Oral administration of monogalactosyl diacylglycerol from spinach inhibits colon tumor growth in mice.

Previously, we observed that purified monogalactosyl diacylglycerol (MGDG), a major glycoglycerolipid from spinach, selectively inhibits the activities of mammalian replicative DNA polymerases (α, δ and ε). However, the function of MGDG following ingestion is not well-known. In the present study, spinach MGDG suppressed the proliferation of Colon26 mouse colon cancer cells with an LD(50) of 24 µg/ml in vitro. γ-cyclodextrin (CD)-MGDG complex was prepared and administered orally following Colon26 mouse tumor adhesion for 26 days. It was observed that 20 mg/kg equivalent (eq.) of the CD-MGDG complex reduced tumor volume by ∼60% compared with that of the vehicle-treated controls. In immunohistochemical analysis, the CD-MGDG complex group showed a decreased number of proliferating cell nuclear antigen (PCNA)-positive cells and reduction of mitosis in the tumor cells compared with the control group. In addition, the CD-MGDG complex increased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive apoptotic cells and inhibited CD31-positive tumor blood vessel growth significantly. These results suggest that MGDG has the potential for cancer prevention and health promotion.

In vivo antitumor effect of liposomes with sialyl Lewis X including monogalactosyl diacylglycerol, a replicative DNA polymerase inhibitor, from spinach.

The glycoglycerolipid monogalactosyl diacylglycerol (MGDG) isolated from spinach selectively inhibits the activities of replicative DNA polymerase species and suppresses the growth of human cancer cell lines, while not affecting normal human cells. Liposomes, carrying surface-bound sialyl Lewis X (SLX) and containing MGDG (SLX-Lipo-MGDG) and the fluorescent dye Cy5.5, were administered intravenously to mice bearing HT-29 human colon adenocarcinoma tumors and liposome distribution observed using fluorescence imaging equipment in vivo. In an in vivo antitumor assay on nude mice bearing HT-29 solid tumors, SLX-Lipo-MGDG was shown to be a stronger and more promising suppressor of solid tumors than MGDG alone. These results suggest that spinach MGDG could be developed into an anticancer compound, SLX-Lipo-MGDG could serve as an effective clinical anticancer drug and that these liposomes may be useful tools as the basis for active targeting drug delivery systems.

Protective effects of glycoglycerolipids extracted from spinach on 5-fluorouracil induced intestinal mucosal injury.

Glycoglycerolipids are mostly found in plants, however the beneficial effects of the glycoglycerolipids on mammalian body have not been understood. In this study, we investigated the effects of glycolipid extracts from spinach, which highly contained glycoglycerolipids, on mucosal injury induced by 5-fluorouracil (5-FU) in rats. Preadministration of glycolipid extracts from spinach (20 mg/kg body weight) prevented villous atrophy, misaligned crypts, and increased inflammatory cytokines in rat jejunum treated with 5-FU (300 mg/kg body weight) compared with the extracts from soybean. The glycolipid extracts from spinach highly contained monogalactosyl-diacylglycerol (MGDG) and diglactosyl-diacylglycerol (DGDG). In Caco-2 cells, MGDG and DGDG inhibited the production of reactive oxygen species induced by phorbol ester. We concluded that glycolipid extracts from spinach has anti-oxidative and anti-inflammatory effects, and the extract may be useful for prevention of drug-induced mucosal injury and other inflammatory diseases. Tokushima

Enhancement of human cancer cell radiosensitivity by conjugated eicosapentaenoic acid - a mammalian DNA polymerase inhibitor.

We previously found that conjugated eicosapentaenoic acid (cEPA) selectively inhibited the activities of mammalian DNA polymerases (pols), and suppressed human cancer cell growth. The aim of the present study was to evaluate the efficacy of concurrent radiation with cEPA in a human colon carcinoma cell line, HCT 116. Furthermore, we examined the most effective timing of irradiation. The post-irradiation addition of cEPA significantly enhanced HCT116 cell radiosensitivity by decreasing the expression of pols beta, delta and epsilon, increasing damaged DNA, such as DNA double-strand breaks, inhibiting clonogenic survival, and inducing apoptosis. However, cells treated by pre-irradiation addition of cEPA did not influence radiosensitive survival and radiation-induced apoptosis. cEPA inhibited the activities of pols needed for DNA repair, thereby DNA damage must be augmented by cEPA and irradiation. These results suggested that the combination of inhibitors of DNA repair-related pols/radiation could be an effective anticancer therapy.

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.

Structure and activity relationship of monogalactosyl diacylglycerols, which selectively inhibited in vitro mammalian replicative DNA polymerase activity and human cancer cell growth.

The glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), containing two alpha-linolenic acids (C18:3), was isolated from bitter melon as a potent and selective inhibitor of mammalian DNA polymerase (pol) species such as pols alpha, gamma, delta, and epsilon with IC(50) values of 17.6-37.2muM. This MGDG also suppressed the growth of six human cancer cell lines, although normal human cell lines were not affected. This compound (i.e., MGDG-C18:3-C18:3) was a stronger inhibitor than both MGDG-C18:1-C18:0 and MGDG-C18:0-C18:0. In this study, we discussed the structure-function relationship in the selective inhibition of mammalian replicative pols and human cancer cell proliferation by MGDGs.

Cell cycle arrest triggered by conjugated eicosapentaenoic acid occurs through several mechanisms including G1 checkpoint activation by induced RPA and ATR expression.

BACKGROUND: Conjugated eicosapentaenoic acid (cEPA) containing conjugated double bonds, which is prepared by alkaline treatment of eicosapentaenoic acid (EPA), selectively inhibited the activities of both mammalian DNA polymerases (pols) and human DNA topoisomerases (topos). METHODS: Human colon carcinoma cell line, HCT116, was cultured and performed drug and small interfering RNA (siRNA) treatment, flow cytometry analysis, BrdU incorporation analysis, and western blot analysis. RESULTS: The levels of bromodeoxyuridine (BrdU) incorporation labeling during DNA synthesis were decreased in time- and dose-dependent manners in HCT116 cells, treated with cEPA. The level of chromatin association of RPA70, a subunit of the single-stranded DNA (ssDNA)-binding protein, was increased following cEPA exposure, suggesting that the replication forks were stalled in response to inhibition of replicative pol activity by cEPA in the cells. cEPA also induced the activation of ataxia-telangiectasia and Rad3-related (ATR) protein in HCT116 cells, and activated the G1 checkpoint pathway in the cells, which was down-regulated by a small interfering RNA (siRNA) against ATR protein. Moreover, caffeine, a known ATR kinase inhibitor, abrogated the cEPA-induced G1 checkpoint in HCT116 cells. GENERAL SIGNIFICANCE: cEPA could inhibit the activity of replicative pols, such as pols alpha, delta and epsilon, affect the DNA replication fork including ssDNA, and then activate the G1 checkpoint pathway by the induction of RPA and ATR expression levels in cancer cells.

Anti-tumor effect of orally administered spinach glycolipid fraction on implanted cancer cells, colon-26, in mice.

We succeeded in purifying a major glycolipid fraction from a green vegetable, spinach. This fraction consists mainly of three glycolipids: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG). In a previous study, we found that the glycolipid fraction inhibited DNA polymerase activity, cancer cell growth and tumor growth with subcutaneous injection. We aimed to clarify oral administration of the glycolipid fraction, suppressing colon adenocarcinoma (colon-26) tumor growth in mice. A tumor graft study showed that oral administration of 20 mg/kg glycolipid fraction for 2 weeks induced a 56.1% decrease in the solid tumor volume (P < 0.05) without any side-effects, such as loss of body weight or major organ failure, in mice. The glycolipid fraction induced the suppression of colon-26 tumor growth with inhibition of angiogenesis and the expression of cell proliferation marker proteins such as Ki-67, proliferating cell nuclear antigen (PCNA), and Cyclin E in the tumor tissue. These results suggest that the orally administered glycolipid fraction from spinach could suppress colon tumor growth in mice by inhibiting the activities of neovascularization and cancer cellular proliferation in tumor tissue.

The inhibitory action of SQDG (sulfoquinovosyl diacylglycerol) from spinach on Cdt1-geminin interaction.

A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural materials containing food components, and found that a glycolipid, sulfoquinovosyl diacylglycerol (SQDG), from spinach can inhibit Cdt1-geminin interaction in vitro, with 50% inhibition observed at concentrations of 1.79mug/ml. Other major glycolipids, such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach, had no influence. Surface plasmon resonance analysis demonstrated that SQDG bound selectively to Cdt1, but did not interact with geminin. Using three-dimensional computer modeling analysis, SQDG was considered to interact with the geminin interaction interface on Cdt1, and the sulfate group of SQDG was assumed to make hydrogen bonds with the residue of Arg346 of Cdt1. These data could help to further understanding of the structure and function of Cdt1. In addition, SQDG could be a clue to developing more appropriate inhibitors of Cdt1-geminin interactions.

Potent antagonists of the CCR2b receptor. Part 3: SAR of the (R)-3-aminopyrrolidine series.

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional assays [Compound 71 CCR2b Binding IC(50) 3.2 nM; MCP-1-Induced Chemotaxis IC(50) 0.83 nM; Ca(2+) Flux IC(50) 7.5 nM].

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 ß, 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 ß-like region of family X pols.

Anti-tumor effects of the glycolipids fraction from spinach which inhibited DNA polymerase activity.

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 oleraceaL.). This glycolipids fraction inhibited the activities of replicative DNA polymerases (pols) such as alpha, delta, and epsilon, and mitochondrial pol gamma with IC50 values of 44.0-46.2 microg/ml, but had no influence on the activity of repair-related pol beta. The fraction also inhibited the proliferation of human cervix carcinoma (HeLa) cells with LD50 values of 57.2 microg/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.

Mechanism of cell cycle arrest and apoptosis induction by conjugated eicosapentaenoic acid, which is a mammalian DNA polymerase and topoisomerase inhibitor.

Conjugated eicosapentaenoic acid (cEPA) selectively inhibited the activities of mammalian DNA polymerases (pols) and human DNA topoisomerases (topos). cEPA inhibited the cell growth of two human leukemia cell lines, NALM-6, which is a p53-wild type, and HL-60, which is a p53-null mutant, with LD50 values of 37.5 and 12.5 microM, respectively. In both cell lines, cEPA arrested in the G1 phase, and increased cyclin E protein levels, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. DNA replication-related proteins such as RPA70, ATR and phosphorylated-Chk1/2 were increased by cEPA treatment in the cell lines, suggesting that cEPA led to DNA replication fork stress inhibiting the activities of pols and topos, and the ATR-dependent DNA damage response pathway could respond to the inhibitor of DNA replication. The compound induced cell apoptosis through both p53-dependent and p53-independent pathways in cell lines NALM-6 and HL-60, respectively. These results suggested the therapeutic potential of cEPA as a leading anti-cancer compound that inhibited activities of pols and topos.

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.

Extracts of Momordica charantia suppress postprandial hyperglycemia in rats.

Momordica charantia (bitter melon) is commonly known as vegetable insulin, but the mechanisms underlying its hypoglycemic effect remain unclear. To address this issue, the effects of bitter melon extracts on postprandial glycemic responses have been investigated in rats. An aqueous extract (AE), methanol fraction (MF) and methanol insoluble fraction (MIF) were prepared from bitter melon. An oral sucrose tolerance test revealed that administration of AE, MF or MIF each significantly suppressed plasma glucose levels at 30 min as compared with the control. In addition, the plasma insulin level at 30 min was also significantly lower after MF administration than in the control in the oral sucrose tolerance test. By contrast, these effects of bitter melon extracts were not observed in the oral glucose tolerance test. In terms of mechanism, bitter melon extracts dose-dependently inhibited the sucrase activity of intestinal mucosa with IC(50) values of 8.3, 3.7 and 12.0 mg/mL for AE, MF and MIF, respectively. The fraction with a molecular weight of less than 1,300 (LT 1,300) obtained from MF inhibited the sucrase activity most strongly in an uncompetitive manner with an IC(50) value of 2.6 mg/mL. Taken together, these results demonstrated that bitter melon suppressed postprandial hyperglycemia by inhibition of alpha-glucosidase activity and that the most beneficial component is present in the LT 1,300 fraction obtained from MF.

Inhibitory action of C22-fatty acids on DNA polymerases and DNA topoisomerases.

We reported previously that unsaturated linear-chain fatty acids of the cis-configuration with a C18-hydrocarbon chain such as linoleic acid (cis-9, 12-octadecadienoic acid, C18:2) could potently inhibit the activity of mammalian DNA polymerases (Biochim Biophys Acta 1308: 256-262, 1996). In this study, we investigated the inhibitory effects of cis-type C22-fatty acids including cis-7,10,13,16,19-docosapentaenoic acid (DPA, C22:5) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA, C22:6) on mammalian DNA polymerases and human DNA topoisomerases. Cis-13,16-docosadienoic acid (C22:2) was the strongest inhibitor of both DNA polymerases and topoisomerases of all C22-fatty acids tested. The inhibitory tendency by the fatty acids on DNA polymerases was the same as that of DNA topoisomerases, and the second strongest inhibitor was cis-13,16,19-docosatrienoic acid (C22:3). The energy-minimized three-dimensional structures of the fatty acids were calculated and it was found that a length of 19-21 Angstrom and width of more than 7 Angstrom in C22-fatty acid structure were important for enzyme inhibition. The three-dimensional structure of the active site of both DNA polymerases and topoisomerases must have a pocket to join C22:2, and this pocket was 19.41 Angstrom long and 9.58 Angstrom wide.

Inhibitory effect of monogalactosyldiacylglycerol, extracted from spinach using supercritical CO2, on mammalian DNA polymerase activity.

We investigated the effective extraction of monogalactosyldiacylglycerol (MGDG) from dried spinach (Spinacia oleracea) using supercritical fluid carbon dioxide (SC-CO(2)) with a modifier/entrainer. The yield of MGDG in the SC-CO(2) extract was not influenced by increasing temperature at a constant pressure, although the total extract yield was decreased. The total extract yield and MGDG yield in the extract from commercially purchased spinach (unknown subspecies), were greatly influenced by lower pressure. In a modifier (i.e., ethanol) concentration range of 2.5-20%, both the extract and MGDG yield increased as the ethanol concentration rose. The highest total extract yield (69.5 mg/g of spinach) and a good MGDG yield (16.3 mg/g of spinach) were obtained at 80 degrees C, 25 MPa, and 20% ethanol. The highest MGDG concentration (76.0% in the extract) was obtained at 80 degrees C, 25 MPa, and 2.5% ethanol, although the total extract yield under these conditions was low (5.2 mg/g of spinach). The optimal conditions for the extraction of MGDG were 80 degrees C, 20 MPa, and 10% ethanol. Of the 11 subspecies of spinach tested under these conditions, "Ujyou" had the highest concentration of MGDG. The total extract yield and MGDG concentration of Ujyou were 20.4 mg of the extract/g of spinach and 70.5%, respectively. The concentration of MGDG was higher in the SC-CO(2) extract than in the extract obtained using solvents such as methanol and n-hexane. The extract of Ujyou, which was the optimal subspecies for the extraction of MGDG, inhibited the activity of calf DNA polymerase alpha with IC(50) values of 145 microg/mL but was not effective against DNA polymerase beta.

Inhibitory effect of sulfoquinovosyl diacylglycerol on prokaryotic DNA polymerase I activity and cell growth of Escherichia coli.

We isolated the glycolipids fraction from spinach (Spinacia oleracea L.) and found that the fraction inhibited the activities of prokaryotic DNA polymerase I from Escherichia coli (E. coli) and cell growth of E. coli. The fraction contained mainly three glycolipids, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), and purified SQDG inhibited these activities, however, purified MGDG and DGDG had no influence. In the tested strains of E. coli, SQDG inhibited the cell proliferation of the JM109 strain. It could be considered that a SQDG-containing thylakoid membrane in plant chloroplasts might have anti-bacterial activity.

Inhibitory effects of glycolipids fraction from spinach on mammalian DNA polymerase activity and human cancer cell proliferation.

We succeeded in purifying the fraction containing the major glycolipids in monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol (SQDG) from dried vegetables. This glycolipids fraction was an inhibitor of DNA polymerase alpha (pol alpha) in vitro and also the proliferation of human cancer cells. In this study, eight common vegetables were investigated in terms of the glycolipids fraction, the amounts of major glycolipids, mammalian DNA polymerase inhibitory activity and antiproliferative activity toward human cancer cells. Green tea possessed the largest amount of glycolipids overall. Spinach contained the largest amount of SQDG, followed by parsley, green onion, chive, sweet pepper, green tea, carrot and garlic. Spinach had the strongest inhibitory effect on pol alpha activity and human cancer cell proliferation. A significant correlation was found between SQDG content and inhibition of DNA polymerase. Therefore, the inhibition of pol alpha activity by SQDG may lead to cell growth suppression. Of the six subspecies of spinach (Spinacia oleracea) tested, "Anna" had the largest amount of SQDG, strongest inhibitory activity toward DNA polymerase and greatest effect on human cancer cell proliferation. Based on these results, the glycolipids fraction from spinach is potentially a source of food material for a novel anticancer activity.