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Clinical Cancer Research Vol. 11, 6040-6049, August 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Green Tea Component, Catechin, Induces Apoptosis of Human Malignant B Cells via Production of Reactive Oxygen Species

Tomonori Nakazato, Keisuke Ito, Yasuo Ikeda and Masahiro Kizaki

Authors' Affiliation: Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan

Requests for reprints: Masahiro Kizaki, Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3785; Fax: 81-3-3353-3515; E-mail: makizaki{at}sc.itc.keio.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Green tea polyphenol, (–)-epigallocatechin-3-gallate, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of (–)-epigallocatechin-3-gallate as a novel therapeutic agent for the patients with B-cell malignancies including multiple myeloma.

Experimental Design: We investigated the effects of (–)-epigallocatechin-3-gallate on the induction of apoptosis in HS-sultan as well as myeloma cells in vitro and further examined the molecular mechanisms of (–)-epigallocatechin-3-gallate-induced apoptosis.

Results: (–)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant B-cell lines in a dose- and time-dependent manner. (–)-Epigallocatechin-3-gallate-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials ({Delta}{psi}m); the release of cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during (–)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan and RPMI8226 cells as well as fresh myeloma cells. Antioxidant, catalase, and Mn superoxide dismutase significantly reduced ROS production and (–)-epigallocatechin-3-gallate-induced apoptosis, suggesting that ROS plays a key role in (–)-epigallocatechin-3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide (As2O3) and (–)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis compared with As2O3 alone via decreased intracellular reduced glutathione levels and increased production of ROS.

Conclusions: (–)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. In addition, (–)-epigallocatechin-3-gallate enhanced As2O3-induced apoptosis in human multiple myeloma cells.

Tea prepared from the dried leaves of Camellia sinensis exists in two forms, green tea and black tea. Recently, green tea attracted much attention due to its beneficial health effects; the polyphenolic compounds present in green tea include (–)-epigallocatechin-3-gallate, (–)-epicatechin-3-gallate, (–)-epigallocatechin, and epicatechin, which have been shown to have cancer chemopreventive effects in many animal tumor models (1). In fact, epidemiologic studies have shown that green tea consumption can reduce the incidence of cancer and metastases (2). Green tea has unique characteristics as an agent, possessing few adverse effects. In addition, it is inexpensive, can be orally consumed, and has a long history as a beverage of general tolerance among all races. Therefore, green tea seems to have the potential of becoming an ideal agent for chemoprevention (3). Moreover, (–)-epigallocatechin-3-gallate has been shown to induce G0-G1 phase cell cycle arrest in human epidermoid carcinoma cells, thereby inhibiting proliferation and inducing apoptosis in many cancer cells in vitro (3, 4).

Multiple myeloma is plasma cell malignancy derived from terminally differentiated neoplastic B cells that remains fatal despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation (5). Severe adverse effects and complications such as serious infection due to anticancer drugs are also major problems in the clinical setting. In particular, side effects of drugs might be fatal in older patients or immunocompromised patients. In addition, repeated episodes of relapse of the disease may lead to refractory or chemotherapy-resistant multiple myeloma. Therefore, novel effective and less toxic therapeutic strategies with new concepts are desired to improve the outcome of patients with multiple myeloma.

It has been suggested that the production of reactive oxygen species (ROS) is a common mechanism in one of the representative pathways of apoptosis (6). Oxidant and its compounds are capable of depleting reduced glutathione (GSH) or damaging the cellular antioxidant defense system and can directly induce apoptosis (7). (–)-Epigallocatechin-3-gallate is generally well known as an antioxidant; however, it can also behave as a pro-oxidant under certain conditions (2, 8). Recently, arsenic trioxide (As2O3) was reported to inhibit the proliferation of human myeloma cells by induction of apoptosis via intracellular production of ROS (9). It has also been reported that GSH is an inhibitor of As2O3-induced cell death either through conjugating As2O3 or sequestering ROS induced by As2O3 (10, 11). Several investigations suggested that ascorbic acid decreases cellular GSH levels and potentiates As2O3-induced cell death of As2O3-resistant myeloma cells (9). Therefore, we hypothesized that (–)-epigallocatechin-3-gallate-induced apoptosis in myeloma cells is enhanced by As2O3 via production of intracellular ROS.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture. Human malignant B-cell lines including myeloma cells (IM9, RPMI8226, and U266) and Burkitt's lymphoma cells (HS-sultan) were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies) in a humidified atmosphere with 5% CO2. These cell lines were obtained from the Japan Cancer Research Resources Bank (Tokyo, Japan). Bone marrow samples from three patients with multiple myeloma were obtained according to appropriate Human Protection Committee validation and with informed consent. Mononuclear cells were separated by lymphoprep (Nycomed Pharma AS, Oslo, Norway). Cells were maintained in RPMI 1640 with 15% fetal bovine serum in a humidified atmosphere with 5% CO2. The morphology was evaluated by cytospin slide preparations with Giemsa staining and the viability was assessed by trypan blue dye exclusion.

Reagents. Various catechin derivatives including epicatechin, (–)-epicatechin-3-gallate, (–)-epigallocatechin, and (–)-epigallocatechin-3-gallate were purchased from WAKO Chemical Co. (Tokyo, Japan). Catalase, Mn superoxide dismutase (Mn-SOD), and As2O3 were obtained from Sigma Chemical Co. (St. Louis, MO). These agents were dissolved in PBS.

Assays for apoptosis. Apoptosis was determined by morphologic change as well as by staining with Annexin V-FITC and propidium iodide labeling. Apoptotic cells were quantified by Annexin V-FITC and propidium iodide double staining by using a staining kit purchased from PharMingen (San Diego, CA). In addition, induction of apoptosis was detected by DNA fragmentation assay. Cells (1 x 106) were harvested and incubated in a lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 10 mmol/L EDTA, 0.5% Triton 100-X] at 4°C. After centrifugation, supernatants were collected and incubated with RNase A (Sigma Chemical) at 50 µg/mL and proteinase K (Sigma Chemical) for 1 hour at 37°C. DNA samples were subjected to 2% agarose gel and were visualized by ethidium bromide staining. The mitochondrial transmembrane potential ({Delta}{psi}m) was determined by flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA). Briefly, cells were washed twice with PBS and incubated with 1 µg/mL rhodamine-123 (Sigma Chemical) at 37°C for 30 minutes. Rhodamine-123 intensity was determined by flow cytometry.

Cell cycle analysis. Cells (1 x 105) were suspended in hypotonic solution [0.1% Triton X-100, 1 mmol/L Tris-HCl (pH 8.0), 3.4 mmol/L sodium citrate, 0.1 mmol/L EDTA] and stained with 50 µg/mL of propidium iodide. The DNA content was analyzed by flow cytometry. The population of cells in each cell cycle phase was determined using ModiFIT software (Becton Dickinson).

Caspase assays. In the caspase inhibitor assay, cells were pretreated with a synthetic pan-caspase inhibitor (20 µmol/L, Z-VAD-FMK) or caspase-3 inhibitor (50 µmol/L, DEVD-CHO), and caspase-8 and caspase-9 inhibitors (50 µmol/L, Z-IETD-FMK and LEHD-CHO, respectively) for 2 hours before addition of (–)-epigallocatechin-3-gallate (20 µmol/L). All inhibitors were purchased from Calbiochem (La Jolla, CA).

Measurement of intracellular superoxide production. To assess the production of superoxide, control and (–)-epigallocatechin-3-gallate-treated cells were incubated with 5 µmol/L dehydroxyethidium (Molecular Probes, Eugene, OR), which is oxidized to the fluorescent intercalator, ethidium by cellular oxidants, particularly superoxide radicals. Cells (1 x 105) were stained with 5 µmol/L dehydroxyethidium for 30 minutes at 37°C and were washed and resuspended in PBS. The oxidative conversion of dehydroxyethidium to ethidium was measured by flow cytometry (Becton Dickinson).

Measurement of intracellular H2O2 production and reduced glutathione levels. To assess the production of H2O2, control and (–)-epigallocatechin-3-gallate-treated cells were incubated with 20 µmol/L dichlorodihydrofluorescein diacetate (Molecular Probes), which is oxidized to the fluorescent compound, dichlorofluorescein by cellular H2O2. Cells (1 x 105) were stained with 20 µmol/L dichlorodihydrofluorescein diacetate for 30 minutes at 37°C. The oxidative conversion of dichlorodihydrofluorescein diacetate to dichlorofluorescein was measured by flow cytometry (Becton Dickinson). To assess the intracellular GSH level, control and (–)-epigallocatechin-3-gallate-treated cells (1 x 105) were stained with 20 µmol/L 5-chloromethyl fluorescein diacetate (Molecular Probes) for 30 minutes at 37°C and analyzed by flow cytometry (Becton Dickinson).

Cell lysate preparation and Western blotting. Cells were collected by centrifugation at 700 x g for 10 minutes and then the pellets were resuspended in lysis buffer [1% NP40, 1 mmol/L phenylmethylsulfonyl fluoride, 40 mmol/L Tris-HCl (pH 8.0), and 150 mmol/L NaCl] at 4°C for 15 minutes. Mitochondrial and cytosolic fractions were prepared with digitonin-nagarse treatment. Protein concentrations were determined using a protein assay DC system (Bio-Rad, Richmond, CA). Cell lysates (20 µg protein per lane) were fractionated in 12.5% SDS polyacrylamide gels before transfer to the membranes (Immobilon-P membranes, Millipore, Bedford, MA) using standard protocol. Antibody binding was detected by using an enhanced chemiluminescence kit for Western blotting detection with hyper-enhanced chemiluminescence film (Amersham, Buckinghamshire, United Kingdom). Blots were stained with Coomassie brilliant blue to confirm equal amounts of protein extract on each lane. The following antibodies were used in this study: anti-caspase 3, anti-caspase 8, anti-caspase 9, anti-cytochrome c (PharMingen), anti-Bcl-2, anti-Bcl-XL, anti-Mcl-1, anti-AIF, anti-ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bax, and anti-Smac/DIABLO (MBL, Nagoya, Japan).

Statistical analysis. Differences in both variables were analyzed for significance by Student's t test. P < 0.05 was considered as statistical significance.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of catechin on cellular proliferation of various human malignant B cells. We first examined whether the green tea poly phenols and the polyphenolic epicatechin derivatives induced inhibition of the growth of myeloma cells (IM9, RPMI8226, and U266) and Burkitt's lymphoma cells (HS-sultan). Among the structurally related catechins [epicatechin, (–)-epicatechin-3-gallate, (–)-epigallocatechin, and (–)-epigallocatechin-3-gallate], (–)-epigallocatechin-3-gallate was the most potent to inhibit the growth of myeloma cells (data not shown); we thus used (–)-epigallocatechin-3-gallate for the series of experiments. (–)-Epigallocatechin-3-gallate inhibited the cellular growth of all malignant B cells in a dose- and time-dependent manner (Fig. 1A); HS-sultan and IM9 cells were the most sensitive to (–)-epigallocatechin-3-gallate with an IC50 of 17 and 20 µmol/L, respectively. In contrast, RPMI8226 cells were less sensitive to (–)-epigallocatechin-3-gallate (Fig. 1A). Interestingly, cell growth was suppressed as early as 6 hours (data not shown), and the typical morphologic appearance of apoptosis was observed in both (–)-epigallocatechin-3-gallate-sensitive HS-sultan and IM9 cells and (–)-epigallocatechin-3-gallate–less sensitive RPMI8226 cells including condensed chromatin and fragmented nuclei with apoptotic bodies (Fig. 1B).



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  		Fig. 1. (–)-Epigallocatechin-3-gallate (EGCG) inhibits the growth of myeloma cells via the induction of apoptosis. A, various myeloma cells [HS-sultan ({bullet}), IM9 ({blacksquare}), RPMI8226 ({diamondsuit}), and U266 ({blacktriangleup})] were treated with various concentrations (0-100 µmol/L) of (–)-epigallocatechin-3-gallate for 24 hours (left) and with 20 µmol/L (–)-epigallocatechin-3-gallate for indicated times (0-72 hours; right). Cell viability was assessed by trypan blue dye exclusion. Points, means of three different experiments; bars, SD (within 10% of the mean). B, morphologic changes characteristic of apoptosis in HS-sultan, IM9, and RPMI8226 cells. HS-sultan and IM9 cells were treated with 20 µmol/L (–)-epigallocatechin-3-gallate, and RPMI8226 cells were incubated with 100 µmol/L (–)-epigallocatechin-3-gallate for 24 hours, and then cytospin slides were prepared and stained with Giemsa. Original magnification, x1,000. C, cell cycle analysis of HS-sultan cells cultured with (–)-epigallocatechin-3-gallate. Cells were cultured with 20 µmol/L (–)-epigallocatechin-3-gallate for 24 hours and stained with propidium iodide (PI). DNA content was analyzed by means of flow cytometry. G0-G1, G2-M, and S indicate cell phase and sub-G1 DNA content refers to apoptotic cells. Each phase was calculated by using ModiFIT program. Representative experiment repeated thrice with similar results. D, agarose gel electrophoresis showing DNA fragmentation in both HS-sultan and IM9 cells treated with 20 µmol/L (–)-epigallocatechin-3-gallate for 6 hours. E, detection of apoptotic cells by Annexin V and propidium iodide double staining. HS-sultan and RPMI8226 cells were cultured with 20 and 100 µmol/L (–)-epigallocatechin-3-gallate, respectively, for 0, 6, and 12 hours, stained with Annexin V-FITC and propidium iodide labeling and analyzed by flow cytometry. Three independent experiments were done and all gave similar results.

 
(–)-Epigallocatechin-3-gallate-induced G1-G0 cell cycle arrest and subsequent apoptosis. The effects of (–)-epigallocatechin-3-gallate on cell cycle progression were investigated using HS-sultan cells. The cells were treated with 20 µmol/L (–)-epigallocatechin-3-gallate for indicated times and analyzed for cell cycle distribution by means of flow cytometry. Cultivation with (–)-epigallocatechin-3-gallate increased the population of cells in the G0-G1 phase with a reduction of cells in the S phase (Fig. 1C). In addition, a strong induction of apoptosis was shown by the appearance of a haplodiploid DNA peak with sub-G1 DNA contents after (–)-epigallocatechin-3-gallate treatment (Fig. 1C). These results indicate that (–)-epigallocatechin-3-gallate led to cell cycle arrest at the G1 phase followed by apoptosis. We then confirmed (–)-epigallocatechin-3-gallate-induced apoptosis by means of DNA ladder formation and Annexin V/propidium iodide staining. Interestingly, DNA ladder formation was confirmed at a time point as early as 6 hours by electrophoresis of genomic DNA extracted from HS-sultan and IM9 cells treated with 20 µmol/L (–)-epigallocatechin-3-gallate (Fig. 1D). Consistent with these results, Annexin V–positive HS-sultan and RPMI8226 cells dramatically increased in a time-dependent manner (Fig. 1E), indicating that (–)-epigallocatechin-3-gallate rapidly induced apoptosis in both HS-sultan and RPMI8226 cells.

Effects of (–)-epigallocatechin-3-gallate on caspase activity. Caspases are believed to play a central role in mediating various apoptotic responses. To address the apoptotic pathway in (–)-epigallocatechin-3-gallate-treated HS-sultan and RPMI8226 cells, we next examined the activation of caspases by Western blot analysis. The down-regulation of procaspase-3 and procaspase-9 were detected after treatment with 20 µmol/L (–)-epigallocatechin-3-gallate for 4 hours in HS-sultan cells (Fig. 2A, left). In addition, expression of activated caspase-3 was increased in RPMI8226 cells in a dose-dependent manner (Fig. 2A, right). Expression levels of procaspase-8 did not change after treatment of (–)-epigallocatechin-3-gallate. Furthermore, to elucidate the functional role of caspases in (–)-epigallocatechin-3-gallate-induced apoptosis, experiments were done with a series of caspase inhibitors. HS-sultan cells were treated with 20 µmol/L (–)-epigallocatechin-3-gallate for 24 hours, either alone or in combination with Z-VAD-FMK (pan-caspase inhibitor), DEVD-CHO (caspase-3-specific inhibitor), Z-IETD-FMK (caspase-8-specific inhibitor), or LEHD-CHO (caspase-9-specific inhibitor). (–)-Epigallocatechin-3-gallate-induced apoptosis was completely blocked by treatment with Z-VAD-FMK, DEVD-CHO, and LEHD-CHO but not caspase-8-specific inhibitor, Z-IETD-FMK (Fig. 2B). These results suggest that (–)-epigallocatechin-3-gallate-induced apoptosis is associated with the activation of caspase-3 and caspase-9 but not caspase-8.



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  		Fig. 2. Effects of (–)-epigallocatechin-3-gallate (EGCG) on caspase activation. A, Western blot analysis of caspase-3, caspase-9, and caspase-8. Total cellular proteins (20 µg per each lane) were separated on 12.5% SDS- polyacrylamide gels and transferred to the membrane. Protein levels of caspases were detected by Western blot analysis using antibodies against anti-caspase-3, caspase-9, and caspase-8 (left). Protein levels of caspase-3 in (–)-epigallocatechin-3-gallate-treated (0-100 µmol/L) RPMI8226 cells were also examined by Western blotting (right). ß-Actin was used to confirm that equal amounts of protein were in each lane. B, effects of caspase inhibitors on (–)-epigallocatechin-3-gallate-treated HS-sultan cells. Inhibition of (–)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan cells was estimated in a coculture with a series of caspase inhibitors. Cells were preincubated with each caspase inhibitor for 2 hours before addition of 20 µmol/L (–)-epigallocatechin-3-gallate. Columns, means of three different experiments; bars, ±SD. Z-VAD-FMK, pan-caspase inhibitor; DEVD-CHO, caspase-3 inhibitor; Z-IETD-FMK, caspase-8 inhibitor; and LEHD-CHO, caspase-9 inhibitor.

 
Expression of apoptosis-associated proteins. To investigate the molecular mechanism of (–)-epigallocatechin-3-gallate-induced apoptosis in HS-sultan and RPMI8226 cells, the expression of several apoptosis-associated proteins were examined. The expression of the antiapoptotic Bcl-2 and Mcl-1 proteins was decreased in a time-dependent manner by the treatment with (–)-epigallocatechin-3-gallate in both (–)-epigallocatechin-3-gallate-sensitive HS-sultan cells and (–)-epigallocatechin-3-gallate–less sensitive RPMI8226 cells (Fig. 3A and B). In contrast, (–)-epigallocatechin-3-gallate did not modulate the levels of proapoptotic Bax and antiapoptotic Bcl-XL proteins in HS-sultan and RPMI8226 cells.



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  		Fig. 3. Expression of the apoptosis-associated proteins. HS-sultan (A) and RPMI8226 (B) cells were treated with various concentrations of (–)-epigallocatechin-3-gallate (0-100 µmol/L) for 24 hours. Cell lysates (20 µg per each lane) were fractionated on 12.5% SDS-polyacrylamide gels and analyzed by Western blotting with antibodies against Bcl-2, Mcl-1, Bcl-XL, Bax, Procaspase-3, and ß-actin proteins.

 
(–)-Epigallocatechin-3-gallate-induced death signaling is mediated through the mitochondrial pathway. Recent studies have suggested that mitochondria play an essential role in death signal transduction (12). Mitochondrial changes, including permeability transition pore opening and the collapse of the {Delta}{psi}m, result in the release of cytochrome c into the cytosol, which subsequently causes apoptosis by the activation of caspases (13). After treatment with (–)-epigallocatechin-3-gallate for 3 hours, low rhodamine-123 staining in HS-sultan and RPMI8226 cells indicated an increase in the loss of {Delta}{psi}m (Fig. 4A). The loss of {Delta}{psi}m appeared in parallel with the activation of caspase-3 and caspase-9, as well as with apoptosis. In addition, (–)-epigallocatechin-3-gallate induced a substantial release of various mitochondrial apoptogenic proteins, cytochrome c, Smac/DIABLO, and AIF from the mitochondria into the cytosol in HS-sultan cells (Fig. 4B). Bax translocation from the cytosol to mitochondria was also detected after (–)-epigallocatechin-3-gallate treatment (Fig. 4B). These results suggest that mitochondrial dysfunction cause the release of cytochrome c, Smac/DIABLO, and AIF into the cytosol; caspase-9 and caspase-3 were then activated thereby propagating the death signal.



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  		Fig. 4. A, flow cytometric analysis of {Delta}{psi}m as estimated by the Rhodamine-123 intensity. HS-sultan and RPMI8226 cells were cultured with 20 and 100 µmol/L (–)-epigallocatechin-3-gallate (EGCG), respectively, for 3 hours with or without 500 units/mL catalase, and Rhodamine-123 fluorescence was analyzed by flow cytometry. B, Western blot analysis of mitochondrial apoptogenic proteins in (–)-epigallocatechin-3-gallate-treated HS-sultan cells. Cells were incubated with 20 µmol/L (–)-epigallocatechin-3-gallate for 4 hours. The cytosolic and mitochondrial proteins were analyzed by Western blotting with anti-cytochrome c, Smac/DIABLO, Bax, and AIF antibodies.

 
Reactive oxygen species production triggers (–)-epigallocatechin-3-gallate-induced apoptosis. Several investigators have reported that (–)-epigallocatechin-3-gallate-induced apoptosis is often associated with the generation of ROS (2, 14). To investigate the role of ROS in (–)-epigallocatechin-3-gallate-induced apoptosis, we used antioxidants, catalase, and Mn-SOD for further experiments. Treatment of HS-sultan cells with catalase or Mn-SOD, completely blocked (–)-epigallocatechin-3-gallate-induced apoptosis (Fig. 5A). We then analyzed the production of intracellular ROS in control and (–)-epigallocatechin-3-gallate-treated cells. Treatment with (–)-epigallocatechin-3-gallate for 1 hour in HS-sultan and RPMI8226 cells showed dramatic oxidation of dehydroxyethidium to ethidium and resulted in the induction of intracellular superoxide compared with control cells (Fig. 5B). We also detected H2O2 production after (–)-epigallocatechin-3-gallate treatment (Fig. 5C). Furthermore, treatment of HS-sultan and RPMI8226 cells with catalase or Mn-SOD completely blocked the generation of intracellular ROS, the loss of {Delta}{psi}m in (–)-epigallocatechin-3-gallate-induced apoptosis (Fig. 4A and Fig. 5A-C). Furthermore, down-regulation of Bcl-2, Mcl-1, and procaspase-3 after (–)-epigallocatechin-3-gallate treatment were completely prevented by catalase pretreatment (Fig. 5D). Our data indicate that the modulation of molecules involved in the redox system may determine the sensitivity of HS-sultan cells to (–)-epigallocatechin-3-gallate.



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  		Fig. 5. (–)-Epigallocatechin-3-gallate (EGCG) induces apoptosis via production of ROS in both HS-sultan and RPMI8226 cells. A, the antioxidant, catalase and Mn-SOD, blocked (–)-epigallocatechin-3-gallate-induced apoptosis in HS-sultan cells. HS-sultan cells were treated with 20 µmol/L (–)-epigallocatechin-3-gallate alone or together with 500 units/mL catalase or 500 units/mL Mn-SOD for 24 hours. Cell viability was measured by trypan blue dye exclusion. Columns, means of at least three different experiments; bars, ±SD. B-C, to determine the intracellular concentration of ROS and H2O2, HS-sultan and RPMI8226 cells were cultured with dehydroxyethidium or dichlorodihydrofluorescein diacetate, and the fluorescence was measured by flow cytometry. HS-sultan and RPMI8226 cells were treated for 1 hour with 20 or 100 µmol/L (–)-epigallocatechin-3-gallate, respectively, with or without 500 units/mL catalase. D, expression of the various apoptosis-associated proteins in HS-sultan cells treated with (–)-epigallocatechin-3-gallate with or without catalase. Cell lysate (20 µg per lane) were fractionated on 12.5% SDS-polyacrylamide gels and analyzed by Western blotting with antibodies against Bcl-2, Mcl-2, and procaspase-3. E, effects of (–)-epigallocatechin-3-gallate on fresh myeloma samples from patients (Pt.1, Pt.2, and Pt.3) with multiple myeloma. Myeloma cells were separated by Lymphoprep sedimentation procedure and subsequently were cultured with 20 µmol/L (–)-epigallocatechin-3-gallate for 8 hours. Apoptosis was evaluated by Annexin V and propidium iodide double staining and showed fold-increase of apoptotic cells in each case. F, intracellular levels of ROS were measured by flow cytometry.

 
(–)-Epigallocatechin-3-gallate induces apoptosis in fresh myeloma cells with the production of reactive oxygen species. We examined the effect of (–)-epigallocatechin-3-gallate on induction of apoptosis and ROS production in fresh myeloma cells from three patients with multiple myeloma. As was the case for myeloma cell lines, (–)-epigallocatechin-3-gallate induced apoptosis in all three fresh myeloma cells and the production of ROS was also detected (Fig. 5E and F).

(–)-Epigallocatechin-3-gallate markedly enhances As2O3-mediated apoptosis in HS-sultan and RPMI8226 myeloma cells. Recently, As2O3 was reported to inhibit the proliferation of human myeloma cells by induction of apoptosis via intracellular production of ROS (8). We further tested the possibility of using an ROS-generating agent, (–)-epigallocatechin-3-gallate, to enhance the activity of As2O3. The combination of low-dose As2O3 (2 µmol/L) and (–)-epigallocatechin-3-gallate (10 µmol/L) resulted in a significant increase in apoptosis compared with low-dose As2O3 or (–)-epigallocatechin-3-gallate treatment alone (~50% increase) in HS-sultan and RPMI8226 cells (Fig. 6A). We also found that the combination of low-dose As2O3 and (–)-epigallocatechin-3-gallate resulted in higher levels of ROS (O2– and H2O2) production than did As2O3 or (–)-epigallocatechin-3-gallate alone in HS-sultan and IM9 cells (Fig. 6B and C). Treatment of both HS-sultan and RPMI8226 cells with catalase completely blocked the combination of As2O3 and (–)-epigallocatechin-3-gallate-induced apoptosis (Fig. 6A). These results suggest that (–)-epigallocatechin-3-gallate increased the production of ROS and potentiated As2O3-induced cytotoxicity in malignant B cells including myeloma cells. It has been reported that GSH is an inhibitor of As2O3-induced cell death either through conjugating As2O3 or sequestering ROS induced by As2O3 (9, 10). Several studies suggested that ascorbic acid decreases cellular GSH levels and potentiates As2O3-mediated cell death of As2O3-resistant myeloma cells (8). To determine the effects of (–)-epigallocatechin-3-gallate and As2O3 on intracellular GSH levels, we measured the intracellular GSH by fluorescence-activated cell sorting analysis. The intracellular GSH levels after treatment with As2O3 plus (–)-epigallocatechin-3-gallate were considerably decreased in both HS-sultan and IM9 cells compared with those of the treatment with As2O3 or (–)-epigallocatechin-3-gallate alone (Fig. 6D). Low-dose As2O3 (2 µmol/L) or (–)-epigallocatechin-3-gallate (10 µmol/L) alone did not modulate the expression of Mcl-1 and Bcl-2, in HS-sultan and RPMI8226 cells, respectively (Fig. 6E; data not shown). However, combination of low-dose As2O3 and (–)-epigallocatechin-3-gallate decreased the levels of Mcl-1 and Bcl-2 in myeloma cells (Fig. 6E). These results suggest that As2O3 and (–)-epigallocatechin-3-gallate combination treatment enhances apoptosis through decreased intracellular GSH levels and increased production of ROS in myeloma cells.



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  		Fig. 6. (–)-Epigallocatechin-3-gallate (EGCG) potentiates As2O3-mediated apoptosis in myeloma cells. A, (–)-epigallocatechin-3-gallate-sensitive HS-sultan (left) and (–)-epigallocatechin-3-gallate-less sensitive RPMI8226 (right) cells were cultured in the absence or the presence of low-dose As2O3 (2 µmol/L), (–)-epigallocatechin-3-gallate (10 µmol/L), or As2O3 + (–)-epigallocatechin-3-gallate for 24 hours. Cell viability was measured by trypan blue dye exclusion. Apoptotic cells were counted by Annexin V and propidium iodide (PI) double staining and analyzed by fluorescence-activated cell sorting in both HS-sultan and RPMI8226 cells. Three independent experiments were done and all gave similar results. B-C, (–)-epigallocatechin-3-gallate potentiates As2O3-mediated ROS production and depletion of intracellular GSH in HS-sultan (white column) and IM9 (black column) cells. HS-sultan and IM9 cells were cultured in the absence or the presence of low-dose As2O3 (2 µmol/L), (–)-epigallocatechin-3-gallate (10 µmol/L), or As2O3 + (–)-epigallocatechin-3-gallate for 3 hours. To determine the intracellular concentration of ROS (O2–, H2O2), HS-sultan and IM9 cells were stained with dehydroxyethidium (for O2–) or dichlorodihydrofluorescein diacetate (for H2O2), and the fluorescence was measured by flow cytometry. D, to assess the intracellular GSH level, HS-sultan (white column) and IM9 (black column) cells were stained with 20 µmol/L 5-chloromethyl fluorescein diacetate and the fluorescence was measured by flow cytometry. Three independent experiments were done and all gave similar results. E, expression of Bcl-2, Mcl-1, and procaspase-3 in myeloma cells. Cell lysates (20 µg per lane) from HS-sultan cells were fractionated on 12.5% SDS-polyacrylamide gels and analyzed by Western blotting with antibodies against Mcl-1, Bcl-2, Procaspase-3, and ß-actin proteins.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Green tea, obtained from the dried leaves of the plant C. sinensis, is a popularly consumed beverage throughout the world. All true teas may be broadly classified as either green tea or black tea. Extensive in vitro cell culture studies, as well as in vivo studies in animal models, have verified the cancer chemopreventive effects of green tea, and specifically, of its individual polyphenols (15). Epidemiologic studies, although inconclusive, have suggested that green tea may reduce the risks associated with many cancers including bladder, prostate, esophagus, and gastric carcinomas (2). Green tea extract, especially its major polyphenolic component (–)-epigallocatechin-3-gallate, is capable of inhibiting the growth of a variety of mouse and human cancer cells via the induction of apoptosis in vitro (1, 16, 17). The mechanical studies of the effect of (–)-epigallocatechin-3-gallate on cell proliferation have shown the regulatory influence of (–)-epigallocatechin-3-gallate on the levels and activities of nuclear factor-{kappa}B, activator protein, cyclin-dependent kinase inhibitor p21CIP1/WAF1, phosphatidylinositol 3-kinase, and mitogen-activated protein kinases (18–22). However, the influence of (–)-epigallocatechin-3-gallate on signaling molecules directly involved in apoptotic pathway has not been fully examined.

Multiple myeloma is a plasma cell neoplasm derived from clonal B lineage cells. Although many therapeutic advances such as combined chemotherapy and hematopoietic stem cell transplantation have been made to improve the survival rate of patients of multiple myeloma, a higher proportion of patients can not be cured and expected the long-term remission due to drug-resistant disease, minimal residual disease, or serious complications such as systemic infection. Therefore, a new potent therapeutic strategy is needed for the treatment of patients with multiple myeloma.

Recently, there have been introduced various novel antimyeloma agents including As2O3 (8, 9), proteasome inhibitor (PS-341; ref. 23), thalidomide and its immunomodulatory derivatives (24, 25), and histone deacetylase inhibitors (26) to overcome drug resistance of the conventional chemotherapy. Recent studies have shown that these antimyeloma agents induce common apoptotic signals: decrease in the mitochondrial transmembrane potential, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage (27, 28). However, these agents also induce differential upstream signaling cascades that lead to caspase activation.

In this study, we showed that (–)-epigallocatechin-3-gallate rapidly induced apoptotic cell death in human malignant B cells in association with the down-regulation of antiapoptotic protein, Bcl-2 and Mcl-1; Bax translocation from the cytosol to mitochondria; the loss of {Delta}{psi}m; the release of mitochondrial apoptogenic proteins such as cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Bax is a proapoptotic member of Bcl-2 family that resides in the cytosol and translocates to mitochondria during induction of apoptosis (29). It has also been reported that chemoresistant myeloma cells express the higher level of antiapoptotic protein, Bcl-2 or Mcl-1 (28, 30). (–)-Epigallocatechin-3-gallate inhibits the expression of Bcl-2 and Mcl-1during induction of apoptosis in HS-sultan cells. Recent reports suggest that alterations in the ratio between proapoptotic and antiapoptotic members of the Bcl-2 family, rather than the absolute expression level of any single Bcl-2 member, can determine apoptotic sensitivity, which would interfere with the availability and translocation of the Bax protein from the cytosol to mitochondria (31). The ratio of Bax/Bcl-2 or Bax/Mcl-1 protein levels is important for cells undergoing (–)-epigallocatechin-3-gallate-induced apoptosis.

Elevation of intracellular ROS production was also shown during (–)-epigallocatechin-3-gallate-induced apoptosis of myeloma and HS-sultan cells. Various studies have shown that stress-induced changes in {Delta}{psi}m correlate with an increase in ROS and the release of mitochondrial cytochrome c and Smac/DIABLO. The role of ROS in mediating apoptosis in various cancer cells is well established (32, 33). The generation of ROS has been linked to the release of Smac or cytochrome c from mitochondria to the cytosol during apoptosis (34). Antioxidant, Mn-SOD, and catalase significantly blocked ROS production, the loss of {Delta}{psi}m, caspase-3 activation, and (–)-epigallocatechin-3-gallate-induced apoptosis in myeloma cells. Previous studies have shown that both catalase and SOD abrogated ROS generation, and SOD inhibited (–)-epigallocatechin-3-gallate-mediated H2O2 generation (35, 36). In addition, it has been reported that ROS directly down-regulates the Bcl-2 and Mcl-1 levels (37). Therefore, catalase and SOD protected the down-regulation of Bcl-2 and Mcl-1 in (–)-epigallocatechin-3-gallate-treated myeloma cells. These results suggest that ROS plays an upstream important mediator during (–)-epigallocatechin-3-gallate-induced apoptosis in B-cell malignancies including myeloma cells.

Among all of the green tea phenolic compounds, (–)-epigallocatechin-3-gallate is the most potent in terms of the bioactivity, and (–)-epigallocatechin-3-gallate contains the most hydroxyl functional groups in its chemical structure. Previous studies on the antioxidative property of (–)-epigallocatechin-3-gallate have shown both the trapping effect of ROS as well as the inhibitory effect of lipid peroxidation (38). However, after neutralizing the peroxyl or other radicals, (–)-epigallocatechin-3-gallate itself could be converted to phenoxyl radical (39). In addition, under normal physiologic pH condition, (–)-epigallocatechin-3-gallate may undergo auto-oxidation to form dimers, accompanying with the generation of ROS intermediates (40, 41). In the recent investigation, the chemical property of (–)-epigallocatechin-3-gallate as a potential pro-oxidant was highlighted by the blocking effects of GSH and NAC against (–)-epigallocatechin-3-gallate-induced apoptosis (42). It has also been reported that (–)-epigallocatechin-3-gallate may induce the production of H2O2 in the culture media (43, 44).

Oxidative damage has been suggested to be a key mechanism by which As2O3 causes cell death (45). As2O3-induced apoptosis has been shown to be associated with the generation of ROS in several experimental models. Antioxidants and free radical scavengers are able to inhibit apoptosis induced by As2O3 (8, 10). These observations suggest the possibility to develop new therapeutic strategies using the free radical-mediated mechanism of As2O3 to selectively kill cancer cells. Based on the ability of both (–)-epigallocatechin-3-gallate and As2O3 to cause free radical generation, we hypothesized that the combination of As2O3 and (–)-epigallocatechin-3-gallate would enhance the cytotoxic activity in myeloma cells. The combination of As2O3 and (–)-epigallocatechin-3-gallate resulted in a significant increase in apoptosis compared with As2O3 or (–)-epigallocatechin-3-gallate treatment alone in all four investigated-malignant B cell lines. We also found that the combination of As2O3 and (–)-epigallocatechin-3-gallate resulted in higher levels of ROS than did of As2O3 or (–)-epigallocatechin-3-gallate alone. Furthermore, treatment of HS-sultan and IM9 cells with catalase or Mn-SOD completely blocked the combination of As2O3 and (–)-epigallocatechin-3-gallate-induced apoptosis. It has been reported that GSH is an inhibitor of As2O3-induced cell death either through conjugating As2O3 or sequestering ROS induced by As2O3 (9, 10). Some studies suggested that ascorbic acid decreases cellular GSH levels and potentiates As2O3-mediated cell death in As2O3-resistant myeloma cells (8). The intracellular GSH levels after the treatment with As2O3 plus (–)-epigallocatechin-3-gallate were considerably decreased in HS-sultan and IM9 cells compared with those of the treatment with As2O3 or (–)-epigallocatechin-3-gallate alone. Previous study has shown that (–)-epigallocatechin-3-gallate was oxidized by H2O2 to form a cytotoxic o-quinone and reacted with GSH to form glutathione conjugates (46). Therefore, it may be possible that oxidant and its components are depleting GSH in cells treated with (–)-epigallocatechin-3-gallate or As2O3. These findings and previous studies indicate that the combination of As2O3 and (–)-epigallocatechin-3-gallate enhances apoptosis through decreased intracellular GSH levels and increased production of ROS in both cells. Our data suggest that (–)-epigallocatechin-3-gallate increased the production of ROS and potentiated As2O3-induced cytotoxicity. Therefore, it is possible that the combination of (–)-epigallocatechin-3-gallate and ROS-generating agents such as As2O3 or 2-methoxyestradiol (known as a SOD inhibitor) would enhance therapeutic activity and overcome drug resistance in myeloma cells.

A component of green tea, catechin, is a natural compound and seems more safe than popular chemotherapeutic agents. In particular, it might be useful in older patients or in immunocompromised patients because of its safety and lack of known toxicity. Because green tea extracts have already entered phase I trials in patients with solid tumors in the United States (47), it would be useful to design similar clinical trials with myeloma patients to evaluate its antimyeloma effects. Recent studies have indicated that green tea is an effective inhibitor of angiogenesis in vivo (48–50). Thus, (–)-epigallocatechin-3-gallate may also have the antiangiogenic effect against multiple myeloma. Furthermore, the combination of (–)-epigallocatechin-3-gallate and ROS-producing agents may provide a new strategy to enhance therapeutic activity and overcome drug resistance. In conclusion, this component of green tea may have potential as a novel therapeutic agent to replace or augment the more cytotoxic agents currently used to treat the myeloma patients.


   Acknowledgments
 
We thank Kaori Saito for her excellent technical assistance.


   Footnotes
 
Grant support: Ministry of Education, Culture, Sports, Science, and Technology of Japan grant 15659231 (M. Kizaki).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/ 5/04; revised 5/11/05; accepted 5/26/05.


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 Results
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