NUTRITION AND CANCER, 57(2), 216223Copyright C 2007, Lawrence Erlbaum Associates, Inc.

Anti-Tumor Effects of the Glycolipids Fraction from Spinachwhich Inhibited DNA Polymerase Activity

Naoki Maeda, Yasuo Kokai, Seiji Ohtani, Hiroeki Sahara, Takahiko Hada, Chisato Ishimaru,Isoko Kuriyama, Yuko Yonezawa, Hiroshi Iijima, Hiromi Yoshida, Noriyuki Sato,

and Yoshiyuki Mizushina

Abstract: We succeeded in purifying the fraction of mono-galactosyl diacylglycerol (MGDG), digalactosyl diacylglyc-erol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG)containing the major glycolipids from a green vegetable,spinach (Spinacia oleracea L.). This glycolipids fraction in-hibited the activities of replicative DNA polymerases (pols)such as , , and , and mitochondrial pol with IC50 valuesof 44.046.2 g/ml, but had no influence on the activity ofrepair-related pol . The fraction also inhibited the prolif-eration of human cervix carcinoma (HeLa) cells with LD50values of 57.2 g/ml. In an in vivo anti-tumor assay onnude mice bearing solid tumors of HeLa cells, the fractionwas shown to be a promising suppressor of solid tumors.Histopathological examination revealed that tumor necrosiswith hemorrhage was significantly enhanced with the glycol-ipids fraction in vivo. The spinach glycolipids fraction mightbe a potent anti-tumor compound, and this fraction may bea healthy food substance with anti-tumor activity.

Introduction

In spite of the many advances in cancer treatment,chemotherapy for solid tumors is still greatly limited by alack of selective anti-cancer drugs and by the recurrence ofdrug-resistant tumors; finding a source of novel chemother-apeutics continues to be a focus of effort. Diets rich in veg-etables are known to reduce cancer risk, implicating edibleplants as potential sources of anti-cancer agents.

Multiple organisms are known to contain at least 14 typesof DNA polymerase (pol) (1), which catalyze both DNAreplication and repair (1,2). Pol inhibitors could therefore beemployed as anti-cancer chemotherapy agents, because theyinhibit cell proliferation. Based on this idea, we have found

N. Maeda, C. Ishimaru, I. Kuriyama, Y. Yonezawa, H. Iijima, H. Yoshida, and Y. Mizushina are affiliated with the Laboratory of Food & NutritionalSciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan. Y. Kokai and S. Ohtani are affiliated with theBiomedical Research Center Laboratory of Biomedical Engineering, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan.H. Sahara is affiliated with the Marine Biomedical Institute, Sapporo Medical University School of Medicine, Oshidomari, Rishirifujij, Hokkaido 097-0101,Japan. T. Hada is affiliated with the Hada Giken Co. Ltd., Yamaguchi-shi, Yamaguchi 753-0047, Japan. H. Yoshida and Y. Mizushina are affiliated with theCooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan. N. Sato is affiliated with the Department ofPathology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan.

many new pol inhibitors over the past 10 yr, e.g., long-chainfatty acids (36), conjugated fatty acids (79), bile acidssuch as lithocholic acid (1012), steroidal glycosides (13,14),steviol derivatives (15), sulfo-glycolipids (1630), catechins(3133), curcumin (3437), vitamin A-related compounds(38), vitamin B6 compounds (39), vitamin D2 and D3 (40),and vitamin E homologs (41), from natural compounds, inparticular food materials.

Of these, sulfo-glycolipids in the class sulfoquinovosyldiacylglycerol (SQDG) from a fern (16) and an alga (17,18)are particularly potent inhibitors of eukaryotic pol. We suc-ceeded in chemically synthesizing SQDG (1922), whichwas the strongest inhibitor of replicative pols such as , ,and in the tested compounds (25). Therefore, this glycolipidshows promise as an agent for cancer chemotherapy.

SQDG is a major glycolipid of the chloroplast membranein plants (42). We have widely screened for the glycolipidsfraction containing SQDG from common vegetables thatshow such inhibitory activity, and found that spinach (Spina-cia oleracea L.) had the largest amount of SQDG and was thestrongest pol inhibitor in the tested vegetables (43). In thisreport, we report that the glycolipids fraction from spinachcan inhibit mammalian pol activities, mammalian culturedcell growth and in vivo solid tumor proliferation, and discusswhether the glycolipids fraction could help to prevent cancer,and be a functional food with anti-cancer activity.

Materials and Methods

Materials

Dried spinach (Spinacia oleracea L.) was obtained fromShinyu Co. Ltd. (Hiroshima, Japan). Diaion HP-20 was

obtained from Mitsubishi Chemical, Inc. (Tokyo, Japan). Nu-cleotides and chemically-synthesized DNA template-primerssuch as [3H]-2-deoxythymidine 5-triphosphate (dTTP, 43Ci/mmol) and poly(dA), oligo(dT)1218 were purchased fromAmersham Biosciences, Inc. (Buckinghamshire, UK). Theantibody for MIB-1 and its staining kit (chemMateENVI-SION kit) were obtained from Dako, Japan (Tokyo, Japan).All other reagents were of analytical grade and were pur-chased from Nacalai Tesque, Inc. (Kyoto, Japan).

DNA Polymerases

Pol was purified from calf thymus by immuno-affinitycolumn chromatography as described previously (44). Pol was purified from a recombinant plasmid expressing ratpol (45). The human pol catalytic gene was cloned intopFastBac. Histidine-tagged enzyme was expressed using theBAC-TO-BAC HT Baculovirus Expression System accord-ing to the suppliers manual (Life technologies, MD) and pu-rified using ProBoundresin (Invitrogen Japan, Tokyo, Japan)(46). Human pols and were purified by the nuclear frac-tionation of human peripheral blood cancer cells (Molt-4)using the second subunit of pols - and -conjugated affinitycolumn chromatography, respectively (47).

DNA Polymerase Assay

The reaction mixtures for pol and pol were describedpreviously (3,4). Those for pol , and pols and were asdescribed by Umeda et al. (46) and Ogawa et al. (48), respec-tively. For the pols, poly(dA)/oligo(dT)1218 (A/T = 2/1)and [3H]-2-deoxythymidine 5-triphosphate ([3H]-dTTP)were used as the DNA template-primer and nucleotide sub-strate, respectively. The glycolipids fraction was dissolved indimethyl sulfoxide at various concentrations and sonicatedfor 30 S. Four microliters of each sonicated sample wasmixed with 16 l of each enzyme (final 0.05 units) in 50 mMTris-HCl (pH 7.5) containing 1 mM dithiothreitol, 50% glyc-erol and 0.1 mM EDTA, and kept at 0C for 10 min. Theseinhibitor-enzyme mixtures (8 l) were added to 16 l ofeach enzyme standard reaction mixtures, and incubation wascarried out at 37C for 60 min. One unit of each pol ac-tivity was defined as the amount of enzyme that catalyzedthe incorporation of 1 nmol of deoxyribonucleoside triphos-phates into synthetic DNA template-primers at 37C for60 min (3,4).

Cell Culture and Measurement of Cell Proliferation

A human cervix carcinoma cell line, HeLa was obtainedfrom the Health Science Research Bank (Osaka, Japan). Thecells were cultured in Eagles minimum essential medium(MEM) supplemented with 10% fetal bovine serum, peni-cillin (100 units/ml), and streptomycin (100 g/ml) at 37Cin a humid atmosphere of 5% CO2/95% air. For the cellproliferation assay, HeLa cells were plated at 3 105 cellsinto each well of 96-well microplates with various concen-

trations of the glycolipids fraction from spinach. The com-pound was dissolved in phosphate-buffered saline (PBS) ata concentration of 10 mM as a stock solution. The stock so-lutions were diluted to the appropriate final concentrationswith growth medium just before use. Cell viability was deter-mined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay (49).

In Vivo Assessment of Anti-tumor Assay

Male BALB/c nu/nu mice, 6 wk of age (2022 g), werepurchased from Japan SLC, Inc. (Shizuoka, Japan). Mice re-ceiving standard laboratory chow and water ad libitum wereacclimatized for 1 wk before the cancer cells implantation.For in vivo experiments, HeLa cells (1 106 cells/mouse)were subcutaneously inoculated nude mice. At 12 days afterimplantation, the tumor sizes in all mice were measured at2-day intervals. Mice bearing solid tumors that had grown to2535 mm3 in volume (tumor volume = length (width)2 0.5) at 12 days after implantation were used for the assess-ment of anti-tumor effect. They were divided randomly into2 groups (n = 5/group). One of the 2 groups was a controlgroup injected with 0.1 ml of PBS alone, and another groupwas injected with the glycolipids fraction from spinach dis-solved in PBS at a dose of 50 mg/kg to the mice. The aboveadministrations all took place between days 12 to 39 subse-quent to implantation. All mice were injected subcutaneously10 times at 2-day intervals with the compound and PBS alone(control). Tumor growth was measured at 2-day intervals for27 days after implantation, and the statistics were analyzedusing Students t-test. At the end of in vivo anti-tumor as-say, some mice treated with the glycolipids fraction and PBSwere separately examined to observe the pathohistologicalfeatures of the tumors and major organs such as lung, heart,spleen, stomach, liver, pancreas, kidney, intestine, and brain.

Pathological Analysis

Tissues or tumors were fixed with 10% formaldehyde inPBS (pH 7.2) and processed for paraffin embedding. Threemicrometers thick sections were stained for hematoxylin-eosin or appropriate antigens by immunohistochemistry. ForMIB-1 staining, the antibody for MIB-1 was reacted withdeparaffinized sections which were antigen-retrieved citratewith a kit. Anti-MIB-1 antibody was diluted 1:100 with PBSand incubated for 30 min, washed, and incubated with anappropriate second antibody.

Results

Preparation of the Glycolipids Fraction from Spinach

As briefly described in the Introduction, we screenedfor and found many pol inhibitors from natural resourcesincluding food materials. Some natural glycolipids such asSQDG were found to be the strongest inhibitors of eukaryoticpols tested over the past 10 yr (1630). We found that spinach

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Figure 1. The method of purifying the glycolipids fraction from driedspinach (Spinacia oleracea L.).

(Spinacia oleracea L.) had the most SQDG in the testedvegetables (43).

Initially, an effective purification method of the glycol-ipids fraction from spinach was established, as shown inFig. 1. The water soluble substances were extracted fromdried spinach (20 g) with 1,000 ml of warm water (60C).The tissue cake containing fat-soluble compounds was addedto 1,000 ml of warm ethanol (60C), and the substances con-taining glycolipids were extracted. The ethanol extract wasdiluted with water to a 70% ethanol solution. The solutionwas subjected to Diaion HP-20 column chromatography (200ml), a hydrophobic type of chromatography, and washed with1,000 ml of 70% ethanol, and then eluted using 95% ethanol(1,000 ml). The 95% ethanol solution was the glycolipidsfraction (23 mg).

In the glycolipids fraction from spinach, 3 major com-pounds were analyzed by thin layer chromatography (TLC),and no other compounds were detected (data not shown).Each of these compounds was completely purified by silicagel column chromatography, and their chemical structureswere determined by 1H-, 13C-, and DEPT (DistortionlessEnhancement by Polarization Transfer) NMR spectroscopicanalyses. These compounds were glycolipids such as mono-galactosyl diacylglycerol (MGDG), digalactosyl diacylglyc-erol (DGDG), and SQDG. The weight percents of MGDG,DGDG, and SQDG in the glycolipids fraction were 72.0%,2.8%, and 25.2%, respectively, and no other glycolipids weredetected. From fatty acid analysis by gas chromatography,the major fatty acids in MGDG were stearic acid (18:0) andoleic acid (18:1), DGDG mostly consisted of palmitic acid

(16:0) and oleic acid (18:1), and SQDG mostly consisted ofpalmitic acid (16:0) and linolenic acid (18:3) (43).

Effects of the Glycolipids Fraction of Spinachon the Activities of Mammalian DNA Polymerasesand Other Enzymes

The glycolipids fraction from spinach containing majornatural glycolipids such as MGDG, DGDG, and SQDG wasinvestigated for its inhibitory effect on mammalian pols

Figure 2. Dose-response curves of the glycolipids fraction from spinach(0100 g/ml) in mammalian DNA polymerases. The enzymes used (0.05units each) were calf thymus pol (circle), rat pol (square), human pol (triangle), human pol (diamond), and human pol (reverse-traingle). Theabsence of the compound was taken as 100%. Data are shown as the means SEM of 3 independent experiments.

Figure 3. Human cancer cell growth inhibition by the glycolipids fractionfrom spinach. Dose-response curve of proliferation inhibition of a humancervix carcinoma cell line, HeLa by the compound. The assay was carriedout under the conditions described in Materials and Methods with thecompounds at the indicated concentrations. Survival rate was determinedby the MTT assay (49). The absence of the compound was taken as 100%.Data are shown as the means SEM of 5 independent experiments.

218 Nutrition and Cancer 2007

to . Pols , , and are representative replicative pols,pol is a repair-related pol, and pol is a mitochon-drial pol (1,2). As shown in Fig. 2, the inhibitory effecton nuclear replicative pols , , and was the strongest inthe tested pols, and the IC50 values were 44.1, 46.2, and44.0 g/ml, respectively. The glycolipids fraction moder-ately inhibited the activity of pol , with 50% inhibition ata dose of 79.8 g/ml. The compound did not influence theactivity of pol at less than 100 g/ml. Since DGDG andMGDG had moderate or no influence on pol activity (data

not shown), SQDG in the fraction might be an inhibitor ofpols.

The glycolipids fraction had no influence at all on theactivities of a higher plant, cauliflower, pols I (-like) and II(-like), or prokaryotic pols such as the Klenow fragment ofE. coli pol I, Taq pol, and T4 pol. The compound also didnot inhibit the activities of other DNA-metabolic enzymessuch as T7 RNA polymerase, T4 polynucleotide kinase, andbovine deoxyribonuclease I. These results suggested that theglycolipids fraction from spinach could selectively inhibit

Figure 4. In vivo study of the anti-tumor effects of the glycolipids fraction from spinach. (a) The inhibitory effect on tumor volume of nude mice. Nude micebearing HeLa solid tumors were injected with PBS as a control group (open square) and the glycolipids fraction (closed circle) at a dose of 50 mg/kg. Data areshown as the means SEM of 5 independent animals. (b) A photograph of nude mice injected with PBS only (right) and 50 mg/kg of the glycolipids fraction(left) bearing HeLa solid tumors at 39 days after.

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the activity of mammalian pols, especially replicative , ,and -types.

Effects of the Glycolipids Fraction of Spinachon Human Cancer Cell Proliferation

The glycolipids fraction, which inhibited mammalian polactivities, might be a suitable anti-cancer agent; therefore,we investigated its influence on a human cervix carcinomacell line, HeLa. Fig. 3 shows inhibition dose-response curvesof the compound on cancer cell growth. Inhibition by thespinach glycolipids fraction was dependent on the dose, andthe LD50 value was 57.2 g/ml. The inhibitory effect onhuman cancer cells was almost the same concentration as thaton replicative pols; therefore, the inhibition of pol activitymight directly affect cell proliferation in cancer cells. Theseresults suggested that SQDG in the glycolipids fraction wasable to penetrate cancer cells and reach the nucleus, inhibitingpol , , and activities.

Effect of the Glycolipids Fraction from Spinachon In Vivo Antitumor Activity

At 12 days after the implantation of HeLa cells, nudemice bearing a solid tumor were injected with the spinachglycolipids fraction (50 mg/kg) at 2-day intervals until39 days. As shown in Fig. 4A, the compound suppressed tu-mor growth from 21 days as compared to the control group,and tumor volume showed a smaller increase at the ratio of54.0% decrease at 39 days (Fig. 4B). Since SQDG containingthe glycolipids fraction inhibited the activities of replicativepols, SQDG might suppress tumor activity. None of the nudemice showed any significant loss of body weight through-out the experimental period. It was also noted that the mainvisceral organs, such as the liver, lung, kidney, spleen, heart,stomach, small intestine, large intestine, pancreas, and testisof all the groups showed no toxic or degenerative histologicalappearance (data not shown); therefore, the glycolipids frac-tion must be of interest as a candidate material for anti-cancertreatment.

As shown in Fig. 5A and B, injected tumors formed asignificant mass in each animal. Although necrosis and hem-orrhage could be detected in the PBS treatment (Fig. 5C), theglycolipids fraction and PBS tend to induce more vigorousnecrosis and hemorrhage compared with PBS (Fig. 5D). InTable 1, histological findings regarding the mass area andtumor area as examined by image analysis software also re-vealed that tumors with the glycolipids fraction treatment hadsmaller areas. The mitotic index shown in Table 2 depicts theeffect of the glycolipids treatment on reducing mitosis in thetumors. This was further examined using the MIB-1 index.MIB-1 antigen provides a good indicator for cell division insitu, providing an objective indication for cell proliferationin specified tissue. Significant decrease of the MIB-1 indexwas observed in tumors treated with the glycolipids fraction

but not in the control treatment. These results suggested thatthe in vivo inhibition of cell proliferation and induction ofnecrosis in the tumor tissue by the glycolipids fraction fromspinach might be caused by the inhibition of replicative polsby the compound.

Discussion

The lipid composition of thylakoid membranes is highlycoserved among higher plants such as spinach (Spinacia ol-

Figure 5. Histopathological examination of tumors treated with the glycol-ipids fraction from spinach. Low magnification (40) appearance of tumorstreated with either PBS as a control (A) or the glycolipids fraction (B). Thebars show as 5 mm. At higher magnification (200), necrosis and hemor-rhage appeared to be evident. The necrotic zone with hemorrhage appearedto be evident. The necrotic zone with hemorrhage tended to be more vigor-ous in tumors treated with the glycolipids fraction (D) compared to that ofthe PBS control (C). The bars show as 200 m. (Continued)

220 Nutrition and Cancer 2007

Figure 5. (Continued)

eracea L.), algae, and cyanobacteria, composed mainly ofthe following three glycolipids, MGDG, DGDG, and SQDG(50). MGDG and DGDG are noncharged lipids, whereasSQDG possesses a negatively charged head group. Thy-lakoid membranes in plant chloroplasts and cyanobacterialcells are unique in possessing photosynthetic electron trans-port and photophosphorylation systems for the conversion oflight to chemical energy. A mutant of Chlamydomonas rein-hardtii, defective in SQDG (hf-2), showed photosystem II(PSII) activity 40% lower than that of wild-type, an increase

Table 1. Histopathological findings of mass area and tumorarea

Area (mm2)Mass area Tumor area

Control 45.25 10.42 21.13 2.91Glycolipids fraction 27.72 2.98 15.45 2.13

Mice were injected with either PBS (control) or the glycolipids fractionin PBS at a dose of 50 mg/kg. Mass area and tumor area were evaluatedwith image scanning software and showed as mm2. Data are shown as themean SEM of 5 independent experiments.

Table 2. Histopathological Findings of Mitosis

Control 17.80 1.92Glycolipids fraction 9.65 0.31

Mice were injected with PBS (control) and the glycolipids fraction in PBS at adose of 50 mg/kg. Mitotic index was calculated at a high magnification field(400). One hundred independent fields were examined for the presenceof mitosis, and the data are shown as the mean SEM of 5 independentexperiments.

in sensitivity of PSII activity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a lower growth rate (5154). Inaccordance with these observations, the incubation of iso-lated thylakoid membranes of hf-2 with SQDG in vitro re-versed the lowered PSII activity; therefore, these results con-cluded that SQDG has the specific function of maintainingPSII properties.

We found previously that spinach contains the mostSQDG in the glycolipids fraction of more than 10 vegetablestested (43); therefore, the spinach glycolipids fraction couldbe developed as an anti-cancer functional food. However, thewater and ethanol extracts from spinach (i.e., water-solubleand fat-soluble fractions, respectively, in Fig. 1) did not in-hibit the activities of pols, human cancer cell growth andtumor activity, although the ethanol extract contained SQDG(data not shown); therefore, it was suggested that some com-pounds avoiding the SQDG bio-activity might be containedin the spinach ethanol extract. It is important to purify the gly-colipids fraction containing SQDG. In this report, we foundthat the glycolipids fraction could be an inhibitor of mam-malian pols and human cancer cell proliferation, and hasanti-tumor activity in vivo. The molecular weight of SQDGcontaining the spinach glycolipids fraction is 800900, thisfraction contains 25.5% of SQDG, and the anti-tumor activ-ity in mice was found at 50 mg/kg; therefore, when an adulthuman (60 kg) eat 3 g of the fraction, SQDG are taken 15 Min the body, and this concentration of SQDG could inhibit theactivity of pols. The glycolipids fraction from spinach couldhelp to prevent cancer disease, and become a functional foodwith anti-cancer activity.

Acknowledgments and Notes

We are grateful to Dr. Masaharu Takemura of Tokyo University ofScience (Tokyo, Japan), Dr. Akio Matsukage of Japan Womens Univer-sity (Tokyo, Japan) and Dr. Kengo Sakaguchi of Tokyo University ofScience (Chiba, Japan) for preparing calf pol , rat pol , and humanpols and , respectively, and for valuable discussions concerning in-hibitors. This work was supported in part by a Grant-in-aid for Kobe-Gakuin University Joint Research (A) (H. Y. and Y. M.) and Coop-erative Research Center of Life Sciences Project for Private Universi-ties: matching fund subsidy from MEXT (Ministry of Education, Culture,Sports, Science and Technology), 2006-2010 (H. Y. and Y. M.). Y. M.acknowledges Grants-in-aid from the Mochida Memorial Foundation forMedical and Pharmaceutical Research (Japan), and the Nakashima Founda-tion (Japan). Address correspondence to Y. Mizushina, Laboratory of Food& Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin

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University, Nishi-ku, Kobe, Hyogo 651-2180, Japan. Phone: +81-78-974-1551 (ext. 3232). FAX: +81-78-974-5689. E-mail: mizushin@nutr.kobegakuin.ac.jp

Submitted 21 June 2006; accepted in final form 17 October 2006.

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