines, cervix carcinoma cells (HeLa) and breast carcinoma cells (MCF-7). The results suggest that these compounds identified inhibition among the families of mammalian pols.

.BETA.-Sitosteryl (6'-O-linoleoyl)-glucoside of Soybean (Glycine max L.) Crude Extract Inhibits Y-Family DNA Polymerases

In the screening of selective DNA polymerase (pol) inhibitors, we isolated an acylated steryl glycoside, β-sitosteryl (6'-O-linoleoyl)-glucoside (compound 1), from the waste extract of soybean (Glycine max L.) oil. This compound exhibited a marked ability to inhibit the activities of eukaryotic Y-family pols (pols η, ι and κ), which are repair-related pols. Among mammalian Y-family pols, the activity of mouse pol κ was most strongly inhibited by compound 1, with an IC(50) value of 10.2 µM. On the other hand, compound 1 had no effect on the activities of other eukaryotic pols such as A-family (pol γ), B-family (pols α, δ, and ε), or X-family (pols β, λ and terminal deoxynucleotidyl transferase) pols. In addition, compound 1 had no effect on prokaryotic pols or other DNA metabolic enzymes such as calf primase of pol α, T7 RNA polymerase, T4 polynucleotide kinase, or bovine deoxyribonuclease I. Compound 1 consists of 3 groups: β-sitosteryl (compound 2), linoleic acid (compound 3), and D-glucose (compound 4). Compound 3 inhibited the activities of all mammalian pols tested, but compounds 2 and 4 did not have any effect on the tested pols. Kinetic studies showed that the inhibition of pol κ activity by compound 1 was noncompetitive with both the DNA template-primer and nucleotide substrate, whereas compound 3-induced inhibition was competitive with the DNA template-primer and noncompetitive with the nucleotide substrate. The relationship between the structure of compound 1 and the selective inhibition of eukaryotic Y-family pols is discussed.