Cytotoxicity of epigallocatechin-3-gallate to LNCaP cells in the presence of Cu²⁺

Cytotoxicity of epigallocatechin-3-gallate to LNCaP cells in the presence of Cu 2+ 1 first-author 1 †E-mail:shrshen@zju.edu.cn †E-mail:shrshen@zju.edu.cn shrshen@zju.edu.cn 2 Department of Tea Science, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310009, China Department of Tea Science, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310009, China Department of Pharmacology, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310009, China Department of Pharmacology, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310009, China Epigallocatechin-3-gallate (EGCG) has shown remarkably anti-cancer activity, with its bioactivity being related to reactive conditions, such as pH and metal ions. The present study investigated the degradation of EGCG and its effect on prostate cancer cell in the presence of Cu 2+ . EGCG was incubated with prostate cancer cells, LNCaP, pretreated with or without Cu 2+ . EGCG in F-12 medium was quantified using HPLC and the viability of cells was assessed by gel electrophoresis, flow cytometry, and electron microscope. The results of HPLC showed that EGCG degraded completely within 12 h in F-12 medium with or without Cu 2+ . Gel electrophoresis and flow cytometry did not detect apoptosis of LNCaP cells when they were incubated with EGCG. Electron microscopy examination revealed that EGCG-Cu 2+ complex led to damage of cytoplasm membrane in LNCaP cells. It was speculated that not EGCG, but its oxide and complex with Cu 2+ , are the bioactive components responsible for its cytotoxicity to LNCaP prostate cancer cells. Copper Epigallocatechin-3-gallate LNCaP cells Tea INTRODUCTION 1 Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, showed remarkably anti-inflammatory and anti-cancer activity in many animal tumor models, cell culture systems, and epidemiological studies (Liao et al., 1995 1995 ; Chung et al., 2001a 2001a ; 2001b 2001b ; Kenshi et al., 1997 1997 ; Yang et al., 1999 1999 ; Lu et al., 2002 2002 ). Many reports proved the structure-activity relationships (Cutter et al., 2001 2001 ; Nihal et al., 2000 2000 ; Hiipakka et al., 2002 2002 ) of EGCG in the biological system. It is known that the structure of EGCG would quickly change in some conditions and that the structural rearrangement would easily occur in vivo. Previous studies investigated the metabolism of EGCG in vivo and suggested that the functional components are its metabolites but not EGCG itself (Unno et al., 1996 1996 ; Unno and Takeo, 1995 1995 ; Nakagawa et al., 1997 1997 ). Copper is one of the essential trace elements in humans and its daily requirement varies with ages. The recommended dietary allowance (RDA) for children is 200–400 μg/day while it is about 900 μg/day for adults. EGCG had been shown to chelate Cu 2+ in various systems (Mira et al., 2002 2002 ; Kimura et al., 1998 1998 ; Yoshioka et al., 2001 2001 ; Kumamoto et al., 2001 2001 ); many previous studies used Cu 2+ to initiate the free-radical oxidation of human low-density lipoproteins. To date, there are no reports of studies to examine the bioactivity of EGCG derivatives and EGCG-Cu 2+ complex. The present study focused on cytotoxicity of EGCG-Cu 2+ to human prostate cancer cell, LNCaP. MATERIALS AND METHODS 1 Materials 2 EGCG was obtained from Sigma (St. Louis, MO, USA); the human prostate cancer cell, LNCaP, was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; ANNEXIN V kit was obtained from Caltag Laboratories (California, USA). All other chemicals were extra-pure grade or analytical grade. Cell culture 2 LNCaP cell line was cultured in F-12 medium (GIBCO, Invitrogen Corporation) supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin. The cells were maintained at 37 °C and 5% CO 2 in a humid environment (Shellab, USA). HPLC analysis and EGCG content studies 2 Cells were seeded onto 25 cm 2 plates at a 1×10 5 density for 48 h, and incubated with different concentrations of EGCG and CuSO 4 (the concentration of CuSO 4 in F-12 medium was 0.00064 μmol/L). Then EGCG content was periodically determined in F-12 medium by HPLC (LC-2010A, Shimadzu, Japan) at 6, 12, 24 h. For the blank control, F-12 medium without cells was also incubated with different concentrations of EGCG and CuSO 4 for 6, 12, 24 h. The samples were analyzed on CLC-ODS column (Shim-pack, Shimadzu, Japan). A gradient elution was carried out in the following solvent systems: mobile phase A, double distilled water/acetonitrile (97:3, containing 0.5% acetic acid); mobile phase B, double distilled water/acetonitrile (70:30, containing 0.5% acetic acid). The linear gradient elution system was: from 100% A to 100% B in 45 min, standing at 100% B for 10 min and returning to 100% A after additional 5 min. The flow was set at 1.0 ml/min. Effluent was monitored at 280 nm (Shimadzu, Japan). Gel electrophoresis and DNA fragmentation studies 2 Cells (1×10 5 ) were seeded onto 25 cm 2 plates for 48 h and incubated with different concentrations of EGCG and CuSO 4 for 24 h. Following this treatment, the cells were washed twice with pH 7.2 PBS (phosphate-buffered saline), followed by addition of 1 ml trypsinase and incubation for 5 min; then collected after centrifugation (300 g, 5 min) at 4 °C, and washed twice with pH 7.2 PBS. The cells were incubated with 200 μl cytolysis solution (0.1 mol/L NaCl, 10 mmol/L Tris-HCl, pH 8.0, 25 mmol/L EDTA, pH 8.0, 0.5% SDS) and proteinase (50 μg/ml) for 10 h at 50 °C. Then 500 μl ethanol was added into the pallets, and DNA was collected by centrifugation (12000 g, 10 min) at 4 °C and DNA was washed twice with 75% ethanol. After ethanol was volatilized entirely, the DNA was incubated with 30 μl TE (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0) and 4 μl RNase A (dissolved with TE, 50 μg/ml) for 1 h at 37 °C. The samples (10 μl) were mixed with 2 μl of loading solution (Bromophenol Blue 0.25%, xylene cyanol FF 0.25%, sucrose 40%), and were loaded onto a 1.2% (w/v) agarose gel (containing 0.5 μg/ml ethidium bromide). The DNA fragments were separated by electrophoresis (Amersham Biosciences) at 5 V/cm for 2.5–3 h in TAE buffer (40 mmol/L Tris-HCl, 20 mmol/L acetic acid, 1 mmol/L Na 2 EDTA). The DNA was visualized by using imaging system (Tianneng Shanghai, China) and photographed by an Alphaimager 2000 digital camera. Flow cytometry and cell damages studies 2 The cells were grown at a density of 2×10 5 in 50 cm 2 plates for 48 h, treated with different concentrations of EGCG and CuSO 4 for 24 h, washed with pH 7.2 PBS and trypsinized followed by centrifugation at 300 g for a period of 10 min, treated with ANNEXIN V kit and then analyzed by flow cytometry (FACS sort, BD, USA). The mortality and the apoptosis ratio of PCA cells were read in flow cytometry. Detection of configuration by electron microscope 2 Cells were treated with different concentrations of EGCG and CuSO 4 , and collected by centrifugation (300 g, 10 min). The samples were fixed overnight in 2.5% glutaraldehyde at 4 °C, buffered at pH 7.0 with phosphate buffer, washed three times, 15 min each time, then postfixed in 1% osmium tetroxide for 1 h, and washed three times with phosphate buffer (pH 7.0), 15 min each time, then dehydrated for 15 min each time, through a graded ethanol series (of 50%, 70%, 80%, 90%, 95%), followed by treatment with 100% ethanol and 100% acetone, 20 min each time. The samples were infiltrated for 1 h in epon-ethanol (1:1, v/v), and infiltrated 3 h in epon-ethanol (3:1, v/v). Then the samples were infiltrated overnight in epon, and the samples immersing in epon were placed into a 70 °C oven overnight. The samples were cut with a Du Pont diamond knife, and a Sorvall Porter-Blum MT2-B ultra-microtome. The samples were examined with an electron microscope (JEM-1200EX, JEOL, Japan). RESULTS AND DISCUSSION 1 Change of EGCG content in F-12 medium 2 EGCG of F-12 medium was quantified using HPLC. The ratio of EGCG to Cu 2+ was chosen in the present study according to our previous study (Yu et al., 2004 2004 ). We found that EGCG declined rapidly at 37 °C in F-12 medium. After being incubated at 37 °C for 6 h, EGCG degraded from 10 μmol/L to 0.63 μmol/L when incubated with LNCaP cells. EGCG in F-12 medium in the absence of LNCaP cells was substantially lower than that in the presence of LNCaP cells. When the initial concentration of EGCG was 200 μmol/L, the degradation pattern was similar to that for 10 μmol/L. It was found that EGCG declined from 200 μmol/L to 5.98 μmol/L in the absence of LNCaP cells and to 9.64 μmol/L when incubated with LNCaP cells. Treatment of F-12 medium with Cu 2+ caused a significant decrease in EGCG content. In the presence of Cu 2+ , EGCG decreased substantially and was completely degraded at 6 h (Table 1 1 ). P < 0.05 P < 0.01 3 Groups 6 Concentration of EGCG (μmol/L) 2 6 h 2 12 h 2 24 h Without cells With cells Without cells With cells Without cells With cells 10 μmol/L EGCG 0 0.63 (±0.1) ** ** 0 0 0 0 200 μmol/L EGCG 5.98 (±0.9) 9.64 (±1.2) * * 0 0 0 0 320 μmol/L CuSO 4 +200 μmol/L EGCG 6.25 (±0.8) 0 ** ** 0 0 0 0 200 μmol/L EGCG+320 μmol/L CuSO 4 0.99 (±0.06) 0 ** ** 0 0 0 0 150 μmol/L EGCG+32 μmol/L CuSO 4 0.74 (±0.03) 0 ** ** 0 0 0 0 32 μmol/L CuSO 4 +150 μmol/L EGCG 0 0 0 0 0 0 Also, the ratio of EGCG to Cu 2+ and the adding order are the factors in degradation of EGCG. At 12 h and 24 h, EGCG was not detected in all plates (Fig. 1 1 ) while EGCG was present in F-12 medium in a very small amount after 6 h under the present experimental conditions. The degradation products of EGCG remain unidentified. Probably, EGCG was oxidized, chelated and polymerized. As a good chelator, EGCG may chelate with amino acid, protein, metal ion and so on. Hatano et al.( 2003 2003 )’s study showed that albumin of fetal bovine serum chelated with EGCG in cell culture system. Probably, chelation of EGCG may be the main reaction existing in the system. It is speculated that interaction of EGCG or Cu 2+ occurs when they are incubated with LNCaP cells. Effects of EGCG on the growth of LNCaP cells 2 EGCG or Cu 2+ showed dose-dependent suppression on growth of LNCaP cells. When CuSO 4 alone increased from 0.0064 to 320 μmol/L, the death rate of LNCaP cells increased from 21.9% to 79.11%. A similar trend was observed for EGCG when it was incubated with LNCaP cells in the absence of CuSO 4 (Table 2 2 ). Groups Mortality of LNCaP cells (%) 0.0064 μmol/L CuSO 4 21.9 (±1.2) 64 μmol/L CuSO 4 31.98 (±1.0) 320 μmol/L CuSO 4 79.11 (±3.5) 50 μmol/L EGCG 14.7 (±0.9) 150 μmol/L EGCG 62.61 (±2.8) 200 μmol/L EGCG 70.28 (±5.0) 320 μmol/L CuSO 4 +200 μmol/L EGCG 79.24 (±2.7) 200 μmol/L EGCG+320 μmol/L CuSO 4 38.51 (±1.1) 150 μmol/L EGCG+32 μmol/L CuSO 4 68.13 (±1.8) 32 μmol/L CuSO 4 +150 μmol/L EGCG 12.44 (±0.5) No synergistic effect was observed when EGCG and Cu 2+ were incubated together with LNCaP cells. The sequence of adding EGCG and Cu 2+ into the medium had significant impact on the mortality of LNCaP cells. As shown in Table 2 2 , the death rate was 79.24% when 320 μmol/L CuSO 4 was added into the medium followed by addition of 200 μmol/L EGCG. In contrast, the death rate was only 38.5% when 200 μmol/L EGCG was introduced into the medium first followed by addition of 320 μmol/L CuSO 4 . The present results indicated that toxicity of EGCG to LNCaP cells was weakened in the presence of Cu 2+ , and that interaction of EGCG with Cu 2+ had a critical role in the system. In fact, our previous report demonstrated clearly that EGCG chelated strongly with Cu 2+ , thus leading to a decrease in its toxicity to LNCaP cells. The results of gel electrophorsis and flow cytometry showed that the EGCG caused death of LNCaP cells were unlikely to be due to apoptosis (Fig. 2 2 , Fig. 3 3 ). a: LNCaP cells; b: LNCaP cells treated with 32 μmol/L CuSO 4 before 150 μmol/L EGCG was added; c: LNCaP cells incubated with 32 μmol/L CuSO 4 after 150 μmol/L EGCG was added; d: LNCaP cells treated with 200 μmol/L EGCG after 320 μmo/L CuSO 4 was added; e: LNCaP cells incubated with 320 μmol/L CuSO 4 after 200 μmol/L EGCG was added Configuration of LNCaP cells detected by electron microscope 2 Ultrastructure of LNCaP cells was scanned by electron microscope. In the control culture, electron microscope revealed that LNCaP cells had intact cytoplasm membrane, clear cytoplasm, intact subcellular organelles, intact nuclear membrane and normal nucleolus (Fig. 4a 4a ). In contrast, after treatment of LNCaP cells with 10 μmol/L EGCG, the nuclear membrane and chromosome were intact but the cytoplasm became more vacuolated, and the cytoplasm membrane was damaged (Fig. 4b 4b ). Similarly, exposure to 0.0064 μmol/L Cu 2+ led to damage of the cytoplasm membrane of LNCaP cells, but there was no significant change on organelle, nucleus, chromosome and cytoplasm (Fig. 4c 4c , Fig. 4d 4d ). When LNCaP cells were treated with 150 μmol/L EGCG and 32 μmol/L Cu 2+ , the damage was similar to that of the cells incubated only with EGCG (Fig. 4e 4e ). The cytoplasm membrane was almost completely destroyed when the cells were treated first with 150 μmol/L EGCG and then with 32 μmol/L Cu 2+ . In this regard, cytoplasm and organelle were hardly detected at the same time while nucleus and chromosome were intact (Fig. 4f 4f ). When 200 μmol/L EGCG was added into the medium after addition of 320 μmol/L CuSO 4 , karyokinesis of LNCaP cells was not inhibited, and nucleus and chromosome were not damaged (Fig. 4g 4g ). There were many organelles observed in the cytoplasm, which was however, vacuolated; and the cytoplasm membrane was not intact. Furthermore, the cytoplasm membrane was destroyed severely when LNCaP cells were treated with 320 μmol/L Cu 2+ first and then with 200 μmol/L EGCG (Fig. 4h 4h ). The present results demonstrated clearly that toxicity of EGCG to LNCaP cells was associated with its damage to the cytoplasm membrane regardless of the presence or absence of Cu 2+ . There is no study to date examining the bioactivity of EGCG-copper complex and the effect of EGCG degradation compounds on cancer cells. EGCG has two rings and many hydroxyl groups, which can potentially participate in chelation with Cu 2+ and be easily oxidized. As shown in this study, mortality of LNCaP cells was increased in the presence of Cu 2+ , suggesting that EGCG-copper complex might be more toxic to LNCaP cells. As a matter of fact, it is difficult for EGCG to cross the cytoplasm membrane owing to its lipo-soluble character. The present results showed that changes of EGCG contents in F-12 medium without LNCaP cells were quite different from those in F-12 medium with LNCaP cells. It is possible that EGCG had interacted with the cytoplasm membrane, and entered the cells. Electron microscopic observation revealed that the cytoplasm membrane was only damaged but that nucleus and organelle remained intact, demonstrating that the first target of EGCG and its derivatives to LNCaP cells was the cytoplasm membrane. This is in agreement with that of Hoshino et al.( 1999 1999 ) who found that the recycling redox reactions occurring between Cu 2+ and Cu + in the microbe system involving catechins and hydrogen peroxide, caused the cytoplasm membrane damages. Some previous studies showed that EGCG could induce apoptosis of cancer cells, including prostate cancer cell (Roy et al., 2003 2003 ; Paschka et al., 1998 1998 ; Gupta et al., 2000 2000 ; 2003 2003 ; Liang et al., 1997 1997 ; Lin, 2002 2002 ). This was in disagreement with the present observation. Probably, the discrepancy was due to different cells and incubation media and conditions. The present study is the first to examine the cytotoxicity of EGCG-Cu 2+ complex on prostate cancer cells. It was concluded that cytotoxicity of EGCG was mediated by its damage to the cytoplasm membrane of LNCaP cells. References LY Chung , TC Cheung , SK Kong , KP Fung , YM Choy , ZY Chan , TT Kwok . Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells . Life Sciences , 2001 . 68 ( 10 ): 1207 - 1214 . . Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells JY Chung , JO Park , H Phyu , ZG Dong , CS Yang . Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3′-digallate . The FASEB Journal , 2001 . 15 2022 - 2024 . . Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3′-digallate H Cutter , LY Wu , CD Kim , J Morre , DM Morre . Is the cancer protective effect correlated with growth inhibitions by green tea (-)-epigallocatechin gallate mediated through an antioxidant mechanism? . Cancer Lett , 2001 . 162 ( 2 ): 149 - 154 . . Is the cancer protective effect correlated with growth inhibitions by green tea (-)-epigallocatechin gallate mediated through an antioxidant mechanism? S Gupta , N Ahmad , AL Nieminent , H Mukhtar . Growth inhibition, cell-cycle dysregulation, and induction of apoptosis by green tea constituent (-)-epigallocatechin-3-gallate in androgen-sensitive and androgen-insensitive human prostate carcinoma cells . Toxicology and Applied Pharmacology , 2000 . 164 ( 1 ): 82 - 90 . . Growth inhibition, cell-cycle dysregulation, and induction of apoptosis by green tea constituent (-)-epigallocatechin-3-gallate in androgen-sensitive and androgen-insensitive human prostate carcinoma cells S Gupta , T Hussain , H Mukhtar . Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells . Archives of Biochemistry and Biophysics , 2003 . 410 ( 1 ): 177 - 185 . . Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells T Hatano , M Hori , RW Hemingway , T Yoshida . Size exclusion chromatographic analysis of polyphenol-serum albumin complexes . Phytochemistry , 2003 . 63 ( 7 ): 817 - 823 . . Size exclusion chromatographic analysis of polyphenol-serum albumin complexes RA Hiipakka , Z Zhang , D Wei , D Qing , S Liao . Structure-activity relationship for inhibition of human 5α-reductases by polyphenols . Biochemistry Pharmacology , 2002 . 63 ( 6 ): 1165 - 1176 . . Structure-activity relationship for inhibition of human 5α-reductases by polyphenols N Hoshino , T Kimura , A Yamaji , T Ando . Damage to the cytoplasmic membrane of Escherichia Coli by catechin-copper (II) complexes . Free Radic Biol Med , 1999 . 27 ( 11-12 ): 1245 - 1250 . . Damage to the cytoplasmic membrane of Escherichia Coli by catechin-copper (II) complexes K Kenshi , T Hiroshi , Y Nozomi , E Satoru , M Shinya , S Shuneki . Inhibitory action of (-)-epigallocatechin gallate on radiation-induced mouse oncogenic transformation . Cancer Lett , 1997 . 112 ( 2 ): 135 - 139 . . Inhibitory action of (-)-epigallocatechin gallate on radiation-induced mouse oncogenic transformation T Kimura , N Hoshino , A Yamaji , F Hayakawa , T Ando . Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide . Lett Appl Microbiol , 1998 . 27 328 - 330 . . Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide M Kumamoto , T Sonda , K Nagayama , M Tabata . Effects of pH and metal ions on antioxidative activities of catechins . Biosci Biotechnol Biochem , 2001 . 65 126 - 132 . . Effects of pH and metal ions on antioxidative activities of catechins YC Liang , SY Lin-shiau , CF Chen , JK Lin . Suppression of extracellular signals and cell proliferation through EGF receptor binding by (-)-epigallocatechin gallate in human A431 epidermoid carcinoma cells . J Cell Biochem , 1997 . 67 ( 1 ): 55 - 65 . . Suppression of extracellular signals and cell proliferation through EGF receptor binding by (-)-epigallocatechin gallate in human A431 epidermoid carcinoma cells S Liao , Y Umekita , J Guo , JM Kokontis , RA Hiipakka . Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate . Cancer Lett , 1995 . 96 ( 2 ): 239 - 243 . . Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate JK Lin . Cancer chemoprevention by tea polyphenols through modulating signal transduction pathways . Arch Pharm Res , 2002 . 25 ( 5 ): 561 - 571 . . Cancer chemoprevention by tea polyphenols through modulating signal transduction pathways YP Lu , YR Lou , JG Xie , QY Peng , J Liao , CS Yang , MT Huang , AH Conney . Topical applications of caffeine or (-)-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice . Proc Natl Acad Sci USA , 2002 . 99 ( 19 ): 12455 - 12460 . . Topical applications of caffeine or (-)-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice L Mira , MT Fernandez , M Santos , R Rocha , MH Florencio , KR Jennings . Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity . Free Radic Res , 2002 . 36 1199 - 1208 . . Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity K Nakagawa , S Okuda , T Miyazawa . Dose-dependent incorporation of tea catechins, (-)-epigallocatechin-3-gallate and (-)-epigallocatechin into human plasma . Biosci Biotechnol Biochem , 1997 . 61 1981 - 1985 . . Dose-dependent incorporation of tea catechins, (-)-epigallocatechin-3-gallate and (-)-epigallocatechin into human plasma A Nihal , G Sanjay , M Hasan . Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor κb in cancer cells versus normal cells . Archives of Biochemistry and Biophysics , 2000 . 376 ( 2 ): 338 - 346 . . Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor κb in cancer cells versus normal cells AG Paschka , RC Butler , YF Young . Induction of apoptosis in prostate cancer cell lines by the green tea component, (-)-epigallocatechin-3-gallate . Cancer Lett , 1998 . 130 ( 1 ): 1 - 7 . . Induction of apoptosis in prostate cancer cell lines by the green tea component, (-)-epigallocatechin-3-gallate M Roy , S Chakrabarty , D Sinha , RK Bhattacharya , M Siddiqi . Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol . Mutation Research , 2003 . 523-524 33 - 41 . . Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol T Unno , T Takeo . Absorption of (-)-epigallocatechin gallate into the circulation system of rates . Biosci Biotechnol Biochem , 1995 . 59 1558 - 1559 . . Absorption of (-)-epigallocatechin gallate into the circulation system of rates T Unno , K Kondo , H Itakura , T Takeo . Analysis of (-)-epigallocatechin gallate in human serum obtained after ingesting green tea . Biosci Biotechnol Biochem , 1996 . 60 2066 - 2068 . . Analysis of (-)-epigallocatechin gallate in human serum obtained after ingesting green tea CS Yang , MJ Lee , LS Chen . Human salivary tea catechin levels and catechin esterase activities: implication in human cancer prevention studies . Cancer Epidemiology Biomarkers & Prevention , 1999 . 8 83 - 89 . . Human salivary tea catechin levels and catechin esterase activities: implication in human cancer prevention studies H Yoshioka , Y Senba , K Saito , T Kimura , F Hayakawa . Spin-trapping study on the hydroxyl radical formed from a tea catechin-Cu (II) system . Biosci Biotechnol Biochem , 2001 . 65 1697 - 1706 . . Spin-trapping study on the hydroxyl radical formed from a tea catechin-Cu (II) system HN Yu , JJ Yin , SR Shen . Growth inhibition of prostate cancer cells by epigallocatechin gallate in the presence of Cu 2+ . J Agric Food Chem , 2004 . 52 ( 3 ): 464 - 466 . . Growth inhibition of prostate cancer cells by epigallocatechin gallate in the presence of Cu2+ 10.1631/jzus.2005.B0125 Project (No. 3047198) supported by the National Natural Science Foundation of China 2004-02-05 2004-08-24 2005-01-20 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 6B