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

Anti-Tumor Effects of the Glycolipids Fraction from Spinach which 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 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 , , and , and mitochondrial pol with IC50 values of 44.046.2 g/ml, but had no inuence on the activity of repair-related pol . The fraction also inhibited the proliferation of human cervix carcinoma (HeLa) cells with LD50 values of 57.2 g/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 signicantly 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.

Introduction In spite of the many advances in cancer treatment, chemotherapy for solid tumors is still greatly limited by a lack of selective anti-cancer drugs and by the recurrence of drug-resistant tumors; nding a source of novel chemotherapeutics continues to be a focus of effort. Diets rich in vegetables are known to reduce cancer risk, implicating edible plants as potential sources of anti-cancer agents. Multiple organisms are known to contain at least 14 types of DNA polymerase (pol) (1), which catalyze both DNA replication and repair (1,2). Pol inhibitors could therefore be employed as anti-cancer chemotherapy agents, because they inhibit cell proliferation. Based on this idea, we have found

many new pol inhibitors over the past 10 yr, e.g., long-chain fatty acids (36), conjugated fatty acids (79), bile acids such 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, in particular food materials. Of these, sulfo-glycolipids in the class sulfoquinovosyl diacylglycerol (SQDG) from a fern (16) and an alga (17,18) are particularly potent inhibitors of eukaryotic pol. We succeeded in chemically synthesizing SQDG (1922), which was the strongest inhibitor of replicative pols such as , , and in the tested compounds (25). Therefore, this glycolipid shows promise as an agent for cancer chemotherapy. SQDG is a major glycolipid of the chloroplast membrane in plants (42). We have widely screened for the glycolipids fraction containing SQDG from common vegetables that show such inhibitory activity, and found that spinach (Spinacia oleracea L.) had the largest amount of SQDG and was the strongest pol inhibitor in the tested vegetables (43). In this report, we report that the glycolipids fraction from spinach can inhibit mammalian pol activities, mammalian cultured cell growth and in vivo solid tumor proliferation, and discuss whether 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 from Shinyu Co. Ltd. (Hiroshima, Japan). Diaion HP-20 was

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

obtained from Mitsubishi Chemical, Inc. (Tokyo, Japan). Nucleotides and chemically-synthesized DNA template-primers such as [3 H]-2 -deoxythymidine 5 -triphosphate (dTTP, 43 Ci/mmol) and poly(dA), oligo(dT)1218 were purchased from Amersham Biosciences, Inc. (Buckinghamshire, UK). The antibody for MIB-1 and its staining kit (chemMateENVISION kit) were obtained from Dako, Japan (Tokyo, Japan). All other reagents were of analytical grade and were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). DNA Polymerases Pol was puried from calf thymus by immuno-afnity column chromatography as described previously (44). Pol was puried from a recombinant plasmid expressing rat pol (45). The human pol catalytic gene was cloned into pFastBac. Histidine-tagged enzyme was expressed using the BAC-TO-BAC HT Baculovirus Expression System according to the suppliers manual (Life technologies, MD) and puried using ProBoundresin (Invitrogen Japan, Tokyo, Japan) (46). Human pols and were puried by the nuclear fractionation of human peripheral blood cancer cells (Molt-4) using the second subunit of pols - and -conjugated afnity column chromatography, respectively (47). DNA Polymerase Assay The reaction mixtures for pol and pol were described previously (3,4). Those for pol , and pols and were as described by Umeda et al. (46) and Ogawa et al. (48), respectively. For the pols, poly(dA)/oligo(dT)1218 (A/T = 2/1) and [3 H]-2 -deoxythymidine 5 -triphosphate ([3 H]-dTTP) were used as the DNA template-primer and nucleotide substrate, respectively. The glycolipids fraction was dissolved in dimethyl sulfoxide at various concentrations and sonicated for 30 S. Four microliters of each sonicated sample was mixed with 16 l of each enzyme (nal 0.05 units) in 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol, 50% glycerol and 0.1 mM EDTA, and kept at 0 C for 10 min. These inhibitor-enzyme mixtures (8 l) were added to 16 l of each enzyme standard reaction mixtures, and incubation was carried out at 37 C for 60 min. One unit of each pol activity was dened as the amount of enzyme that catalyzed the incorporation of 1 nmol of deoxyribonucleoside triphosphates into synthetic DNA template-primers at 37 C for 60 min (3,4). Cell Culture and Measurement of Cell Proliferation A human cervix carcinoma cell line, HeLa was obtained from the Health Science Research Bank (Osaka, Japan). The cells were cultured in Eagles minimum essential medium (MEM) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 g/ml) at 37 C in a humid atmosphere of 5% CO2 /95% air. For the cell proliferation assay, HeLa cells were plated at 3 105 cells into each well of 96-well microplates with various concenVol. 57, No. 2

trations of the glycolipids fraction from spinach. The compound was dissolved in phosphate-buffered saline (PBS) at a concentration of 10 mM as a stock solution. The stock solutions were diluted to the appropriate nal concentrations with growth medium just before use. Cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl2H-tetrazolium bromide) assay (49). In Vivo Assessment of Anti-tumor Assay Male BALB/c nu/nu mice, 6 wk of age (2022 g), were purchased from Japan SLC, Inc. (Shizuoka, Japan). Mice receiving standard laboratory chow and water ad libitum were acclimatized 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 after implantation, the tumor sizes in all mice were measured at 2-day intervals. Mice bearing solid tumors that had grown to 2535 mm3 in volume (tumor volume = length (width)2 0.5) at 12 days after implantation were used for the assessment of anti-tumor effect. They were divided randomly into 2 groups (n = 5/group). One of the 2 groups was a control group injected with 0.1 ml of PBS alone, and another group was injected with the glycolipids fraction from spinach dissolved in PBS at a dose of 50 mg/kg to the mice. The above administrations all took place between days 12 to 39 subsequent to implantation. All mice were injected subcutaneously 10 times at 2-day intervals with the compound and PBS alone (control). Tumor growth was measured at 2-day intervals for 27 days after implantation, and the statistics were analyzed using Students t-test. At the end of in vivo anti-tumor assay, some mice treated with the glycolipids fraction and PBS were separately examined to observe the pathohistological features of the tumors and major organs such as lung, heart, spleen, stomach, liver, pancreas, kidney, intestine, and brain. Pathological Analysis Tissues or tumors were xed with 10% formaldehyde in PBS (pH 7.2) and processed for parafn embedding. Three micrometers thick sections were stained for hematoxylineosin or appropriate antigens by immunohistochemistry. For MIB-1 staining, the antibody for MIB-1 was reacted with deparafnized sections which were antigen-retrieved citrate with a kit. Anti-MIB-1 antibody was diluted 1:100 with PBS and incubated for 30 min, washed, and incubated with an appropriate second antibody. Results Preparation of the Glycolipids Fraction from Spinach As briey described in the Introduction, we screened for and found many pol inhibitors from natural resources including food materials. Some natural glycolipids such as SQDG were found to be the strongest inhibitors of eukaryotic pols tested over the past 10 yr (1630). We found that spinach 217

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

Effects of the Glycolipids Fraction of Spinach on the Activities of Mammalian DNA Polymerases and Other Enzymes The glycolipids fraction from spinach containing major natural glycolipids such as MGDG, DGDG, and SQDG was investigated for its inhibitory effect on mammalian pols

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

(Spinacia oleracea L.) had the most SQDG in the tested vegetables (43). Initially, an effective purication method of the glycolipids fraction from spinach was established, as shown in Fig. 1. The water soluble substances were extracted from dried spinach (20 g) with 1,000 ml of warm water (60 C). The tissue cake containing fat-soluble compounds was added to 1,000 ml of warm ethanol (60 C), and the substances containing glycolipids were extracted. The ethanol extract was diluted with water to a 70% ethanol solution. The solution was subjected to Diaion HP-20 column chromatography (200 ml), a hydrophobic type of chromatography, and washed with 1,000 ml of 70% ethanol, and then eluted using 95% ethanol (1,000 ml). The 95% ethanol solution was the glycolipids fraction (23 mg). In the glycolipids fraction from spinach, 3 major compounds were analyzed by thin layer chromatography (TLC), and no other compounds were detected (data not shown). Each of these compounds was completely puried by silica gel column chromatography, and their chemical structures were determined by 1 H-, 13 C-, and DEPT (Distortionless Enhancement by Polarization Transfer) NMR spectroscopic analyses. These compounds were glycolipids such as monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (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 were detected. From fatty acid analysis by gas chromatography, the major fatty acids in MGDG were stearic acid (18:0) and oleic acid (18:1), DGDG mostly consisted of palmitic acid 218

Figure 2. Dose-response curves of the glycolipids fraction from spinach (0100 g/ml) in mammalian DNA polymerases. The enzymes used (0.05 units each) were calf thymus pol (circle), rat pol (square), human pol (triangle), human pol (diamond), and human pol (reverse-traingle). The absence 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 fraction from spinach. Dose-response curve of proliferation inhibition of a human cervix carcinoma cell line, HeLa by the compound. The assay was carried out under the conditions described in Materials and Methods with the compounds at the indicated concentrations. Survival rate was determined by the MTT assay (49). The absence of the compound was taken as 100%. Data are shown as the means SEM of 5 independent experiments.

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to . Pols , , and are representative replicative pols, pol is a repair-related pol, and pol is a mitochondrial pol (1,2). As shown in Fig. 2, the inhibitory effect on nuclear replicative pols , , and was the strongest in the tested pols, and the IC50 values were 44.1, 46.2, and 44.0 g/ml, respectively. The glycolipids fraction moderately inhibited the activity of pol , with 50% inhibition at a dose of 79.8 g/ml. The compound did not inuence the activity of pol at less than 100 g/ml. Since DGDG and MGDG had moderate or no inuence on pol activity (data

not shown), SQDG in the fraction might be an inhibitor of pols. The glycolipids fraction had no inuence at all on the activities of a higher plant, cauliower, pols I ( -like) and II ( -like), or prokaryotic pols such as the Klenow fragment of E. coli pol I, Taq pol, and T4 pol. The compound also did not inhibit the activities of other DNA-metabolic enzymes such as T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I. These results suggested that the glycolipids 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 mice bearing 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 are shown 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 Spinach on Human Cancer Cell Proliferation The glycolipids fraction, which inhibited mammalian pol activities, might be a suitable anti-cancer agent; therefore, we investigated its inuence on a human cervix carcinoma cell line, HeLa. Fig. 3 shows inhibition dose-response curves of the compound on cancer cell growth. Inhibition by the spinach glycolipids fraction was dependent on the dose, and the LD50 value was 57.2 g/ml. The inhibitory effect on human cancer cells was almost the same concentration as that on replicative pols; therefore, the inhibition of pol activity might directly affect cell proliferation in cancer cells. These results suggested that SQDG in the glycolipids fraction was able to penetrate cancer cells and reach the nucleus, inhibiting pol , , and activities.

but not in the control treatment. These results suggested that the in vivo inhibition of cell proliferation and induction of necrosis in the tumor tissue by the glycolipids fraction from spinach might be caused by the inhibition of replicative pols by the compound.

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

Effect of the Glycolipids Fraction from Spinach on In Vivo Antitumor Activity At 12 days after the implantation of HeLa cells, nude mice bearing a solid tumor were injected with the spinach glycolipids fraction (50 mg/kg) at 2-day intervals until 39 days. As shown in Fig. 4A, the compound suppressed tumor growth from 21 days as compared to the control group, and tumor volume showed a smaller increase at the ratio of 54.0% decrease at 39 days (Fig. 4B). Since SQDG containing the glycolipids fraction inhibited the activities of replicative pols, SQDG might suppress tumor activity. None of the nude mice showed any signicant loss of body weight throughout the experimental period. It was also noted that the main visceral organs, such as the liver, lung, kidney, spleen, heart, stomach, small intestine, large intestine, pancreas, and testis of all the groups showed no toxic or degenerative histological appearance (data not shown); therefore, the glycolipids fraction must be of interest as a candidate material for anti-cancer treatment. As shown in Fig. 5A and B, injected tumors formed a signicant mass in each animal. Although necrosis and hemorrhage could be detected in the PBS treatment (Fig. 5C), the glycolipids fraction and PBS tend to induce more vigorous necrosis and hemorrhage compared with PBS (Fig. 5D). In Table 1, histological ndings regarding the mass area and tumor area as examined by image analysis software also revealed that tumors with the glycolipids fraction treatment had smaller areas. The mitotic index shown in Table 2 depicts the effect of the glycolipids treatment on reducing mitosis in the tumors. This was further examined using the MIB-1 index. MIB-1 antigen provides a good indicator for cell division in situ, providing an objective indication for cell proliferation in specied tissue. Signicant decrease of the MIB-1 index was observed in tumors treated with the glycolipids fraction 220

Figure 5. Histopathological examination of tumors treated with the glycolipids fraction from spinach. Low magnication (40) appearance of tumors treated with either PBS as a control (A) or the glycolipids fraction (B). The bars show as 5 mm. At higher magnication (200), necrosis and hemorrhage appeared to be evident. The necrotic zone with hemorrhage appeared to be evident. The necrotic zone with hemorrhage tended to be more vigorous in tumors treated with the glycolipids fraction (D) compared to that of the PBS control (C). The bars show as 200 m. (Continued)

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Table 2. Histopathological Findings of Mitosis

Control Glycolipids fraction 17.80 1.92 9.65 0.31

Mice were injected with PBS (control) and the glycolipids fraction in PBS at a dose of 50 mg/kg. Mitotic index was calculated at a high magnication eld (400). One hundred independent elds were examined for the presence of mitosis, and the data are shown as the mean SEM of 5 independent experiments.

Figure 5. (Continued)

eracea L.), algae, and cyanobacteria, composed mainly of the following three glycolipids, MGDG, DGDG, and SQDG (50). MGDG and DGDG are noncharged lipids, whereas SQDG possesses a negatively charged head group. Thylakoid membranes in plant chloroplasts and cyanobacterial cells are unique in possessing photosynthetic electron transport and photophosphorylation systems for the conversion of light to chemical energy. A mutant of Chlamydomonas reinhardtii, defective in SQDG (hf-2), showed photosystem II (PSII) activity 40% lower than that of wild-type, an increase Table 1. Histopathological ndings of mass area and tumor area
Area (mm2 ) Mass area Control Glycolipids fraction 45.25 10.42 27.72 2.98 Tumor area 21.13 2.91 15.45 2.13

in sensitivity of PSII activity to 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU), and a lower growth rate (5154). In accordance with these observations, the incubation of isolated thylakoid membranes of hf-2 with SQDG in vitro reversed the lowered PSII activity; therefore, these results concluded that SQDG has the specic function of maintaining PSII properties. We found previously that spinach contains the most SQDG in the glycolipids fraction of more than 10 vegetables tested (43); therefore, the spinach glycolipids fraction could be developed as an anti-cancer functional food. However, the water and ethanol extracts from spinach (i.e., water-soluble and fat-soluble fractions, respectively, in Fig. 1) did not inhibit the activities of pols, human cancer cell growth and tumor activity, although the ethanol extract contained SQDG (data not shown); therefore, it was suggested that some compounds avoiding the SQDG bio-activity might be contained in the spinach ethanol extract. It is important to purify the glycolipids fraction containing SQDG. In this report, we found that the glycolipids fraction could be an inhibitor of mammalian pols and human cancer cell proliferation, and has anti-tumor activity in vivo. The molecular weight of SQDG containing the spinach glycolipids fraction is 800900, this fraction contains 25.5% of SQDG, and the anti-tumor activity in mice was found at 50 mg/kg; therefore, when an adult human (60 kg) eat 3 g of the fraction, SQDG are taken 15 M in the body, and this concentration of SQDG could inhibit the activity of pols. The glycolipids fraction from spinach could help to prevent cancer disease, and become a functional food with anti-cancer activity.

Acknowledgments and Notes

We are grateful to Dr. Masaharu Takemura of Tokyo University of Science (Tokyo, Japan), Dr. Akio Matsukage of Japan Womens University (Tokyo, Japan) and Dr. Kengo Sakaguchi of Tokyo University of Science (Chiba, Japan) for preparing calf pol , rat pol , and human pols and , respectively, and for valuable discussions concerning inhibitors. This work was supported in part by a Grant-in-aid for KobeGakuin University Joint Research (A) (H. Y. and Y. M.) and Cooperative Research Center of Life Sciences Project for Private Universities: 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 for Medical and Pharmaceutical Research (Japan), and the Nakashima Foundation (Japan). Address correspondence to Y. Mizushina, Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin

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

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University, Nishi-ku, Kobe, Hyogo 651-2180, Japan. Phone: +81-78974-1551 (ext. 3232). FAX: +81-78-974-5689. E-mail: mizushin@nutr. kobegakuin.ac.jp Submitted 21 June 2006; accepted in nal form 17 October 2006.

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