Effects of glycolipids from spinach on mammalian DNA polymerases.

We purified the major glycolipids in the class of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) from a green vegetable, spinach (Spinacia oleracea L.). MGDG was an inhibitor of the growth of NUGC-3 human gastric cancer cells, but DGDG and SQDG had no such cytotoxic effect. Therefore, we studied MGDG and its monoacyglycerol-form, monogalactosyl monoacylglycerol (MGMG), in detail. MGMG with one fatty acid molecule was obtained from MGDG with two fatty acid molecules by hydrolyzing with a pancreatic lipase. MGMG was also found to prevent the cancer cell growth. MGDG was a potent inhibitor of replicative DNA polymerases such as alpha, delta and epsilon. MGMG inhibited the activities of all mammalian DNA polymerases including repair-related DNA polymerase beta with IC(50) values of 8.5-36 microg/mL, and the inhibition by MGMG was stronger than that by MGDG. Both MGDG and MGMG could halt the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The relationship between the DNA polymerase inhibition and the cell growth effect by these glycolipids is discussed.

Molecular action mode of Hippospongic acid A, an inhibitor of gastrulation of starfish embryos.

Hippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp. We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A. In this study, we examined its action mode in vitro. HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as alpha, beta, delta, epsilon, eta, kappa, and lambda, in the IC(50) range of 5.9-17.6 microM, and interestingly also those of human DNA topoisomerases I and II (IC(50) = 15-25 microM). HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes. HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes. Since DNA polymerase beta is the smallest, we used it to analyze the biochemical relationship with HA-A. Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase beta, and HA-A inhibited the ssDNA binding activity. HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells. The LD(50) value was 9.5 microM, i.e. in the same range as for the enzyme inhibition. Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A. We also discussed the in vivo role of HA-A.

Inhibitory action of emulsified sulfoquinovosyl acylglycerol on mammalian DNA polymerases.

We reported previously that sulfoquinovosyl diacylglycerol and sulfoquinovosyl monoacylglycerol (SQDG/SQMG) are potent inhibitors of mammalian DNA polymerases and DNA topoisomerase II, and can be potent immunosuppressive agents and anticancer chemotherapy agents [Matsumoto, Y., Sahara, H., Fujita T., Shimozawa, K., Takenouchi, M., Torigoe, T., Hanashima, S., Yamazaki, T., Takahashi, S., Sugawara, F., et al., An Immunosuppressive Effect by Synthetic Sulfonolipids Deduced from Sulfonoquinovosyl Diacylglycerols of Sea Urchin, Transplantation 74, 261-267 (2002); Sahara, H., Hanashima, S., Yamazaki, T., Takahashi, S., Sugawara, F., Ohtani, S., Ishikawa, M., Mizushina, Y., Ohta, K., Shimozawa, K., et al., Anti-tumor Effect of Chemically Synthesized Sulfolipids Based on Sea Urchin's Natural Sulfonoquinovosylmonoacylglycerols, Jpn. J. Cancer Res. 93, 85-92 (2002)]. In those experiments, the in vivo effectiveness greatly depended on the degree of water solubility of SQDG/SQMG. In the present work, we studied the emulsification of SQDG/SQMG in terms of their use in in vivo experiments. Lipid emulsions containing SQDG/SQMG (oil-in-water emulsions) in which the particle size was smaller than 100 nm were designed and synthesized, and then the biochemical modes of emulsified SQDG/SQMG were studied in comparison with those of SQDG/SQMG solubilized by DMSO. Emulsified SQDG/SQMG are also selective mammalian DNA polymerase inhibitors and potent antineoplastic agents but do not inhibit the DNA topoisomerase II activity. The growth inhibition effect of emulsified SQMG to NUGC-3 cancer cells was twofold stronger than DMSO-soluble SQMG (69 and 151 microM, respectively). From these results, the properties of lipid emulsions containing SQDG/SQMG and their possible use in in vivo experiments including clinical use are discussed.

Selective inhibition of mammalian DNA polymerase alpha by vitamin D2 and D3.

As described previously (H. Togashi et al. Biochem Pharmacol. 1998;56:583-590), the irradiated products of provitamin D(2) (ergosterol) inhibit the activities of eukaryotic DNA polymerases. In this report, therefore, we investigated whether vitamin D and its related compounds inhibited the activities of DNA polymerases. As expected, vitamin D(2) and vitamin D(3) were found to be selective inhibitors of mammalian DNA polymerase alpha (pol alpha) with IC(50) values of 123 and 96 micro M, respectively. On the other hand, provitamin D(2), provitamin D(3), and the active form of vitamin D(3) such as 1alpha,25-dihydroxyvitamin D(3) could not influence any of the DNA polymerase activities. Interestingly, vitamin D(3)-3beta-sulfate was a much stronger pol alpha inhibitor with an IC(50) value of 7.1 micro M. Vitamin D(2), vitamin D(3), and vitamin D(3)-3beta-sulfate could prevent the growth of NUGC-3 human gastric cancer cells with LD(50) values of 133, 77, and 44 micro M, respectively, but provitamin D(2) and provitamin D(3) could not. The cells were halted at the G1 phase in the cell cycle by these compounds.

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 lanostane-type 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 (IC(50) = 4.6 microM), while the compound moderately inhibited the activities of DNA polymerases alpha, beta, gamma, delta, epsilon, eta, iota, kappa and lambda only from mammals, to similar extents. Another compound, dehydrotrametenonic acid, also showed moderate inhibitory effects against topo II (IC(50) = 37.5 microM) and weak effects against all the polymerases tested. Both compounds showed no inhibitory effect against topo I, higher plant (cauliflower) DNA polymerase I (alpha-like polymerase) or II (beta-like 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 T 7 RNA polymerase, T4 polynucleotide kinase and bovine deoxyribonuclease 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 LD(50) values were 63.6 and 38.4 microM, 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.

The inhibitory action of pyrrolidine alkaloid, 1,4-dideoxy-1,4-imino-D-ribitol, on eukaryotic DNA polymerases.

The pyrrolidine alkaloids mimicking the structures of pentose with nitrogen in the ring are known to be inhibitors of glycosidases. We report here that a compound belonging to this category is an inhibitor of eukaryotic DNA polymerases. Among the eight naturally occurring pyrrolidine alkaloids we tested, only one compound, 1,4-dideoxy-1,4-imino-D-ribitol (DRB), which was purified from the mulberry tree (Morus alba), strongly inhibited the activities of eukaryotic DNA polymerases with IC50 values of 21-35 microM, and had almost no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by DRB was competitive with respect to dNTP substrate. Whereas DNA polymerase alpha inhibition was noncompetitive with the template-primer, the inhibition of DNA polymerase beta was found to be competitive with the template-primer. The K(i) values of DNA polymerases alpha and beta for the template-primer were smaller than those for dNTP substrate. Therefore, the affinity of DRB was suggested to be higher at the template-primer binding site than at the dNTP substrate-binding site, although DRB is an analogue of deoxyribose consisting of dNTP. Computational analyses of the eight pyrrolidine alkaloids revealed a remarkable difference in the distribution of positive and negative electrostatic charges on the surface of molecules. The relationship between the structure of DRB and the inhibition of eukaryotic DNA polymerases is discussed.

Pyridoxal 5'-phosphate is a selective inhibitor in vivo of DNA polymerase alpha and epsilon.

Vitamin B(6) compounds such as pyridoxal 5(')-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), which reportedly have anti-angiogenic and anti-cancer effects, were thought to be inhibitors of some types of eukaryotic DNA polymerases. PL moderately inhibited only the activities of calf DNA polymerase alpha (pol alpha), while PN and PM had no inhibitory effects on any of the polymerases tested. On the other hand, PLP, a phosphated form of PL, was potentially a strong inhibitor of pol alpha and epsilon from phylogenetic-wide organisms including mammals, fish, insects, plants, and protists. PLP did not suppress the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as deoxyribonuclease I. For pol alpha and epsilon, PLP acted non-competitively with the DNA template-primer and competitively with the nucleotide substrate. Since PL was converted to PLP in vivo after being incorporated into human cancer cells, the anti-angiogenic and anti-cancer effects caused by PL must have been caused by the inhibition of pol alpha and epsilon activities after conversion to PLP.

A sulphoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibitor.

Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.