Takahiko Hada

Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins

Arachidonate 12-lipoxygenases of porcine and bovine leukocytes were different in substrate specificity and immunogenicity from the enzyme of bovine platelets (Arch. Biochem. Biophys. (1988) 266, 613). In order to extend the comparative studies on the two types of 12-lipoxygenase, we purified the enzyme from the cytosol of human platelets by immunoaffinity chromatography to a specific activity of about 0.3 mumol/min per mg protein at 37 degrees C. The purified enzyme was active with eicosapolyenoic acids and docosahexaenoic acid. Linoleic and linolenic acids were poor substrates in contrast to the high reactivity of the leukocyte enzymes with these octadecapolyenoic acids. The finding that the human platelet enzyme catalyzed 15-oxygenation of 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, raised a question if lipoxins were produced by incubation of the enzyme with leukotriene A4. However, the leukotriene A4 was scarcely transformed to lipoxin isomers by 12-lipoxygenases of human and bovine platelets. In sharp contrast, the porcine and bovine leukocyte enzymes converted leukotriene A4 to various lipoxin isomers by the reaction rates of 3% and 2% of the arachidonate 12-oxygenation. Thus, 12-lipoxygenases of human and bovine platelets were catalytically distinct from the porcine and bovine leukocyte enzymes in terms of their reactivities not only with linoleic and linolenic acids, but also with leukotriene A4 as lipoxin precursor.

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.

Localization of arachidonate 12-lipoxygenase in canine brain tissues.

Abstract: The cytosol fraction from a thoroughly imgated canine cerebrum was subjected to immunoaffinity chromatography using a monoclonal antibody against porcine leukocyte 12‐lipoxygenase. Arachidonate 12‐lipoxygenase eluted from the column with some retardation. The enzyme, with a specific activity of 9 nmol/min/mg of protein, converted arachidonic acid to 12(S)‐hydroperoxy‐5,8,10,14‐eicosatet‐raenoic acid. The enzyme was active not only with arachidonic acid, but also with linoleic and α‐linolenic acids. In contrast, 12‐lipoxygenase of canine platelets was almost inactive with linoleic and α‐linolenic acids, and the platelet enzyme was also distinguished from the cerebral enzyme in terms of reactivity with the anti‐12‐lipoxygenase antibody. 12‐Lipoxygenase activity was also detected in the cytosol fractions of other parts of canine brain: basal ganglia, hippocampus, cerebellum, olfactory bulb, and medulla oblongata.

Conjugated Docosahexaenoic Acid Is a Potent Inducer of Cell Cycle Arrest and Apoptosis and Inhibits Growth of Colo 201 Human Colon Cancer Cells

Abstract: The effect of conjugated docosahexaenoic acid (CDHA) on the inhibition of colon cancer cell growth was examined in the colo 201 human colon cancer cell line, and the effect was compared with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). CDHA was a more potent tumor cell growth inhibitor than DHA and EPA by colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (IC50 for 72 h: 31.6 μM, 46.8 μM, and 56.6 μM, respectively). CDHA inhibited cell cycle progression, due to accumulation of cells in G1 phase, which involved increased p21Cip1/Waf1 and decreased cyclin D1, cyclin E, and proliferating cell nuclear antigen expression; the p53 and cyclin A levels were unchanged. Induction of apoptosis was confirmed by the appearance of sub-G1 populations, and apoptosis cascade involved upregulation of the apoptosis-enhancing proteins (Bak and Bcl-ϰS) and downregulation of the apoptosis-suppressing proteins (Bcl-ϰL and Bcl-2). CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins, similar to the effects of DHA. CDHA at a dietary dose of 1.0% significantly inhibited growth of colo 201 cells transplanted in nude mice.

Arachidonate 12-lipoxygenase of rat pineal glands : catalytic properties and primary structure deduced from its cDNA

When a crude extract of rat pineal glands (the 1000 x g supernatant of a homogenate) was incubated with arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was found as a major product. The 12-lipoxygenase of rat pineal gland also reacted with linoleic and alpha-linolenic acids at 35% and 101% the rate of arachidonate 12-oxygenation, respectively. Upon Western blot analysis using polyclonal antibody against porcine leukocyte 12-lipoxygenase, the cytosol fraction of rat pineal gland showed a positive band with a molecular weight of approx. 74 kDa. A full-length cDNA for this enzyme was cloned from a cDNA library of rat pineal gland and the identity of the 12-lipoxygenase cDNA was confirmed by its expression in E. coli. The amino acid sequence of the enzyme was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75,305. The enzyme showed 72% identity of amino acid sequence with porcine leukocyte 12-lipoxygenase and 73% with bovine tracheal 12-lipoxygenase, but only 59% with human platelet 12-lipoxygenase. Taken together, the high reactivity with C-18 fatty acids, the immunoreactivity and the amino acid homology data indicate that the rat pineal 12-lipoxygenase is more closely related to leukocyte 12-lipoxygenase than to platelet 12-lipoxygenase. Upon RNA blot analysis, by far the highest content of 12-lipoxygenase mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain. The predominant presence of 12-lipoxygenase mRNA in pineal gland was confirmed by in situ hybridization of rat brain. Significant amounts of 12-lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.

Antithrombotic Effects of Odorless Garlic Powder Both in Vitro and in Vivo

Antithrombotic activities of odorless garlic powder were demonstrated in blood fibrinolytic and coagulation systems. Though the odorless garlic preparation did not influence tissue-type plasminogen activator (t-PA) or its inhibitor secretions from human umbilical vein endothelial cells, it enhanced plasmin generation by t-PA on fibrin film and in chromogenic assays by 1.8-fold and 8.7-fold respectively. The coagulation system was considerably reduced after the administration of the garlic in a rat in situ loop model, indicating that increased levels of thrombin-antithrombin III (TAT) complex in the control group were significantly reduced to normal (sham) in the garlic group (p<0.05), which was associated with decreasing tendencies towards prolonged or increased values of coagulation parameters in the control group. These findings suggest that odorless garlic not only activates fibrinolytic activity by accelerating t-PA-mediated plasminogen activation, but also suppresses the coagulation system by downregulating thrombin formation, suggesting a beneficial role in preventing pathological thrombus formation in such cardiovascular disorders.

Conjugated docosahexaenoic acid suppresses KPL-1 human breast cancer cell growth in vitro and in vivo: potential mechanisms of action

IntroductionThe present study was conducted to examine the effect of conjugated docosahexaenoic acid (CDHA) on cell growth, cell cycle progression, mode of cell death, and expression of cell cycle regulatory and/or apoptosis-related proteins in KPL-1 human breast cancer cell line. This effect of CDHA was compared with that of docosahexaenoic acid (DHA).MethodsKPL-1 cell growth was assessed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; cell cycle progression and mode of cell death were examined by flow cytometry; and levels of expression of p53, p21Cip1/Waf1, cyclin D1, Bax, and Bcl-2 proteins were examined by Western blotting analysis. In vivo tumor growth was examined by injecting KPL-1 cells subcutaneously into the area of the right thoracic mammary fat pad of female athymic mice fed a CDHA diet.ResultsCDHA inhibited KPL-1 cells more effectively than did DHA (50% inhibitory concentration for 72 hours: 97 μmol/l and 270 μmol/l, respectively). With both CDHA and DHA growth inhibition was due to apoptosis, as indicated by the appearance of a sub-G1 fraction. The apoptosis cascade involved downregulation of Bcl-2 protein; Bax expression was unchanged. Cell cycle progression was due to G0/G1 arrest, which involved increased expression of p53 and p21Cip1/Waf1, and decreased expression of cyclin D1. CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins in a manner similar to that of parent DHA. In the athymic mouse system 1.0% dietary CDHA, but not 0.2%, significantly suppressed growth of KPL-1 tumor cells; CDHA tended to decrease regional lymph node metastasis in a dose dependent manner.ConclusionCDHA inhibited growth of KPL-1 human breast cancer cells in vitro more effectively than did DHA. The mechanisms of action involved modulation of apoptosis cascade and cell cycle progression. Dietary CDHA at 1.0% suppressed KPL-1 cell growth in the athymic mouse system.

Antiproliferative activity of guava leaf extract via inhibition of prostaglandin endoperoxide H synthase isoforms

Prostaglandin endoperoxide H synthase (PGHS) is a key enzyme for the synthesis of prostaglandins (PGs) which play important roles in inflammation and carcinogenesis. Because the extract from Psidium guajava is known to have a variety of beneficial effects on our body including the anti-inflammatory, antioxidative and antiproliferative activities, we investigated whether the extract inhibited the catalytic activity of the two PGHS isoforms using linoleic acid as an alternative substrate. The guava leaf extract inhibited the cyclooxygenase reaction of recombinant human PGHS-1 and PGHS-2 as assessed by conversion of linoleic acid to 9- and 13-hydroxyoctadecadienoic acids (HODEs). The guava leaf extract also inhibited the PG hydroperoxidase activity of PGHS-1, which was not affected by nonsteroidal anti-inflammatory drugs (NSAIDs). Quercetin which was one of the major components not only inhibited the cyclooxygenase activity of both isoforms but also partially inhibited the PG hydroperoxidase activity. Overexpression of human PGHS-1 and PGHS-2 in the human colon carcinoma cells increased the DNA synthesis rate as compared with mock-transfected cells which did not express any isoforms. The guava leaf extract not only inhibited the PGE(2) synthesis but also suppressed the DNA synthesis rate in the PGHS-1- and PGHS-2-expressing cells to the same level as mock-transfected cells. These results demonstrate the antiproliferative activity of the guava leaf extract which is at least in part caused by inhibition of the catalytic activity of PGHS isoforms.

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.

Inhibitory effect on replicative DNA polymerases, human cancer cell proliferation, and in vivo anti-tumor activity by glycolipids from spinach.

We succeeded in purifying a major glycolipids fraction (i.e., Fraction-II) in the class of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) from spinach using hydrophobic column chromatography. Fraction-II inhibited the activities of replicative DNA polymerases (pols) such as alpha, delta and epsilon, and mitochondrial pol gamma with IC(50) values of 43-79 microg/ml, but had no influence on the activity of repair-related pols beta and lambda. MGDG, DGDG, SQDG were purified from Fraction-II of spinach using silica gel column chromatography, and SQDG was the strongest inhibitor of mammalian pols in the three glycolipids. Therefore, SQDG and its related compounds were chemically synthesized, and the sulfate group and fatty acid moiety of the compound were suggested to be important for pol inhibition. These glycolipids showed no effect even on the activities of plant pols, prokaryotic pols and other DNA metabolic enzymes such as T4 polynucleotide kinase, T7 RNA polymerase and deoxyribonuclease I. Fraction-II also inhibited the proliferation of human cervix carcinoma (HeLa) cells with LD(50) values of 57 microg/ml, and could halt the cell cycle at the G1-phase, and subsequently induced severe apoptosis. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, Fraction-II was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that tumor necrosis with hemorrhage was significantly enhanced with Fraction-II in vivo. The spinach Fraction-II containing SQDG might be a potent anti-tumor compound, and may be a healthy food substance with anti-tumor activity.