Yuko Yonezawa

DNA polymerase γ inhibition by vitamin K3 induces mitochondria-mediated cytotoxicity in human cancer cells

Among the vitamin K (VK) compounds, VK3 exhibits distinct cytotoxic activity in cancer cells and is thought to affect redox cycling; however, the underlying mechanisms remain unclear. Here we demonstrate that VK3 selectively inhibits DNA polymerase (pol) γ, the key enzyme responsible for mitochondrial DNA replication and repair. VK3 at 30 µM inhibited pol γ by more than 80%, caused impairment of mitochondrial DNA replication and repair, and induced a significant increase in reactive oxygen species (ROS), leading to apoptosis. At a lower concentration (3 µM), VK3 did not cause a significant increase in ROS, but was able to effectively inhibit cell proliferation, which could be reversed by supplementing glycolytic substrates. The cytotoxic action of VK3 was independent of p53 tumor suppressor gene status. Interestingly, VK3 only inhibited pol γ but did not affect other pol including human pol α, pol β, pol δ, and pol ɛ. VK1 and VK2 exhibited no inhibitory effect on any of the pol tested. These data together suggest that the inhibition of pol γ by VK3 is relatively specific, and that this compound seems to exert its anticancer activity by two possible mechanisms in a concentration‐dependent manner: (1) induction of ROS‐mediated cell death at high concentrations; and (2) inhibition of cell proliferation at lower concentrations likely through the suppression of mitochondrial respiratory function. These findings may explain various cytotoxic actions induced by VK3, and may pave the way for the further use of VK3. (Cancer Sci 2008; 99: 1040–1048)

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.

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.

Diallyl sulfides: Selective inhibitors of family X DNA polymerases from garlic (Allium sativum L.)

Diallyl sulfides, organosulfur compounds isolated from garlic (Allium sativum L.), selectively inhibit the activities of mammalian family X DNA polymerases (pols), such as pol β, pol λ and terminal deoxynucleotidyl transferase (TdT), in vitro. The purified fraction (i.e., Sample-A) consisted of diallyl trisulfide, diallyl tetrasulfide and diallyl pentasulfide (molecular ratio: 5.3:3:1). Commercially purchased diallyl sulfides also inhibited the activities of family X pols, and the order of their effect was as follows: Sample-A>diallyl trisulfide>diallyl disulfide>diallyl monosulfide, suggesting that the number of sulfur atoms in the compounds might play an important structural role in enzyme inhibition. The suppression of human cancer cell (promyelocytic leukaemia cell line, HL-60) growth had the same tendency as the inhibition of pol X family among the compounds. Diallyl sulfides were suggested to bind to the pol β-like region of family X pols.

Inhibitory effect of coenzyme Q1 on eukaryotic DNA polymerase γ and DNA topoisomerase II activities on the growth of a human cancer cell line

Coenzyme Q (CoQ) is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ having shorter isoprenoid chains, especially CoQ1 and CoQ2, selectively inhibited the in vitro activity of eukaryotic DNA polymerase (pol) γ, which is a mitochondrial pol. These compounds did not influence the activities of nuclear DNA replicative pols such as α, δ and ɛ, and nuclear DNA repair‐related pols such as β, η, ι, κ and λ. CoQ also inhibited DNA topoisomerase II (topo II) activity, although the enzymatic characteristics, including modes of action, amino acid sequences and three‐dimensional structures, were markedly different from those of pol γ. These compounds did not inhibit the activities of procaryotic pols such as Escherichia coli pol I, and other DNA metabolic enzymes such as human immunodeficiency virus reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. CoQ1, which has the shortest isoprenoid chains, had the strongest inhibitory effect on pol γ and topo II activities among CoQ1–CoQ10, with 50% inhibitory concentration (IC50) values of 12.2 and 15.5 µM, respectively. CoQ1 could prevent the growth of human promyelocytic leukemia cells, HL‐60, and the 50% lethal dose (LD50) value was 14.0 µM. The cells were halted at S phase and G1 phase in the cell cycle, and suppressed mitochondrial proliferation. From these results, the relationship between the inhibition of pol γ/topo II and cancer cell growth by CoQ is discussed. (Cancer Sci 2006; 97: 716–723)

Inhibitory action of conjugated C18-fatty acids on DNA polymerases and DNA topoisomerases

We reported previously that unsaturated linear-chain FA of the cis-configuration with a C18-hydrocarbon chain such as linoleic acid (18∶2Δ9c, 12c) could potently inhibit the activities of mammalian DNA polymerases and DNA topoisomerases, but their saturated forms could not. There are chemically two classes of unsaturated FA, normal and conjugated, but only the conjugated forms show potent antitumor activity. In this report, we study the inhibitory effects of chemically synthesized conjugated C18-FA on mammalian DNA polymerases and DNA topoisomerases as compared with normal unsaturated FA. The conjugated α-eleostearic acid (18∶3Δ9c, 11t, 13t) was the strongest of all the FA tested. For the inhibition, the conjugated form is crucially important. The energy-minimized 3-D structures of the FA were calculated, and both a length of less than 20 Å and a width of 8.13–9.24 Å in the C18-FA structure were found to be important for enzyme inhibition. The 3-D structure of the active site of both DNA polymerases and topoisomerases must have had a pocket to join α-eleostearic acid, and this pocket was 12.03 Å long and 9.24 Å wide.