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Chapter 33
Inhibition of Carcinogenesis by Tea Aqueous NonDialyzates Fractionated from Crude Tea Extracts 1
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Yoshiyuki Nakamura , Mamoru Isemura , Sumio Hayakawa , Kouichi Saeki , Yu Koyama , Takashi Kimura , Masanori Kuroyanagi , Tadataka Noro , Kyoji Yoshino , and Toshihiro Tsuneyoshi 2
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1School of Pharmaceutical Sciences and Graduate University of Food and Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan 3Hiroshima Prefectural University, Syobara, Hiroshima 727-0023, Japan Numazu College of Technology, Numazu, Shizuoka 410-8501, Japan Shizuoka Institute of Science and Technology, Fukuroi, Shizuoka 437-8555, Japan 4
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High molecular weight tea fractions TNDs (tea non -dialysates) were found to induce apoptosis in mouse JB6 cells, human leukemia U937 cells, stomach cancer M K N - 4 5 cells, and colon cancer WiDr cells. Black tea non-dialysate caused the decrease in the expression of mRNAs for caspase 1 and TNF-α and the increase in m R N A expression for several cell surface proteins related to apoptosis in U937 cells. Peroral administration of green tea non-dialyzate (0.05 % in water) resulted in the decrease in the polyp number i n the stomach and colon of APC knockout mice and the increase i n the apoptosis index in the small tumors. These data suggest that drinking of tea is beneficial for prevention o f cancer of the digestive tract on the basis of apoptosis-inducing activities of its constituents.
© 2003 American Chemical Society Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Many animal studies have shown that tea and tea components have anticancer activities (1, 2). Green tea and black tea catechin compounds such as epigallocatechin gallate (EGCG) and theaflavin have been investigated most intensively to reveal molecular basis for their anti-tumor activities (3-8). However, tea infusion contains many compounds other than catechins and we have been interested in water-soluble high molecular weight fractions of tea extracts. We have previously reported that the green tea non-dialyzate (GTND), a green tea high molecular weight fraction, could inhibit carcinogenesis induced by chemical carcinogen, A/-ethyl-A^-mtro-iV-mtrosoguamdine in the mouse duodenum. For example, the 0.05 % GTND-treated group had a half of the number of tumors compared with the untreated control group (9). This contribution describes the results of further studies on the anti-tumor activities of high molecular weight tea fractions TNDs (tea non-dialysates), with special reference to its apoptosis-inducing activity.
Preparation of TNDs Leaves of green tea, black tea, Oolong tea or Pu-erh tea were extracted with hot water. The extracts were then extracted with chloroform, ethyl acetate and butanol, and the residual aqueous layer was dialyzed exhaustively against water and non-dialyzable fractions were freeze-dried and used as TNDs. The yields were 0.42, 2.01, 1.78, and 4.52% for green tea G T N D , black tea B T N D , Oolong tea OTND, and Pu-erh tea P T N D , respectively (10). These high molecular weight fractions are considered to be a complex mixture of tannins and contain several monosaccharides such as glucose and galactose and polyphenols. As polyphenolic compounds, kaempherol, quercetin, gallic acid, and catechins were identified (10).
Anti-tumor Promotion Activity of TNDs Murine JB6 cells cannot grow in soft agar. However, after treatment with 12-0-tetradecanoylphorbol-13-acetate they can grow in soft agar. When B T N D was added to this system, the number of the colonies was reduced dosedependently. The reduction of the colony number was concentration-dependent and 30 pg/ml of B T N D inhibited colony formation almost completely (10). Other TNDs also inhibited neoplastic transformation induced by T P A in a concentration-dependent manner.
Apoptosis-inducing Activity of TNDs It was noticed that B T N D and E G C G induce apoptosis in the transformant of JB6 cells. These transformant cells were prepared by the treatment of JB6 cells with A^-ethyl-A^-nitro-/V-nitrosoguanidine. Apoptotic cells were detected
Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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as fluorescent cells by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method (Figure 1). With B T N D at 80 pg/ml, 12% of the cell population underwent apoptosis, while no apparent apoptosis was observed for the untreated cells. Other TNDs also induced apoptosis, but the effects were weaker than B T N D . JB6 cells before transformation did not show any apoptotic feature under these conditions. Thus, the effects of B T N D and E G C G on mouse tumor cells appear to be stronger than those on the normal counterpart. TNDs and E G C G were also found to inhibit cell proliferation by inducing apoptosis in human leukemia U937 cells, human stomach cancer M K N 4 5 cells, and human colon cancer WiDr cells (11).
Effects on Expression of Apoptosis-related mRNAs It is generally accepted that immediate-early response oncogenes are activated in the course of apoptosis (12,13). The reverse transcriptionpolymerase chain reaction (RT-PCR) method detected the increase in the levels of these oncogenes, particularly in c-fos, by treatment with E G C G or B T N D (Figure 2). The expression of mRNAs related to apoptosis was also examined using R T - P C R with primer sets for several proteins, called Multiplex supplied by Maxim Biotech, Inc. (South San Francisco, C A ; Figure 3). E G C G did not affect so much the expression of mRNAs for these, while B T N D caused drastic changes of these m R N A levels. The mRNAs for ICE or caspase 1 and TNF-α were greatly reduced, but there was no change in bcl-2 (Figure 3, a and b). There were also several other changes. The m - R N A for caspase 8 (Flice) was increased and mRNAs for cell-membrane-associated proteins, Fas, Fasligand, Fadd and Tradd were also increased (Figure 3, c and d). However the mechanism of apoptosis induction by TNDs has not yet been elucidated. The data, however, suggest that the apoptosis induction mechanism of B T N D is different from that of E G C G .
Carcinogenesis in APC Knockout Mice The A P C 1309 knockout mice with a mutated adenomatous polyposis coli gene were generously supplied by the Japanese Foundation for Cancer Research, Cell Biology Department, Cancer Institute, Tokyo, Japan. B y 15weeks of age, they developed polyps of about 30 in average in the gastrointestinal tract. The oral administration of E G C G (0.05% w/v in water) or G T N D (0.05% w/v in water) was started at 4 weeks of age. Table I shows the number of tumors larger than 0.5 mm in size in each gastrointestinal segment of APC1309 knockout mice. E G C G reduced the number of tumors in the stomach and the colon by 60 and 66%, respectively, but failed to do so in the small intestine. However, when the total tumor number was compared, no significant difference was observed between the E G C G and the control groups. In these
Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Figure 1. JB6 transformants cultured on cover slips were incubated with (b) or without (a) BIND at 80 μg/mlfor 24 h. Apoptotic cells labeledfluorescently by the TUNEL method were detected using excitation at 492 nm and emission at 513 nm.
Figure 2. Effect of EGCG and BIND on expression of immediate-early response proto- oncogenes.
Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Figure 3. Effect of EGCG and BTND on mRNA expression. RT-PCR products from mRNA of control U937 cells (lane 1), cells incubated -with EGCG at 200 μΜ (lane 2) or with 1.2 mg/ml of BTND (lane 3) were examined by agarose gel electrophoresis (a and c), and fluorescence intensity (ordinate) determined by Fluorolmager (Molecular Dynamics Japan Inc., Yokyo, Japan) is expressed in percentage relative to that for glyceraldehyde-3-phosphate dehydrogenase, GAPDH (b and d). Primers used were sets for GAPDH, ICE, TNF-a, NF-kB, bcl-2, andI-κΒ (a) and for GAPDH, Flice, Fas, FasL, Fadd, and Tradd (c). M, DNA size marker.
Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Table I. Number of Tumors in Each Gastrointestinal Segment in A P C 1309 Knockout Mice Gastrointestinal
segment &
Stomach
Small intestine
Colon
Total
Number of tumors per mouse
Control
EGCG group
1.89 ± 1 . 3 4
0.75 ± 0 . 9 7
1.04±1.04
(p
