- Email: [email protected]
Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells
Chemico-Biological Interactions 122 (1999) 59 – 71
www.elsevier.com/locate/chembiont
Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells David Opare Kennedy, Miho Matsumoto, Akiko Kojima, Isao Matsui-Yuasa * Department of Food and Nutrition, Faculty of Human Life Science, Osaka City Uni6ersity, 3 -3 -138 Sugimoto, Sumiyoshi-ku, Osaka 558 -8585, Japan Received 9 February 1999; received in revised form 3 May 1999; accepted 17 May 1999
Abstract Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea, and several mechanisms have been proposed for these effects. In this study the relationship between cellular sulfhydryl (SH) groups and the cytotoxicity of green tea polyphenols in Ehrlich ascites tumor cells was examined. It was found that in the presence of green tea extract (GTE) (100 mg/ml) and one of its polyphenolic components, epigallocatechin (EGC; 100 mM), both cellular non-protein (GSH) and protein-sulfhydryl (PSH) levels were significantly decreased and this was associated with a decrease in cell viability. Replenishing the thiol levels by using N-acetylcysteine (NAC) caused a recovery in cell viability, but this recovery was dependent on the time of thiol replenishment in the presence of EGC (initial 15 min). These results identify SH groups as a novel target of green tea polyphenols cytotoxicity in tumor cells, and a regulatory role for green tea in terms of reducing sulfhydryls in tumor inhibition. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Green tea polyphenols; Glutathione; Protein sulfhydryl; Cell death; Ehrlich ascites tumor cells
* Corresponding author. Tel./fax: + 81-6-66052810. E-mail address: [email protected] (I. Matsui-Yuasa) 0009-2797/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 1 1 4 - 3
60
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
1. Introduction Commercial green tea is made by steaming or drying fresh tea leaves at elevated temperatures. Its chemical composition is similar to that of fresh tea leaves. Green tea contains polyphenols, which include flavanols, flavandiols, flavonoids, and phenolic acids. Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea. The most noteworthy properties of tea polyphenols that may affect carcinogenesis are their antioxidative activities, inhibitory action against nitrosation reactions, modulation of carcinogen enzymes, trapping of ultimate carcinogens, and inhibition of cell proliferation-related activities [1]. The major green tea polyphenols, which are ( − )-epigallocatechin-3-gallate (EGCG), (− )-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), (− ) epicatechin (EC) and ( +)-catechin, have been found to have these activities. (+ )-Catechin and (−) epicatechin are the structural parent compounds of green tea polyphenols. Sulfhydryl groups in both proteins and non-proteins are involved in the maintenance of various cellular functions, including many enzymatic activities [2]. Thus, perturbation of intracellular thiol homeostasis has been found to be critically involved in the development of chemically induced cell damage. Glutathione (GSH), the major non-protein thiol in mammalian cells, is involved in many cellular functions including amino acid transport [3] and thiol-disulfide balance [4]. GSH has also been suggested as a potential regulator of protein synthesis [5], DNA synthesis and cell proliferation [6]. Free sulfhydryl groups in proteins (PSH) play the role of highly reactive functional groups in biological systems and participate in several different reactions such as alkylation, arylation, oxidation, thiol-disulfide exchange, etc. Therefore the modification of PSH groups can result in severe functional damage, including loss of enzyme activity [7]. Some selected PSH inhibitors were found to induce immunity against some cancers in mice while commonly used antitumor agents showed no such capability [8]. In this study the role of cellular sulfhydryl groups in the effect of green tea polyphenols in Ehrlich ascites tumor cells was examined. This included measurement of GSH and PSH under the effect of green tea extract and its polyphenolic constituents, as well as the effect on cell viability.
2. Materials and methods
2.1. Chemicals Catechin, EC and EGC were purchased from Sigma (St. Louis, MO). Green tea extract (GTE), and ECG were purchased purified from Taiyo Kagaku, Japan. The polyphenols were purchased at a minimum purity of 98%. EGCG and 5,5%-dithiobis-2-nitrobenzoic acid (DTNB) were purchased from Wako (Osaka, Japan). Analysis show that the major components of GTE are (% w/w) gallo-catechin (12.8), EGC (16.5) and EGCG (21.3). It also contains caffeine, sugars, amino acids and
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
61
ash as minor components. Structures of the polyphenols are shown in Fig. 1. Fetal calf serum (FCS) was purchased from JRH Biosciences (Lenexa, Australia). Other chemicals used in this study were special grade commercial products.
2.2. Cell culture Ehrlich ascites tumor cells were cultured in a humidified atmosphere of 5% CO2 in air at 37°C for 3 – 4 days in Eagle’s minimum essential medium containing 10% FCS, then washed and cultured again at a concentration of 1× 106/ml in fresh medium. Tea polyphenols were dissolved in ethanol and diluted in the cultured medium immediately before use (final ethanol concentration B 0.25%). In all the experiments control cultures were made up of medium, ethanol and the cells.
2.3. Assay of cell 6iability Cell viability was determined by the Trypan blue exclusion analysis. Cells (1× 106/ml) treated with the tea polyphenols at various concentrations were incubated in a humidified atmosphere of 5% CO2 in air at 37°C overnight in Eagle’s minimum essential medium containing 10% FCS. To a cell suspension was added an equal volume of 0.4% Trypan blue reagent (Sigma) and percentages of viable cells evaluated under the field microscope. Assays were performed in triplicate.
Fig. 1. Structures of green tea polyphenols.
62
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
2.4. Cellular GSH and protein-SH assays Cellular GSH (non-protein thiols) and protein-SH concentrations were measured as described in [9]. After incubating with GTE and the polyphenols for both dose and time dependent studies, cells (1× 106) were harvested and an extract was obtained by treating with 0.5 ml 2% 5-sulfosalicylic acid, and centrifuged at 3000 rpm for 10 min. The supernatant obtained was used for the assay of intracellular GSH concentration and the cell pellet suspended in 1.2 ml 0.5 M Tris–HCl, pH 7.6 was used for the PSH assay. DTNB (at a final concentration of 100 mM) was then added and after 20 min the absorbance was measured at 412 nm. Data were expressed as nmol SH as per 1 × 106 cells, calculated on the basis of a GSH calibration curve.
2.5. Statistical analysis All data are represented as means9S.D. (standard deviation from the mean) and statistical evaluations were made using Student’s t-test with P-value B 0.05 indicating statistically significant difference.
3. Results
3.1. Effect of green tea extract and its polyphenols on cell 6iability The effect of the green tea extract (GTE) and its five polyphenolic components (Fig. 1) on cell viability was examined in Ehrlich ascites tumor cells by the Trypan blue method. After 3 – 4 days’ culture in Eagle’s minimum essential medium containing 10% FCS, the cells were diluted and incubated again in fresh medium with or without the different polyphenols. Cell viability was measured 24 h later. As presented in Fig. 2, GTE decreased cell viability significantly as compared with non-treated cells (50 and 100 mg/ml: PB 0.005). Of the individual polyphenols of GTE, EGC (50 and 100 mM, P B0.005 and PB 0.0005, respectively) and EGCG (100 mM, P B 0.05) decreased cell viability. Based on these results obtained, 100 mM of the polyphenols was used for subsequent determinations.
3.2. Changes in cellular glutathione and protein sulfhydryl le6els The effect of the polyphenols on cellular GSH and PSH levels was examined. It was found that at 8 h of incubation, GTE (100 mg/ml) and EGC (100 mM) significantly decreased cellular PSH (Fig. 3A) as well as GSH (Fig. 3B). A parallel correlation between decreased cell viability and decreased cellular PSH and GSH by both GTE and EGC has therefore been observed, whereas the other polyphenols showed no such effects. Further analyses showed this effect of EGC on PSH and GSH reductions to be both dose (Fig. 4) and time (Fig. 5A and B, respectively) dependent.
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
63
Fig. 2. Effect of (A) green tea extract (GTE) and (B) its polyphenols on Ehrlich ascites tumor cell viability. After 3–4 days’ culture in Eagle’s minimum essential medium containing 10% FCS, the cells were diluted and incubated again in fresh medium with or without GTE and the different polyphenols. Cell viability was measured 24 h later by the Trypan blue exclusion method. Green tea extract and polyphenols were dissolved in ethanol (final concentration of ethanol was B 0.25%). Each point is the mean ( 9 S.D.) of three experiments. *PB 0.05; **PB 0.005 compared with control.
3.3. Effect of N-acetylcysteine on cellular thiol le6els in the presence of EGC In order to ascertain that the effect of EGC on cell viability was directly related to the reductions in cellular thiol levels, a series of studies were done using N-acetylcysteine (NAC), the acetylated variant of the amino acid L-cysteine and an excellent source of sulfhydryl groups. As shown in Fig. 6, when both EGC and NAC were added to cells at the beginning of the incubation period, cell viability
64
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
Fig. 3. Effect of green tea extract (GTE) and its polyphenols on (A) protein sulfhydryl and (B) cellular glutathione (non-protein sulfhydryls) in Ehrlich ascites tumor cells. Cells (1 × 106) were collected after 8-h incubation, and an extract was obtained by treating with 0.5 ml 2% 5-sulfosalicylic acid, and centrifuged at 3000 rpm for 10 min. The supernatant obtained was used for the assay of intracellular GSH (non-protein thiols) concentration and the cell pellet suspended in 1.2 ml 0.5 M Tris – HCl, pH 7.6 was used for the protein-SH assay. DTNB (at a final concentration of 100 mM) was then added and after 20 min the absorbance was measured at 412 nm. None-treated samples contained cells, medium and ethanol only. Data were expressed as nmol per 106 cells, calculated on the basis of a GSH calibration curve, and are the mean of three experiments. *PB 0.05; **PB0.005, compared with ‘None’.
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
65
measured after 24 h improved significantly. However, late addition of NAC (60 min after initially adding EGC) did not improve cell viability to any significant extent. It was also found that NAC caused a significant recovery in PSH levels and this correlated with the improved cell viability observed under the same treatment.
3.4. Effect of green tea extract on GSH and PSH in primary hepatocytes Hepatocytes were used as a normal cell model and after treatment with GTE under similar conditions as the tumor cells, GSH and PSH levels were determined. It was found that at all time points measured from 0 to 22 h GTE-treated cells had significantly higher levels of GSH and PSH compared with non-treated control cells (Table 1).
4. Discussion The results obtained in the present study demonstrate for the first time, to the best of our knowledge, that a green tea extract (GTE) exhibits cytotoxicity to Ehrlich ascites tumor cells in a cellular thiol-dependent way.
Fig. 4. Dose-dependent effect of EGC (100 mM) on protein sulfhydryl and cellular glutathione (non-protein sulfhydryls) in Ehrlich ascites tumor cells. Cells (1 ×106) were treated as in Section 2 for 8 h. Data were expressed as % of control, calculated on the basis of a GSH calibration curve, and are the mean of three determinations. Control values at 8 h for PSH and GSH obtained were 85.0 and 20.6 nmol/106 cells, respectively.
66
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
Fig. 5. Time-course effect of EGC (100 mM) on (A) protein sulfhydryl and (B) cellular glutathione (non-protein sulfhydryls) in Ehrlich ascites tumor cells. Cells (1 × 106) were treated as in Section 2. Data were expressed as % of control (zero time), calculated on the basis of a GSH calibration curve and are the mean of three determinations. Values at zero time for PSH and GSH obtained were 47.0 and 9.4 nmol/106 cells, respectively.
GTE (100 mg/ml) caused a significant reduction in the viability of the tumor cells, and further analyses revealed that this effect was attributable to one of GTE polyphenolic components, (− )-epigallocatechin (EGC). Furthermore, both GTE and EGC (100 mM) caused significant reductions in both non-protein sulfhydryls (GSH) and protein sulfhydryl (PSH) levels. The effect of EGC on the reductions in cellular thiol levels was found to be both dose and time dependent. EGCG also had a slight effect on cell viability but like the other polyphenols, which had no effect on cell viability, did not have any effect on GSH or PSH reductions. The unique
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
67
effect of one catechin over other structurally related catechins is not new in tea polyphenol research. Several studies have reported differential inhibitory or enhancement effect of structurally related catechins. Most recently, Watanabe et al. [10] found an IC50 value of acetyl-CoA carboxylase inhibitory activity of EGCG and ECG to be 300 mM, whereas ( + )-catechin, EC, EGC and gallic acid had no effect. The inhibitory activity was attributable to the presence of the 3-O-gallate group of the catechin structure. Bioavailability studies show that the levels of tea catechins found in human plasma after ingestion of 1.5 and 3.0 g of tea reached a saturation point, Cmax, of
Fig. 6. Effect of EGC (100 mM; 8 h) and addition of N-acetylcysteine (NAC; 6 mM) on (A) cell viability and (B) PSH levels in Ehrlich ascites tumor cells. NAC was added at different times and cell treatment was as described in Section 2. None-treated samples contained cells, medium and ethanol only. Data were expressed as % of control. *PB 0.05; **PB 0.005, compared with EGC-treated samples. Graph shows mean of three determinations.
68
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
1.1, 1.9 and 0.7 mg/ml for EGCG, EGC and EC, respectively. Increasing the tea dose to 4.5 g did not cause any significant increase in Cmax, suggesting a saturation phenomenon [11]. The concentration required to achieve the effects observed in the present study may seem high (100 mg/ml for GTE, 50–100 mM for EGC) relative to human tissue concentrations under normal dietary conditions, but are well within the range achievable in animal chemoprevention studies, some of which have employed higher catechin concentrations [12–14]. It was also demonstrated that replenishing the cellular thiol stores with N-acetylcysteine (NAC), the acetylated variant of the amino acid L-cysteine, and an excellent source of sulfhydryl groups, caused a recovery of cell viability in the presence of EGC. This reversibility was however, dependent on an early addition of NAC (when added until 30 min after EGC addition), as later additions could not reverse the effect of EGC. The correlation with cell viability did not hold after 60 min of adding EGC before NAC, and we conclude that the effect of EGC after this time was irreversible and that longer depletions of cellular PSH might have caused irreversible cell damage and an associated cell death. The recovery in cellular thiol levels in relation to improved cell viability was found to be significantly associated with a recovery in PSH levels. Inhibition of carcinogenesis in the skin, lung, esophagus, forestomach and colon in several rodent models [15 – 18], has been attributed to green tea extract and some of its components and several mechanisms by which green tea polyphenols exert their anticarcinogenic effects have been proposed. Green tea polyphenols have been shown to be efficient antioxidants, and the concept that tea components may inhibit carcinogenesis through antioxidative activities is supported by many findings. H2O2 formation induced by 12-O-tetradecanoylphorbol 13-acetate in HeLa cells was inhibited by EGCG, and the oxidation of lard as evaluated by the active oxygen method was suppressed as well [19,20]. Activation of protein tyrosine kinase activity by EGC via phosphorylation of two proteins, p42 and p45, was also found to be associated with decreased cell viability [21]. Green tea and its individual epicatechin derivatives inhibited skin tumor promoter-mediated induction of epidermal ornithine decarboxylase (ODC) in SENCAR mice [22]. The present study has shown that the green tea polyphenol, EGC, exhibits cytotoxicity to Ehrlich ascites tumor cells via decreasing cellular thiol levels, particularly protein sulfhydryl. Although the reaction of polyphenols with GSH is known, the correlation between thiol depletion, PSH reduction, and toxicity is novel, to the best of our knowledge. Polyphenolic-glutathione conjugates and their metabolites retain the electrophilic and redox properties of the parent polyphenol, and indeed, the reactivity of the thioether metabolites frequently exceeds that of the parent polyphenol. These conjugates have been found to contribute to the nephrotoxicity, nephrocarcinogenicity, and neurotoxicity of a variety of polyphenols [23,24]. Sulfhydryl groups show more reactivity than any other group in biologic systems with the exception of hydrogen bond associations, because of the ease with which anion of SH group donates electrons to even weakly electrophilic reagents. Chemical significance of SH groups stems from this extraordinary reactivity as nucleophile as well as in free
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
69
Table 1 Effect of green tea extract (GTE; 100 mg/ml) on protein sulfhydryl and glutathione (non-protein sulfhydryl) in isolated primary cultured rat hepatocytes Time (h)
0 2 4 8 22
Protein-SH (nmol/106 cells)
Glutathione (nmol/106 cells)
Control
GTE
Control
GTE
16.39 0.1 14.89 4.1 13.89 2.3 13.19 3.8 12.39 0.4
18.0 90.9* 17.4 9 0.2* 15.8 9 1.4* 22.0 93.3*
2.7 9 0 2.8 9 1.3 2.2 9 0.5 3.8 91.3 2.8 91.0
5.1 9 1.1* 5.4 90.1* 7.5 9 0.8* 5.0 91.2*
* Statistically different compared with corresponding control levels (Student’s t-test, PB0.05).
radical reactions and in formation of intermolecular complexes whose effects are exquisitely sensitive to modulations of intimate, submicroscopic chemical milieu [25]. A proposed mechanism of direct toxicity, such as for acetaminophen, in a cell has been linked to a decrease in the activity of several early target proteins, and these early target proteins appeared to be proteins with accessible nucleophilic sulfhydryl groups with a high concentration of the protein in the cell [26]. Eventually the cell machinery is overwhelmed and the cell can then no longer function, or there is a disruption of the redox balance within the cell due to the decreased function of numerous proteins. On the importance of sulfhydryl groups in tumor studies, it was found that selected SH inhibitors in chemical sensitivity tests were considerably more active against a variety of human and animal cancers than were other commonly used antitumor agents [27]. In related experiments with isolated primary cultured rat hepatocytes, 100 mg/ml GTE at 22 h of incubation caused a significant increase in both cellular levels of GSH (186%) and PSH (135%) compared with control samples, contrasting the effect observed in Ehrlich ascites tumor cells. This suggests that green tea sulfhydryl-dependent effects differ in normal and tumor cells (Table 1). On the role of thiols in cancer cells, a ubiquitous protein, thioredoxin, found over-expressed in some human tumors and which stimulates cancer cell growth, exists either in reduced form (thioredoxin-(SH)2) with a dithiol, or in oxidized form (thioredoxin-S2) [28,29]. Thioredoxin-(SH)2 is a powerful protein disulfide reductase that participates in many thiol-dependent, cellular-reductive processes. Molecular studies have provided proof-of-principle that the thioredoxin system is a rational target for anticancer drug development, and initial approach in studies was to develop agents which might selectively inhibit the thioredoxin system and thioredoxin-dependent cell proliferation. It is thought that the effect of green tea extract and its polyphenol in Ehrlich ascites tumor cells observed in this study might be related to an inhibition of thioredoxin, and that green tea might inhibit the thioredoxin system and thioredoxin-dependent cell proliferation. This subject is currently under intense scrutiny in our laboratory.
70
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
Acknowledgements This investigation was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a grant from the Co-operative Kyoto Prefectural Tea Association of Japan.
References [1] C.S. Yang, Z.-Y. Wang, Tea and cancer, J. Natl. Cancer Inst. 85 (1993) 1038 – 1049. [2] J.S. Stamler, A. Slivka, Biological chemistry of thiols in the vasculature and in vascular-related disease, Nutr. Rev. 54 (1996) 1–30. [3] A. Meister, On the enzymology of amino acid transport, Science 180 (1973) 33 – 39. [4] D.M. Ziegler, Role of reversible oxidation-reduction of enzyme thiols disulfides in metabolic regulation, Ann. Rev. Biochem. 54 (1985) 305 – 329. [5] N.S. Kosower, E.M. Kosower, The glutathione status of cells, Int. Rev. Cytol. 54 (1978) 109 – 160. [6] M. Suthanthiran, M.E. Anderson, V.K. Sharma, A. Meister, Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens, Proc. Natl. Acad. Sci. USA 87 (1990) 3343 – 3347. [7] S. Orrenius, Biochemical mechanisms of cytotoxicity, Trends Pharmacol. Sci. 6 (1985) 15 – 20. [8] F.E. Knock, R.M. Galt, Y.T. Oester, R. Sylvester, H. Rebechini-Zasadny, Selected sulfhydryl inhibitors capable of inducing immunity against cancer in mice, Oncology 36 (1979) 197 – 201. [9] E. Beutler, O. Duron, B.M. Kelly, Improved method for the determination of blood glutathione, J. Lab. Clin. Med. 61 (1963) 882–888. [10] J. Watanabe, J. Kawabata, R. Niki, Isolation and identification of acetyl-CoA carboxylase inhibitors from green tea (Camellia sinensis), Biosci. Biotechnol. Biochem. 62 (1998) 532 – 534. [11] C.S. Yang, L. Chen, M.J. Lee, D. Balentine, M.C. Kuo, S.P. Schantz, Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers, Cancer Epidemiol. Biomarkers Prev. 7 (1998) 351–354. [12] Y. Sagesaka-Mitane, M. Miwa, S. Okada, Platelet aggregation inhibitors in hot water extract of green tea, Chem. Pharm. Bull. Tokyo 38 (1990) 790 – 793. [13] M.M. Chan, D. Fong, C.T. Ho, H.I. Huang, Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea, Biochem. Pharmacol. 54 (1997) 1281 – 1286. [14] R. Yu, J.-J. Jiao, J.-L. Duh, K. Gudehithlu, T.-H. Tan, A.-N.T. Kong, Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated Phase II enzyme gene expression, Carcinogenesis 18 (1997) 451–456. [15] Z.Y. Wang, W.A. Khan, D.R. Bickers, H. Mukhtar, Protection against polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by green tea polyphenols, Carcinogenesis 10 (1989) 411–415. [16] Z.Y. Wang, M.T. Huang, T. Ferraro, C.Q. Wong, Y.R. Lou, M. Iatropoulos, C.S. Yang, A.H. Conney, Inhibitory effect of green tea in drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in the skin of SKH-1 mice, Cancer Res. 52 (1992) 1162 – 1170. [17] R. Agarwal, S.K. Katiyar, S.I. Zaidi, H. Mukhtar, Inhibition of skin tumor promoter-caused inhibition of epidermal ornithine decarboxylase in SENCAR mice by polyphenolic fraction isolated from green tea and its individual epicatechin derivatives, Cancer Res. 52 (1992) 3582 – 3588. [18] H. Nishida, M. Omori, Y. Fukutomi, M. Ninomiya, S. Nishiwaki, M. Suganuma, H. Moriwaki, Y. Muto, Inhibitory effects of ( − )- epigallocatechin gallate on spontaneous hepatoma in C3H/HeNCrj mice and human hepatoma-derived PLC/PRF/5 cells, Jpn. J. Cancer Res. 85 (1994) 221 – 225.
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
71
[19] T. Matsuzaki, Y. Hara, Antioxidative activity of tea leaf catechins, J. Agric. Chem. Soc. Jpn. 59 (1985) 126–134. [20] R. Bhimani, K. Frenkel, Suppression of H2O2 production and oxidative DNA damage in HeLa cells by ( −)-epigallocatechin gallate (EGCG), Proc. Am. Assoc. Cancer Res. 32 (1991) 126. [21] D.O. Kennedy, S. Nishimura, T. Hasuma, Y. Yano, S. Otani, I. Matsui-Yuasa, Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro, Chem.-Biol. Interact. 110 (1998) 159 – 172. [22] I. Holm, L. Persson, L. Stjernborg, L. Thorsson, O. Heby, Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro, Biochem. J. 258 (1989) 343 – 350. [23] T.J. Monks, S.S. Lau, The pharmacology and toxicology of polyphenolic-glutathione conjugates, Annu. Rev. Pharmacol. Toxicol. 38 (1998) 225 – 229. [24] R.T. Miller, S.S. Lau, T.J. Monks, Metabolism of 5-(glutathion-S-yl)- alpha-methyldopamine following intracerebroventricular administration to male Sprague – Dawley rats, Chem. Res. Toxicol. 8 (1995) 634–641. [25] F.E. Knock, Possible role of accessibility of protein-SH groups to the cancer state, Arch. Geschwulstforsch. 51 (1981) 75–79. [26] N.R. Pumford, N.C. Halmes, J.A. Hinson, Covalent binding of xenobiotics to specific proteins in the liver, Drug Metab. Rev. 29 (1997) 39 – 57. [27] F.E. Knock, R.M. Galt, Y.T. Oester, R. Sylvester, In vitro estimate of sensitivity of individual human tumors to antitumor agents, Oncology 30 (1974) 1 – 22. [28] A. Holmgren, Thioredoxin, Ann. Rev. Biochem. 54 (1985) 237 – 271. [29] G. Powis, D.L. Kirkpatrick, M. Angulo, A. Baker, Thioredoxin redox control of cell growth and death and the effects of inhibitors, Chem-Biol. Interact. 111 – 112 (1998) 23 – 34.
. .
www.elsevier.com/locate/chembiont
Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells David Opare Kennedy, Miho Matsumoto, Akiko Kojima, Isao Matsui-Yuasa * Department of Food and Nutrition, Faculty of Human Life Science, Osaka City Uni6ersity, 3 -3 -138 Sugimoto, Sumiyoshi-ku, Osaka 558 -8585, Japan Received 9 February 1999; received in revised form 3 May 1999; accepted 17 May 1999
Abstract Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea, and several mechanisms have been proposed for these effects. In this study the relationship between cellular sulfhydryl (SH) groups and the cytotoxicity of green tea polyphenols in Ehrlich ascites tumor cells was examined. It was found that in the presence of green tea extract (GTE) (100 mg/ml) and one of its polyphenolic components, epigallocatechin (EGC; 100 mM), both cellular non-protein (GSH) and protein-sulfhydryl (PSH) levels were significantly decreased and this was associated with a decrease in cell viability. Replenishing the thiol levels by using N-acetylcysteine (NAC) caused a recovery in cell viability, but this recovery was dependent on the time of thiol replenishment in the presence of EGC (initial 15 min). These results identify SH groups as a novel target of green tea polyphenols cytotoxicity in tumor cells, and a regulatory role for green tea in terms of reducing sulfhydryls in tumor inhibition. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Green tea polyphenols; Glutathione; Protein sulfhydryl; Cell death; Ehrlich ascites tumor cells
* Corresponding author. Tel./fax: + 81-6-66052810. E-mail address: [email protected] (I. Matsui-Yuasa) 0009-2797/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 1 1 4 - 3
60
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
1. Introduction Commercial green tea is made by steaming or drying fresh tea leaves at elevated temperatures. Its chemical composition is similar to that of fresh tea leaves. Green tea contains polyphenols, which include flavanols, flavandiols, flavonoids, and phenolic acids. Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea. The most noteworthy properties of tea polyphenols that may affect carcinogenesis are their antioxidative activities, inhibitory action against nitrosation reactions, modulation of carcinogen enzymes, trapping of ultimate carcinogens, and inhibition of cell proliferation-related activities [1]. The major green tea polyphenols, which are ( − )-epigallocatechin-3-gallate (EGCG), (− )-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), (− ) epicatechin (EC) and ( +)-catechin, have been found to have these activities. (+ )-Catechin and (−) epicatechin are the structural parent compounds of green tea polyphenols. Sulfhydryl groups in both proteins and non-proteins are involved in the maintenance of various cellular functions, including many enzymatic activities [2]. Thus, perturbation of intracellular thiol homeostasis has been found to be critically involved in the development of chemically induced cell damage. Glutathione (GSH), the major non-protein thiol in mammalian cells, is involved in many cellular functions including amino acid transport [3] and thiol-disulfide balance [4]. GSH has also been suggested as a potential regulator of protein synthesis [5], DNA synthesis and cell proliferation [6]. Free sulfhydryl groups in proteins (PSH) play the role of highly reactive functional groups in biological systems and participate in several different reactions such as alkylation, arylation, oxidation, thiol-disulfide exchange, etc. Therefore the modification of PSH groups can result in severe functional damage, including loss of enzyme activity [7]. Some selected PSH inhibitors were found to induce immunity against some cancers in mice while commonly used antitumor agents showed no such capability [8]. In this study the role of cellular sulfhydryl groups in the effect of green tea polyphenols in Ehrlich ascites tumor cells was examined. This included measurement of GSH and PSH under the effect of green tea extract and its polyphenolic constituents, as well as the effect on cell viability.
2. Materials and methods
2.1. Chemicals Catechin, EC and EGC were purchased from Sigma (St. Louis, MO). Green tea extract (GTE), and ECG were purchased purified from Taiyo Kagaku, Japan. The polyphenols were purchased at a minimum purity of 98%. EGCG and 5,5%-dithiobis-2-nitrobenzoic acid (DTNB) were purchased from Wako (Osaka, Japan). Analysis show that the major components of GTE are (% w/w) gallo-catechin (12.8), EGC (16.5) and EGCG (21.3). It also contains caffeine, sugars, amino acids and
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
61
ash as minor components. Structures of the polyphenols are shown in Fig. 1. Fetal calf serum (FCS) was purchased from JRH Biosciences (Lenexa, Australia). Other chemicals used in this study were special grade commercial products.
2.2. Cell culture Ehrlich ascites tumor cells were cultured in a humidified atmosphere of 5% CO2 in air at 37°C for 3 – 4 days in Eagle’s minimum essential medium containing 10% FCS, then washed and cultured again at a concentration of 1× 106/ml in fresh medium. Tea polyphenols were dissolved in ethanol and diluted in the cultured medium immediately before use (final ethanol concentration B 0.25%). In all the experiments control cultures were made up of medium, ethanol and the cells.
2.3. Assay of cell 6iability Cell viability was determined by the Trypan blue exclusion analysis. Cells (1× 106/ml) treated with the tea polyphenols at various concentrations were incubated in a humidified atmosphere of 5% CO2 in air at 37°C overnight in Eagle’s minimum essential medium containing 10% FCS. To a cell suspension was added an equal volume of 0.4% Trypan blue reagent (Sigma) and percentages of viable cells evaluated under the field microscope. Assays were performed in triplicate.
Fig. 1. Structures of green tea polyphenols.
62
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
2.4. Cellular GSH and protein-SH assays Cellular GSH (non-protein thiols) and protein-SH concentrations were measured as described in [9]. After incubating with GTE and the polyphenols for both dose and time dependent studies, cells (1× 106) were harvested and an extract was obtained by treating with 0.5 ml 2% 5-sulfosalicylic acid, and centrifuged at 3000 rpm for 10 min. The supernatant obtained was used for the assay of intracellular GSH concentration and the cell pellet suspended in 1.2 ml 0.5 M Tris–HCl, pH 7.6 was used for the PSH assay. DTNB (at a final concentration of 100 mM) was then added and after 20 min the absorbance was measured at 412 nm. Data were expressed as nmol SH as per 1 × 106 cells, calculated on the basis of a GSH calibration curve.
2.5. Statistical analysis All data are represented as means9S.D. (standard deviation from the mean) and statistical evaluations were made using Student’s t-test with P-value B 0.05 indicating statistically significant difference.
3. Results
3.1. Effect of green tea extract and its polyphenols on cell 6iability The effect of the green tea extract (GTE) and its five polyphenolic components (Fig. 1) on cell viability was examined in Ehrlich ascites tumor cells by the Trypan blue method. After 3 – 4 days’ culture in Eagle’s minimum essential medium containing 10% FCS, the cells were diluted and incubated again in fresh medium with or without the different polyphenols. Cell viability was measured 24 h later. As presented in Fig. 2, GTE decreased cell viability significantly as compared with non-treated cells (50 and 100 mg/ml: PB 0.005). Of the individual polyphenols of GTE, EGC (50 and 100 mM, P B0.005 and PB 0.0005, respectively) and EGCG (100 mM, P B 0.05) decreased cell viability. Based on these results obtained, 100 mM of the polyphenols was used for subsequent determinations.
3.2. Changes in cellular glutathione and protein sulfhydryl le6els The effect of the polyphenols on cellular GSH and PSH levels was examined. It was found that at 8 h of incubation, GTE (100 mg/ml) and EGC (100 mM) significantly decreased cellular PSH (Fig. 3A) as well as GSH (Fig. 3B). A parallel correlation between decreased cell viability and decreased cellular PSH and GSH by both GTE and EGC has therefore been observed, whereas the other polyphenols showed no such effects. Further analyses showed this effect of EGC on PSH and GSH reductions to be both dose (Fig. 4) and time (Fig. 5A and B, respectively) dependent.
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
63
Fig. 2. Effect of (A) green tea extract (GTE) and (B) its polyphenols on Ehrlich ascites tumor cell viability. After 3–4 days’ culture in Eagle’s minimum essential medium containing 10% FCS, the cells were diluted and incubated again in fresh medium with or without GTE and the different polyphenols. Cell viability was measured 24 h later by the Trypan blue exclusion method. Green tea extract and polyphenols were dissolved in ethanol (final concentration of ethanol was B 0.25%). Each point is the mean ( 9 S.D.) of three experiments. *PB 0.05; **PB 0.005 compared with control.
3.3. Effect of N-acetylcysteine on cellular thiol le6els in the presence of EGC In order to ascertain that the effect of EGC on cell viability was directly related to the reductions in cellular thiol levels, a series of studies were done using N-acetylcysteine (NAC), the acetylated variant of the amino acid L-cysteine and an excellent source of sulfhydryl groups. As shown in Fig. 6, when both EGC and NAC were added to cells at the beginning of the incubation period, cell viability
64
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
Fig. 3. Effect of green tea extract (GTE) and its polyphenols on (A) protein sulfhydryl and (B) cellular glutathione (non-protein sulfhydryls) in Ehrlich ascites tumor cells. Cells (1 × 106) were collected after 8-h incubation, and an extract was obtained by treating with 0.5 ml 2% 5-sulfosalicylic acid, and centrifuged at 3000 rpm for 10 min. The supernatant obtained was used for the assay of intracellular GSH (non-protein thiols) concentration and the cell pellet suspended in 1.2 ml 0.5 M Tris – HCl, pH 7.6 was used for the protein-SH assay. DTNB (at a final concentration of 100 mM) was then added and after 20 min the absorbance was measured at 412 nm. None-treated samples contained cells, medium and ethanol only. Data were expressed as nmol per 106 cells, calculated on the basis of a GSH calibration curve, and are the mean of three experiments. *PB 0.05; **PB0.005, compared with ‘None’.
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
65
measured after 24 h improved significantly. However, late addition of NAC (60 min after initially adding EGC) did not improve cell viability to any significant extent. It was also found that NAC caused a significant recovery in PSH levels and this correlated with the improved cell viability observed under the same treatment.
3.4. Effect of green tea extract on GSH and PSH in primary hepatocytes Hepatocytes were used as a normal cell model and after treatment with GTE under similar conditions as the tumor cells, GSH and PSH levels were determined. It was found that at all time points measured from 0 to 22 h GTE-treated cells had significantly higher levels of GSH and PSH compared with non-treated control cells (Table 1).
4. Discussion The results obtained in the present study demonstrate for the first time, to the best of our knowledge, that a green tea extract (GTE) exhibits cytotoxicity to Ehrlich ascites tumor cells in a cellular thiol-dependent way.
Fig. 4. Dose-dependent effect of EGC (100 mM) on protein sulfhydryl and cellular glutathione (non-protein sulfhydryls) in Ehrlich ascites tumor cells. Cells (1 ×106) were treated as in Section 2 for 8 h. Data were expressed as % of control, calculated on the basis of a GSH calibration curve, and are the mean of three determinations. Control values at 8 h for PSH and GSH obtained were 85.0 and 20.6 nmol/106 cells, respectively.
66
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
Fig. 5. Time-course effect of EGC (100 mM) on (A) protein sulfhydryl and (B) cellular glutathione (non-protein sulfhydryls) in Ehrlich ascites tumor cells. Cells (1 × 106) were treated as in Section 2. Data were expressed as % of control (zero time), calculated on the basis of a GSH calibration curve and are the mean of three determinations. Values at zero time for PSH and GSH obtained were 47.0 and 9.4 nmol/106 cells, respectively.
GTE (100 mg/ml) caused a significant reduction in the viability of the tumor cells, and further analyses revealed that this effect was attributable to one of GTE polyphenolic components, (− )-epigallocatechin (EGC). Furthermore, both GTE and EGC (100 mM) caused significant reductions in both non-protein sulfhydryls (GSH) and protein sulfhydryl (PSH) levels. The effect of EGC on the reductions in cellular thiol levels was found to be both dose and time dependent. EGCG also had a slight effect on cell viability but like the other polyphenols, which had no effect on cell viability, did not have any effect on GSH or PSH reductions. The unique
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
67
effect of one catechin over other structurally related catechins is not new in tea polyphenol research. Several studies have reported differential inhibitory or enhancement effect of structurally related catechins. Most recently, Watanabe et al. [10] found an IC50 value of acetyl-CoA carboxylase inhibitory activity of EGCG and ECG to be 300 mM, whereas ( + )-catechin, EC, EGC and gallic acid had no effect. The inhibitory activity was attributable to the presence of the 3-O-gallate group of the catechin structure. Bioavailability studies show that the levels of tea catechins found in human plasma after ingestion of 1.5 and 3.0 g of tea reached a saturation point, Cmax, of
Fig. 6. Effect of EGC (100 mM; 8 h) and addition of N-acetylcysteine (NAC; 6 mM) on (A) cell viability and (B) PSH levels in Ehrlich ascites tumor cells. NAC was added at different times and cell treatment was as described in Section 2. None-treated samples contained cells, medium and ethanol only. Data were expressed as % of control. *PB 0.05; **PB 0.005, compared with EGC-treated samples. Graph shows mean of three determinations.
68
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
1.1, 1.9 and 0.7 mg/ml for EGCG, EGC and EC, respectively. Increasing the tea dose to 4.5 g did not cause any significant increase in Cmax, suggesting a saturation phenomenon [11]. The concentration required to achieve the effects observed in the present study may seem high (100 mg/ml for GTE, 50–100 mM for EGC) relative to human tissue concentrations under normal dietary conditions, but are well within the range achievable in animal chemoprevention studies, some of which have employed higher catechin concentrations [12–14]. It was also demonstrated that replenishing the cellular thiol stores with N-acetylcysteine (NAC), the acetylated variant of the amino acid L-cysteine, and an excellent source of sulfhydryl groups, caused a recovery of cell viability in the presence of EGC. This reversibility was however, dependent on an early addition of NAC (when added until 30 min after EGC addition), as later additions could not reverse the effect of EGC. The correlation with cell viability did not hold after 60 min of adding EGC before NAC, and we conclude that the effect of EGC after this time was irreversible and that longer depletions of cellular PSH might have caused irreversible cell damage and an associated cell death. The recovery in cellular thiol levels in relation to improved cell viability was found to be significantly associated with a recovery in PSH levels. Inhibition of carcinogenesis in the skin, lung, esophagus, forestomach and colon in several rodent models [15 – 18], has been attributed to green tea extract and some of its components and several mechanisms by which green tea polyphenols exert their anticarcinogenic effects have been proposed. Green tea polyphenols have been shown to be efficient antioxidants, and the concept that tea components may inhibit carcinogenesis through antioxidative activities is supported by many findings. H2O2 formation induced by 12-O-tetradecanoylphorbol 13-acetate in HeLa cells was inhibited by EGCG, and the oxidation of lard as evaluated by the active oxygen method was suppressed as well [19,20]. Activation of protein tyrosine kinase activity by EGC via phosphorylation of two proteins, p42 and p45, was also found to be associated with decreased cell viability [21]. Green tea and its individual epicatechin derivatives inhibited skin tumor promoter-mediated induction of epidermal ornithine decarboxylase (ODC) in SENCAR mice [22]. The present study has shown that the green tea polyphenol, EGC, exhibits cytotoxicity to Ehrlich ascites tumor cells via decreasing cellular thiol levels, particularly protein sulfhydryl. Although the reaction of polyphenols with GSH is known, the correlation between thiol depletion, PSH reduction, and toxicity is novel, to the best of our knowledge. Polyphenolic-glutathione conjugates and their metabolites retain the electrophilic and redox properties of the parent polyphenol, and indeed, the reactivity of the thioether metabolites frequently exceeds that of the parent polyphenol. These conjugates have been found to contribute to the nephrotoxicity, nephrocarcinogenicity, and neurotoxicity of a variety of polyphenols [23,24]. Sulfhydryl groups show more reactivity than any other group in biologic systems with the exception of hydrogen bond associations, because of the ease with which anion of SH group donates electrons to even weakly electrophilic reagents. Chemical significance of SH groups stems from this extraordinary reactivity as nucleophile as well as in free
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
69
Table 1 Effect of green tea extract (GTE; 100 mg/ml) on protein sulfhydryl and glutathione (non-protein sulfhydryl) in isolated primary cultured rat hepatocytes Time (h)
0 2 4 8 22
Protein-SH (nmol/106 cells)
Glutathione (nmol/106 cells)
Control
GTE
Control
GTE
16.39 0.1 14.89 4.1 13.89 2.3 13.19 3.8 12.39 0.4
18.0 90.9* 17.4 9 0.2* 15.8 9 1.4* 22.0 93.3*
2.7 9 0 2.8 9 1.3 2.2 9 0.5 3.8 91.3 2.8 91.0
5.1 9 1.1* 5.4 90.1* 7.5 9 0.8* 5.0 91.2*
* Statistically different compared with corresponding control levels (Student’s t-test, PB0.05).
radical reactions and in formation of intermolecular complexes whose effects are exquisitely sensitive to modulations of intimate, submicroscopic chemical milieu [25]. A proposed mechanism of direct toxicity, such as for acetaminophen, in a cell has been linked to a decrease in the activity of several early target proteins, and these early target proteins appeared to be proteins with accessible nucleophilic sulfhydryl groups with a high concentration of the protein in the cell [26]. Eventually the cell machinery is overwhelmed and the cell can then no longer function, or there is a disruption of the redox balance within the cell due to the decreased function of numerous proteins. On the importance of sulfhydryl groups in tumor studies, it was found that selected SH inhibitors in chemical sensitivity tests were considerably more active against a variety of human and animal cancers than were other commonly used antitumor agents [27]. In related experiments with isolated primary cultured rat hepatocytes, 100 mg/ml GTE at 22 h of incubation caused a significant increase in both cellular levels of GSH (186%) and PSH (135%) compared with control samples, contrasting the effect observed in Ehrlich ascites tumor cells. This suggests that green tea sulfhydryl-dependent effects differ in normal and tumor cells (Table 1). On the role of thiols in cancer cells, a ubiquitous protein, thioredoxin, found over-expressed in some human tumors and which stimulates cancer cell growth, exists either in reduced form (thioredoxin-(SH)2) with a dithiol, or in oxidized form (thioredoxin-S2) [28,29]. Thioredoxin-(SH)2 is a powerful protein disulfide reductase that participates in many thiol-dependent, cellular-reductive processes. Molecular studies have provided proof-of-principle that the thioredoxin system is a rational target for anticancer drug development, and initial approach in studies was to develop agents which might selectively inhibit the thioredoxin system and thioredoxin-dependent cell proliferation. It is thought that the effect of green tea extract and its polyphenol in Ehrlich ascites tumor cells observed in this study might be related to an inhibition of thioredoxin, and that green tea might inhibit the thioredoxin system and thioredoxin-dependent cell proliferation. This subject is currently under intense scrutiny in our laboratory.
70
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
Acknowledgements This investigation was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a grant from the Co-operative Kyoto Prefectural Tea Association of Japan.
References [1] C.S. Yang, Z.-Y. Wang, Tea and cancer, J. Natl. Cancer Inst. 85 (1993) 1038 – 1049. [2] J.S. Stamler, A. Slivka, Biological chemistry of thiols in the vasculature and in vascular-related disease, Nutr. Rev. 54 (1996) 1–30. [3] A. Meister, On the enzymology of amino acid transport, Science 180 (1973) 33 – 39. [4] D.M. Ziegler, Role of reversible oxidation-reduction of enzyme thiols disulfides in metabolic regulation, Ann. Rev. Biochem. 54 (1985) 305 – 329. [5] N.S. Kosower, E.M. Kosower, The glutathione status of cells, Int. Rev. Cytol. 54 (1978) 109 – 160. [6] M. Suthanthiran, M.E. Anderson, V.K. Sharma, A. Meister, Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens, Proc. Natl. Acad. Sci. USA 87 (1990) 3343 – 3347. [7] S. Orrenius, Biochemical mechanisms of cytotoxicity, Trends Pharmacol. Sci. 6 (1985) 15 – 20. [8] F.E. Knock, R.M. Galt, Y.T. Oester, R. Sylvester, H. Rebechini-Zasadny, Selected sulfhydryl inhibitors capable of inducing immunity against cancer in mice, Oncology 36 (1979) 197 – 201. [9] E. Beutler, O. Duron, B.M. Kelly, Improved method for the determination of blood glutathione, J. Lab. Clin. Med. 61 (1963) 882–888. [10] J. Watanabe, J. Kawabata, R. Niki, Isolation and identification of acetyl-CoA carboxylase inhibitors from green tea (Camellia sinensis), Biosci. Biotechnol. Biochem. 62 (1998) 532 – 534. [11] C.S. Yang, L. Chen, M.J. Lee, D. Balentine, M.C. Kuo, S.P. Schantz, Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers, Cancer Epidemiol. Biomarkers Prev. 7 (1998) 351–354. [12] Y. Sagesaka-Mitane, M. Miwa, S. Okada, Platelet aggregation inhibitors in hot water extract of green tea, Chem. Pharm. Bull. Tokyo 38 (1990) 790 – 793. [13] M.M. Chan, D. Fong, C.T. Ho, H.I. Huang, Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea, Biochem. Pharmacol. 54 (1997) 1281 – 1286. [14] R. Yu, J.-J. Jiao, J.-L. Duh, K. Gudehithlu, T.-H. Tan, A.-N.T. Kong, Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated Phase II enzyme gene expression, Carcinogenesis 18 (1997) 451–456. [15] Z.Y. Wang, W.A. Khan, D.R. Bickers, H. Mukhtar, Protection against polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by green tea polyphenols, Carcinogenesis 10 (1989) 411–415. [16] Z.Y. Wang, M.T. Huang, T. Ferraro, C.Q. Wong, Y.R. Lou, M. Iatropoulos, C.S. Yang, A.H. Conney, Inhibitory effect of green tea in drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in the skin of SKH-1 mice, Cancer Res. 52 (1992) 1162 – 1170. [17] R. Agarwal, S.K. Katiyar, S.I. Zaidi, H. Mukhtar, Inhibition of skin tumor promoter-caused inhibition of epidermal ornithine decarboxylase in SENCAR mice by polyphenolic fraction isolated from green tea and its individual epicatechin derivatives, Cancer Res. 52 (1992) 3582 – 3588. [18] H. Nishida, M. Omori, Y. Fukutomi, M. Ninomiya, S. Nishiwaki, M. Suganuma, H. Moriwaki, Y. Muto, Inhibitory effects of ( − )- epigallocatechin gallate on spontaneous hepatoma in C3H/HeNCrj mice and human hepatoma-derived PLC/PRF/5 cells, Jpn. J. Cancer Res. 85 (1994) 221 – 225.
D.O. Kennedy et al. / Chemico-Biological Interactions 122 (1999) 59–71
71
[19] T. Matsuzaki, Y. Hara, Antioxidative activity of tea leaf catechins, J. Agric. Chem. Soc. Jpn. 59 (1985) 126–134. [20] R. Bhimani, K. Frenkel, Suppression of H2O2 production and oxidative DNA damage in HeLa cells by ( −)-epigallocatechin gallate (EGCG), Proc. Am. Assoc. Cancer Res. 32 (1991) 126. [21] D.O. Kennedy, S. Nishimura, T. Hasuma, Y. Yano, S. Otani, I. Matsui-Yuasa, Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro, Chem.-Biol. Interact. 110 (1998) 159 – 172. [22] I. Holm, L. Persson, L. Stjernborg, L. Thorsson, O. Heby, Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro, Biochem. J. 258 (1989) 343 – 350. [23] T.J. Monks, S.S. Lau, The pharmacology and toxicology of polyphenolic-glutathione conjugates, Annu. Rev. Pharmacol. Toxicol. 38 (1998) 225 – 229. [24] R.T. Miller, S.S. Lau, T.J. Monks, Metabolism of 5-(glutathion-S-yl)- alpha-methyldopamine following intracerebroventricular administration to male Sprague – Dawley rats, Chem. Res. Toxicol. 8 (1995) 634–641. [25] F.E. Knock, Possible role of accessibility of protein-SH groups to the cancer state, Arch. Geschwulstforsch. 51 (1981) 75–79. [26] N.R. Pumford, N.C. Halmes, J.A. Hinson, Covalent binding of xenobiotics to specific proteins in the liver, Drug Metab. Rev. 29 (1997) 39 – 57. [27] F.E. Knock, R.M. Galt, Y.T. Oester, R. Sylvester, In vitro estimate of sensitivity of individual human tumors to antitumor agents, Oncology 30 (1974) 1 – 22. [28] A. Holmgren, Thioredoxin, Ann. Rev. Biochem. 54 (1985) 237 – 271. [29] G. Powis, D.L. Kirkpatrick, M. Angulo, A. Baker, Thioredoxin redox control of cell growth and death and the effects of inhibitors, Chem-Biol. Interact. 111 – 112 (1998) 23 – 34.
. .