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Green Tea Component Catechin Induces Apoptosis of Human Green Tea Extract
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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
Abstract Purpose: Green tea polyphenol, (À)-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 (À)-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 (À)-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 (À)-epigallocatechin-3-gallate-induced apoptosis.
Results: (À)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant
B-cell lines in a dose- and time-dependent manner. (À)-Epigallocatechin-3-gallate-induced
apoptosis was in association with the loss of mitochondrial transmembrane potentials (Dw 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 (À)-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 (À)-epigallocatechin-
3-gallate-induced apoptosis, suggesting that ROS plays a key role in (À)-epigallocatechin-
3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide
(As2O3) and (À)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis
compared with As2O3 alone via decreased intracellular reduced glutathione levels and
increased production of ROS.
Conclusions: (À)-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, (À)-epigallocatechin-3-gallate enhanced
As2O3-induced apoptosis in human multiple myeloma cells.
Tea prepared from the dried leaves of Camellia sinensis exists in be orally consumed, and has a long history as a beverage of
two forms, green tea and black tea. Recently, green tea attracted general tolerance among all races. Therefore, green tea seems to
much attention due to its beneficial health effects; the have the potential of becoming an ideal agent for chemo-
polyphenolic compounds present in green tea include (À)- prevention (3). Moreover, (À)-epigallocatechin-3-gallate has
epigallocatechin-3-gallate, (À)-epicatechin-3-gallate, (À)-epi- been shown to induce G0-G1 phase cell cycle arrest in human
gallocatechin, and epicatechin, which have been shown to epidermoid carcinoma cells, thereby inhibiting proliferation
have cancer chemopreventive effects in many animal tumor and inducing apoptosis in many cancer cells in vitro (3, 4).
models (1). In fact, epidemiologic studies have shown that Multiple myeloma is plasma cell malignancy derived from
green tea consumption can reduce the incidence of cancer and terminally differentiated neoplastic B cells that remains fatal
metastases (2). Green tea has unique characteristics as an agent, despite the use of high-dose chemotherapy with hematopoietic
possessing few adverse effects. In addition, it is inexpensive, can 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
Authors’ Affiliation: Division of Hematology, Department of Internal Medicine,
particular, side effects of drugs might be fatal in older patients
Keio University School of Medicine,Tokyo, Japan or immunocompromised patients. In addition, repeated
Received 11/5/04; revised 5/11/05; accepted 5/26/05. episodes of relapse of the disease may lead to refractory or
Grant support: Ministry of Education, Culture, Sports, Science, andTechnology of chemotherapy-resistant multiple myeloma. Therefore, novel
Japan grant 15659231 (M. Kizaki).
effective and less toxic therapeutic strategies with new concepts
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 are desired to improve the outcome of patients with multiple
with 18 U.S.C. Section 1734 solely to indicate this fact. myeloma.
Requests for reprints: Masahiro Kizaki, Division of Hematology, Department of It has been suggested that the production of reactive oxygen
Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, species (ROS) is a common mechanism in one of the
Shinjuku-ku,Tokyo 160-8582, Japan. Phone: 81-3-5363-3785; Fax: 81-3-3353-
3515; E-mail: makizaki@ sc.itc.keio.ac.jp.
representative pathways of apoptosis (6). Oxidant and its
F 2005 American Association for Cancer Research. compounds are capable of depleting reduced glutathione
doi:10.1158/1078-0432.CCR-04-2273 (GSH) or damaging the cellular antioxidant defense system
Clin Cancer Res 2005;11(16) August 15, 2005 6040 www.aacrjournals.org
Catechin Induces Apoptosis of Malignant B Cells
and can directly induce apoptosis (7). (À)-Epigallocatechin- 3-gallate (20 Amol/L). All inhibitors were purchased from Calbiochem
3-gallate is generally well known as an antioxidant; however, it (La Jolla, CA).
can also behave as a pro-oxidant under certain conditions Measurement of intracellular superoxide production. To assess the
production of superoxide, control and (À)-epigallocatechin-3-gallate-
(2, 8). Recently, arsenic trioxide (As2O3) was reported to inhibit
treated cells were incubated with 5 Amol/L dehydroxyethidium
the proliferation of human myeloma cells by induction of
(Molecular Probes, Eugene, OR), which is oxidized to the fluorescent
apoptosis via intracellular production of ROS (9). It has also intercalator, ethidium by cellular oxidants, particularly superoxide
been reported that GSH is an inhibitor of As2O3-induced cell radicals. Cells (1 Â 105) were stained with 5 Amol/L dehydroxy-
death either through conjugating As2O3 or sequestering ROS ethidium for 30 minutes at 37jC and were washed and resuspended in
induced by As2O3 (10, 11). Several investigations suggested PBS. The oxidative conversion of dehydroxyethidium to ethidium was
that ascorbic acid decreases cellular GSH levels and potentiates measured by flow cytometry (Becton Dickinson).
As2O3-induced cell death of As2O3-resistant myeloma cells (9). Measurement of intracellular H2O2 production and reduced gluta-
Therefore, we hypothesized that (À)-epigallocatechin-3-gallate- thione levels. To assess the production of H2O2, control and (À)-
induced apoptosis in myeloma cells is enhanced by As2O3 via epigallocatechin-3-gallate-treated cells were incubated with 20 Amol/L
dichlorodihydrofluorescein diacetate (Molecular Probes), which is
production of intracellular ROS.
oxidized to the fluorescent compound, dichlorofluorescein by cellular
H2O2. Cells (1 Â 105) were stained with 20 Amol/L dichlorodihydro-
Materials and Methods fluorescein diacetate for 30 minutes at 37jC. The oxidative conversion
of dichlorodihydrofluorescein diacetate to dichlorofluorescein was
Cells and cell culture. Human malignant B-cell lines including measured by flow cytometry (Becton Dickinson). To assess the
myeloma cells (IM9, RPMI8226, and U266) and Burkitt’s lymphoma intracellular GSH level, control and (À)-epigallocatechin-3-gallate-
cells (HS-sultan) were cultured in RPMI 1640 (Life Technologies, Grand treated cells (1 Â 105) were stained with 20 Amol/L 5-chloromethyl
Island, NY) supplemented with 10% fetal bovine serum (Life fluorescein diacetate (Molecular Probes) for 30 minutes at 37jC and
Technologies) in a humidified atmosphere with 5% CO2. These cell analyzed by flow cytometry (Becton Dickinson).
lines were obtained from the Japan Cancer Research Resources Bank Cell lysate preparation and Western blotting. Cells were collected by
(Tokyo, Japan). Bone marrow samples from three patients with centrifugation at 700 Â g for 10 minutes and then the pellets were
multiple myeloma were obtained according to appropriate Human resuspended in lysis buffer [1% NP40, 1 mmol/L phenylmethylsulfonyl
Protection Committee validation and with informed consent. Mono- fluoride, 40 mmol/L Tris-HCl (pH 8.0), and 150 mmol/L NaCl] at 4jC
nuclear cells were separated by lymphoprep (Nycomed Pharma AS, for 15 minutes. Mitochondrial and cytosolic fractions were prepared
Oslo, Norway). Cells were maintained in RPMI 1640 with 15% fetal with digitonin-nagarse treatment. Protein concentrations were deter-
bovine serum in a humidified atmosphere with 5% CO2. The mined using a protein assay DC system (Bio-Rad, Richmond, CA). Cell
morphology was evaluated by cytospin slide preparations with Giemsa lysates (20 Ag protein per lane) were fractionated in 12.5% SDS
staining and the viability was assessed by trypan blue dye exclusion. polyacrylamide gels before transfer to the membranes (Immobilon-P
Reagents. Various catechin derivatives including epicatechin, (À)- membranes, Millipore, Bedford, MA) using standard protocol. Anti-
epicatechin-3-gallate, (À)-epigallocatechin, and (À)-epigallocatechin- body binding was detected by using an enhanced chemiluminescence
3-gallate were purchased from WAKO Chemical Co. (Tokyo, Japan). kit for Western blotting detection with hyper-enhanced chemilumines-
Catalase, Mn superoxide dismutase (Mn-SOD), and As2O3 were cence film (Amersham, Buckinghamshire, United Kingdom). Blots were
obtained from Sigma Chemical Co. (St. Louis, MO). These agents were stained with Coomassie brilliant blue to confirm equal amounts of
dissolved in PBS. protein extract on each lane. The following antibodies were used in this
Assays for apoptosis. Apoptosis was determined by morphologic study: anti-caspase 3, anti-caspase 8, anti-caspase 9, anti-cytochrome c
change as well as by staining with Annexin V-FITC and propidium (PharMingen), anti-Bcl-2, anti-Bcl-XL, anti-Mcl-1, anti-AIF, anti-h-actin
iodide labeling. Apoptotic cells were quantified by Annexin V-FITC and (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bax, and anti-Smac/
propidium iodide double staining by using a staining kit purchased DIABLO (MBL, Nagoya, Japan).
from PharMingen (San Diego, CA). In addition, induction of apoptosis Statistical analysis. Differences in both variables were analyzed for
was detected by DNA fragmentation assay. Cells (1 Â 106) were significance by Student’s t test. P < 0.05 was considered as statistical
harvested and incubated in a lysis buffer [10 mmol/L Tris-HCl (pH 7.4), significance.
10 mmol/L EDTA, 0.5% Triton 100-X] at 4jC. After centrifugation,
supernatants were collected and incubated with RNase A (Sigma
Chemical) at 50 Ag/mL and proteinase K (Sigma Chemical) for 1 hour
Results
at 37jC. DNA samples were subjected to 2% agarose gel and were
Effects of catechin on cellular proliferation of various human
visualized by ethidium bromide staining. The mitochondrial trans-
membrane potential (Dw m) was determined by flow cytometry malignant B cells. We first examined whether the green tea poly
(FACSCalibur; Becton Dickinson, San Jose, CA). Briefly, cells were phenols and the polyphenolic epicatechin derivatives induced
washed twice with PBS and incubated with 1 Ag/mL rhodamine-123 inhibition of the growth of myeloma cells (IM9, RPMI8226,
(Sigma Chemical) at 37jC for 30 minutes. Rhodamine-123 intensity and U266) and Burkitt’s lymphoma cells (HS-sultan). Among
was determined by flow cytometry. the structurally related catechins [epicatechin, (À)-epicatechin-
Cell cycle analysis. Cells (1 Â 105) were suspended in hypotonic 3-gallate, (À)-epigallocatechin, and (À)-epigallocatechin-
solution [0.1% Triton X-100, 1 mmol/L Tris-HCl (pH 8.0), 3.4 mmol/L 3-gallate], (À)-epigallocatechin-3-gallate was the most potent
sodium citrate, 0.1 mmol/L EDTA] and stained with 50 Ag/mL of to inhibit the growth of myeloma cells (data not shown); we
propidium iodide. The DNA content was analyzed by flow cytometry. thus used (À)-epigallocatechin-3-gallate for the series of experi-
The population of cells in each cell cycle phase was determined using
ments. (À)-Epigallocatechin-3-gallate inhibited the cellular
ModiFIT software (Becton Dickinson).
Caspase assays. In the caspase inhibitor assay, cells were pretreated growth of all malignant B cells in a dose- and time-dependent
with a synthetic pan-caspase inhibitor (20 Amol/L, Z-VAD-FMK) or manner (Fig. 1A); HS-sultan and IM9 cells were the most
caspase-3 inhibitor (50 Amol/L, DEVD-CHO), and caspase-8 and sensitive to (À)-epigallocatechin-3-gallate with an IC50 of
caspase-9 inhibitors (50 Amol/L, Z-IETD-FMK and LEHD-CHO, 17 and 20 Amol/L, respectively. In contrast, RPMI8226 cells
respectively) for 2 hours before addition of (À)-epigallocatechin- were less sensitive to (À)-epigallocatechin-3-gallate (Fig. 1A).
www.aacrjournals.org 6041 Clin Cancer Res 2005;11(16) August 15, 2005
Cancer Therapy: Preclinical
Fig. 1. (À)-Epigallocatechin-3-gallate (EGCG) inhibits the growth of myeloma cells via the induction of apoptosis. A , various myeloma cells [HS-sultan (.), IM9 (n),
RPMI8226 (x), and U266 (E)] were treated with various concentrations (0-100 Amol/L) of (À)-epigallocatechin-3-gallate for 24 hours (left) and with 20 Amol/L
(À)-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 Amol/L (À)-epigallocatechin-3-gallate, and RPMI8226 cells were incubated with 100 Amol/L (À)-epigallocatechin-3-gallate for 24 hours, and then cytospin slides were
prepared and stained with Giemsa. Original magnification, Â1,000. C, cell cycle analysis of HS-sultan cells cultured with (À)-epigallocatechin-3-gallate. Cells were cultured
with 20 Amol/L (À)-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 Amol/L (À)-epigallocatechin-3-gallate
for 6 hours. E, detection of apoptotic cells byAnnexinV and propidium iodide double staining. HS-sultan and RPMI8226 cells were cultured with 20 and 100 Amol/L
(À)-epigallocatechin-3-gallate, respectively, for 0, 6, and 12 hours, stained with AnnexinV-FITC and propidium iodide labeling and analyzed by flow cytometry. Three
independent experiments were done and all gave similar results.
Clin Cancer Res 2005;11(16) August 15, 2005 6042 www.aacrjournals.org
Catechin Induces Apoptosis of Malignant B Cells
Interestingly, cell growth was suppressed as early as 6 hours epigallocatechin-3-gallate-treated HS-sultan and RPMI8226
(data not shown), and the typical morphologic appearance cells, we next examined the activation of caspases by Western
of apoptosis was observed in both (À)-epigallocatechin- blot analysis. The down-regulation of procaspase-3 and
3-gallate-sensitive HS-sultan and IM9 cells and (À)-epigalloca- procaspase-9 were detected after treatment with 20 Amol/L
techin-3-gallate – less sensitive RPMI8226 cells including (À)-epigallocatechin-3-gallate for 4 hours in HS-sultan cells
condensed chromatin and fragmented nuclei with apoptotic (Fig. 2A, left). In addition, expression of activated caspase-3 was
bodies (Fig. 1B). increased in RPMI8226 cells in a dose-dependent manner
(À)-Epigallocatechin-3-gallate-induced G1-G0 cell cycle arrest (Fig. 2A, right). Expression levels of procaspase-8 did not
and subsequent apoptosis. The effects of (À)-epigallocatechin- change after treatment of (À)-epigallocatechin-3-gallate. Fur-
3-gallate on cell cycle progression were investigated using thermore, to elucidate the functional role of caspases in (À)-
HS-sultan cells. The cells were treated with 20 Amol/L (À)- epigallocatechin-3-gallate-induced apoptosis, experiments were
epigallocatechin-3-gallate for indicated times and analyzed for done with a series of caspase inhibitors. HS-sultan cells were
cell cycle distribution by means of flow cytometry. Cultivation treated with 20 Amol/L (À)-epigallocatechin-3-gallate for 24
with (À)-epigallocatechin-3-gallate increased the population of hours, either alone or in combination with Z-VAD-FMK (pan-
cells in the G0-G1 phase with a reduction of cells in the S phase caspase inhibitor), DEVD-CHO (caspase-3-specific inhibitor),
(Fig. 1C). In addition, a strong induction of apoptosis was Z-IETD-FMK (caspase-8-specific inhibitor), or LEHD-CHO
shown by the appearance of a haplodiploid DNA peak with (caspase-9-specific inhibitor). (À)-Epigallocatechin-3-gallate-
sub-G1 DNA contents after (À)-epigallocatechin-3-gallate induced apoptosis was completely blocked by treatment with
treatment (Fig. 1C). These results indicate that (À)-epigallo- Z-VAD-FMK, DEVD-CHO, and LEHD-CHO but not caspase-
catechin-3-gallate led to cell cycle arrest at the G1 phase 8-specific inhibitor, Z-IETD-FMK (Fig. 2B). These results suggest
followed by apoptosis. We then confirmed (À)-epigallocate- that (À)-epigallocatechin-3-gallate-induced apoptosis is associ-
chin-3-gallate-induced apoptosis by means of DNA ladder ated with the activation of caspase-3 and caspase-9 but not
formation and Annexin V/propidium iodide staining. Inter- caspase-8.
estingly, DNA ladder formation was confirmed at a time Expression of apoptosis-associated proteins. To investigate
point as early as 6 hours by electrophoresis of genomic the molecular mechanism of (À)-epigallocatechin-3-gallate-
DNA extracted from HS-sultan and IM9 cells treated with induced apoptosis in HS-sultan and RPMI8226 cells, the
20 Amol/L (À)-epigallocatechin-3-gallate (Fig. 1D). Consis- expression of several apoptosis-associated proteins were exam-
tent with these results, Annexin V – positive HS-sultan and ined. The expression of the antiapoptotic Bcl-2 and Mcl-1
RPMI8226 cells dramatically increased in a time-dependent proteins was decreased in a time-dependent manner by the
manner (Fig. 1E), indicating that (À)-epigallocatechin- treatment with (À)-epigallocatechin-3-gallate in both (À)-
3-gallate rapidly induced apoptosis in both HS-sultan and epigallocatechin-3-gallate-sensitive HS-sultan cells and (À)-
RPMI8226 cells. epigallocatechin-3-gallate – less sensitive RPMI8226 cells
Effects of (À)-epigallocatechin-3-gallate on caspase activity. (Fig. 3A and B). In contrast, (À)-epigallocatechin-3-gallate did
Caspases are believed to play a central role in mediating various not modulate the levels of proapoptotic Bax and antiapoptotic
apoptotic responses. To address the apoptotic pathway in (À)- Bcl-XL proteins in HS-sultan and RPMI8226 cells.
Fig. 2. Effects of (À)-epigallocatechin-
3-gallate (EGCG) on caspase activation.
A,Western blot analysis of caspase-3,
caspase-9, and caspase-8. Total cellular
proteins (20 Ag 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 (À)-epigallocatechin-3-gallate-treated
(0-100 Amol/L) RPMI8226 cells were also
examined by Western blotting (right).
h-Actin was used to confirm that equal
amounts of protein were in each lane.
B, effects of caspase inhibitors on
(À)-epigallocatechin-3-gallate-treated
HS-sultan cells. Inhibition of
(À)-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 Amol/L (À)-epigallocatechin-
3-gallate. Columns, means of three different
experiments; bars, FSD. Z-VAD-FMK,
pan-caspase inhibitor; DEVD-CHO,
caspase-3 inhibitor; Z-IETD-FMK, caspase-8
inhibitor; and LEHD-CHO, caspase-9
inhibitor.
www.aacrjournals.org 6043 Clin Cancer Res 2005;11(16) August 15, 2005
Cancer Therapy: Preclinical
Fig. 3. Expression of the apoptosis-
associated proteins. HS-sultan (A) and
RPMI8226 (B) cells were treated
with various concentrations of
(À)-epigallocatechin-3-gallate
(0-100 Amol/L) for 24 hours. Cell lysates
(20 Ag per each lane) were fractionated on
12.5% SDS-polyacrylamide gels and
analyzed byWestern blotting with antibodies
against Bcl-2, Mcl-1, Bcl-XL, Bax,
Procaspase-3, and h-actin proteins.
(À)-Epigallocatechin-3-gallate-induced death signaling is caspase-9 and caspase-3 were then activated thereby propagat-
mediated through the mitochondrial pathway. Recent studies ing the death signal.
have suggested that mitochondria play an essential role in death Reactive oxygen species production triggers (À)-epigallocatechin-
signal transduction (12). Mitochondrial changes, including 3-gallate-induced apoptosis. Several investigators have reported
permeability transition pore opening and the collapse of the that (À)-epigallocatechin-3-gallate-induced apoptosis is often
Dw m, result in the release of cytochrome c into the cytosol, associated with the generation of ROS (2, 14). To investigate
which subsequently causes apoptosis by the activation of the role of ROS in (À)-epigallocatechin-3-gallate-induced
caspases (13). After treatment with (À)-epigallocatechin- apoptosis, we used antioxidants, catalase, and Mn-SOD for
3-gallate for 3 hours, low rhodamine-123 staining in HS-sultan further experiments. Treatment of HS-sultan cells with catalase
and RPMI8226 cells indicated an increase in the loss of Dw m or Mn-SOD, completely blocked (À)-epigallocatechin-3-
(Fig. 4A). The loss of Dw m appeared in parallel with the gallate-induced apoptosis (Fig. 5A). We then analyzed the
activation of caspase-3 and caspase-9, as well as with apoptosis. production of intracellular ROS in control and (À)-epigalloca-
In addition, (À)-epigallocatechin-3-gallate induced a substan- techin-3-gallate-treated cells. Treatment with (À)-epigallocate-
tial release of various mitochondrial apoptogenic proteins, chin-3-gallate for 1 hour in HS-sultan and RPMI8226 cells
cytochrome c, Smac/DIABLO, and AIF from the mitochondria showed dramatic oxidation of dehydroxyethidium to ethidium
into the cytosol in HS-sultan cells (Fig. 4B). Bax translocation and resulted in the induction of intracellular superoxide com-
from the cytosol to mitochondria was also detected after (À)- pared with control cells (Fig. 5B). We also detected H2O2 pro-
epigallocatechin-3-gallate treatment (Fig. 4B). These results duction after (À)-epigallocatechin-3-gallate treatment (Fig. 5C).
suggest that mitochondrial dysfunction cause the release Furthermore, treatment of HS-sultan and RPMI8226 cells with
of cytochrome c, Smac/DIABLO, and AIF into the cytosol; catalase or Mn-SOD completely blocked the generation of
Fig. 4. A , flow cytometric analysis of Dw m
as estimated by the Rhodamine-123
intensity. HS-sultan and RPMI8226 cells
were cultured with 20 and 100 Amol/L
(À)-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
(À)-epigallocatechin-3-gallate-treated
HS-sultan cells. Cells were incubated with
20 Amol/L (À)-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.
Clin Cancer Res 2005;11(16) August 15, 2005 6044 www.aacrjournals.org
Catechin Induces Apoptosis of Malignant B Cells
intracellular ROS, the loss of Dw m in (À)-epigallocatechin- induction of apoptosis and ROS production in fresh
3-gallate-induced apoptosis (Fig. 4A and Fig. 5A-C). Further- myeloma cells from three patients with multiple myeloma.
more, down-regulation of Bcl-2, Mcl-1, and procaspase-3 after As was the case for myeloma cell lines, (À)-epigallocate-
(À)-epigallocatechin-3-gallate treatment were completely pre- chin-3-gallate induced apoptosis in all three fresh myeloma
vented by catalase pretreatment (Fig. 5D). Our data indicate cells and the production of ROS was also detected (Fig.
that the modulation of molecules involved in the redox system 5E and F).
may determine the sensitivity of HS-sultan cells to (À)- (À)-Epigallocatechin-3-gallate markedly enhances As2O3-
epigallocatechin-3-gallate. mediated apoptosis in HS-sultan and RPMI8226 myeloma
(À)-Epigallocatechin-3-gallate induces apoptosis in fresh cells. Recently, As2O3 was reported to inhibit the proliferation
myeloma cells with the production of reactive oxygen species. of human myeloma cells by induction of apoptosis via
We examined the effect of (À)-epigallocatechin-3-gallate on intracellular production of ROS (8). We further tested the
Fig. 5. (À)-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 (À)-epigallocatechin-3-gallate-induced apoptosis in HS-sultan cells. HS-sultan cells were treated with 20 Amol/L (À)-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, FSD. 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 1hour with 20 or 100 Amol/L
(À)-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
(À)-epigallocatechin-3-gallate with or without catalase. Cell lysate (20 Ag 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 (À)-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 Amol/L (À)-epigallocatechin-3-gallate for
8 hours. Apoptosis was evaluated byAnnexinV 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.
www.aacrjournals.org 6045 Clin Cancer Res 2005;11(16) August 15, 2005
Cancer Therapy: Preclinical
possibility of using an ROS-generating agent, (À)-epigalloca- (18 – 22). However, the influence of (À)-epigallocatechin-
techin-3-gallate, to enhance the activity of As2O3. The 3-gallate on signaling molecules directly involved in apoptotic
combination of low-dose As2O3 (2 Amol/L) and (À)-epigallo- pathway has not been fully examined.
catechin-3-gallate (10 Amol/L) resulted in a significant increase Multiple myeloma is a plasma cell neoplasm derived from
in apoptosis compared with low-dose As2O3 or (À)-epigallo- clonal B lineage cells. Although many therapeutic advances
catechin-3-gallate treatment alone (f50% increase) in HS- such as combined chemotherapy and hematopoietic stem cell
sultan and RPMI8226 cells (Fig. 6A). We also found that the transplantation have been made to improve the survival rate of
combination of low-dose As2O3 and (À)-epigallocatechin- patients of multiple myeloma, a higher proportion of patients
À
3-gallate resulted in higher levels of ROS (O2 and H2O2) can not be cured and expected the long-term remission due
production than did As2O3 or (À)-epigallocatechin-3-gallate to drug-resistant disease, minimal residual disease, or serious
alone in HS-sultan and IM9 cells (Fig. 6B and C). Treatment of complications such as systemic infection. Therefore, a new
both HS-sultan and RPMI8226 cells with catalase completely potent therapeutic strategy is needed for the treatment of
blocked the combination of As2O3 and (À)-epigallocatechin-3- patients with multiple myeloma.
gallate-induced apoptosis (Fig. 6A). These results suggest that Recently, there have been introduced various novel anti-
(À)-epigallocatechin-3-gallate increased the production of myeloma agents including As2O3 (8, 9), proteasome inhibitor
ROS and potentiated As2O3-induced cytotoxicity in malignant (PS-341; ref. 23), thalidomide and its immunomodulatory
B cells including myeloma cells. It has been reported that derivatives (24, 25), and histone deacetylase inhibitors (26) to
GSH is an inhibitor of As2O3-induced cell death either overcome drug resistance of the conventional chemotherapy.
through conjugating As2O3 or sequestering ROS induced by Recent studies have shown that these antimyeloma agents
As2O3 (9, 10). Several studies suggested that ascorbic acid induce common apoptotic signals: decrease in the mitochon-
decreases cellular GSH levels and potentiates As2O3-mediated drial transmembrane potential, caspase-3 activation, and
cell death of As2O3-resistant myeloma cells (8). To determine poly(ADP-ribose) polymerase cleavage (27, 28). However,
the effects of (À)-epigallocatechin-3-gallate and As2O3 on these agents also induce differential upstream signaling
intracellular GSH levels, we measured the intracellular GSH cascades that lead to caspase activation.
by fluorescence-activated cell sorting analysis. The intracellular In this study, we showed that (À)-epigallocatechin-3-gallate
GSH levels after treatment with As2O3 plus (À)-epigallocatechin- rapidly induced apoptotic cell death in human malignant B
3-gallate were considerably decreased in both HS-sultan cells in association with the down-regulation of antiapoptotic
and IM9 cells compared with those of the treatment with protein, Bcl-2 and Mcl-1; Bax translocation from the cytosol to
As2O3 or (À)-epigallocatechin-3-gallate alone (Fig. 6D). Low- mitochondria; the loss of Dw m; the release of mitochondrial
dose As2O3 (2 Amol/L) or (À)-epigallocatechin-3-gallate apoptogenic proteins such as cytochrome c, Smac/DIABLO,
(10 Amol/L) alone did not modulate the expression of Mcl-1 and AIF from mitochondria into the cytosol; and the
and Bcl-2, in HS-sultan and RPMI8226 cells, respectively activation of caspase-3 and caspase-9. Bax is a proapoptotic
(Fig. 6E; data not shown). However, combination of low-dose member of Bcl-2 family that resides in the cytosol and
As2O3 and (À)-epigallocatechin-3-gallate decreased the levels translocates to mitochondria during induction of apoptosis
of Mcl-1 and Bcl-2 in myeloma cells (Fig. 6E). These results (29). It has also been reported that chemoresistant myeloma
suggest that As2O3 and (À)-epigallocatechin-3-gallate combi- cells express the higher level of antiapoptotic protein, Bcl-2 or
nation treatment enhances apoptosis through decreased intra- Mcl-1 (28, 30). (À)-Epigallocatechin-3-gallate inhibits the
cellular GSH levels and increased production of ROS in expression of Bcl-2 and Mcl-1during induction of apoptosis in
myeloma cells. HS-sultan cells. Recent reports suggest that alterations in the
ratio between proapoptotic and antiapoptotic members of the
Discussion Bcl-2 family, rather than the absolute expression level of any
single Bcl-2 member, can determine apoptotic sensitivity,
Green tea, obtained from the dried leaves of the plant C. which would interfere with the availability and translocation
sinensis, is a popularly consumed beverage throughout the of the Bax protein from the cytosol to mitochondria (31).
world. All true teas may be broadly classified as either green tea The ratio of Bax/Bcl-2 or Bax/Mcl-1 protein levels is important
or black tea. Extensive in vitro cell culture studies, as well as for cells undergoing (À)-epigallocatechin-3-gallate-induced
in vivo studies in animal models, have verified the cancer apoptosis.
chemopreventive effects of green tea, and specifically, of its Elevation of intracellular ROS production was also shown
individual polyphenols (15). Epidemiologic studies, although during (À)-epigallocatechin-3-gallate-induced apoptosis of
inconclusive, have suggested that green tea may reduce the myeloma and HS-sultan cells. Various studies have shown
risks associated with many cancers including bladder, prostate, that stress-induced changes in Dw m correlate with an increase
esophagus, and gastric carcinomas (2). Green tea extract, in ROS and the release of mitochondrial cytochrome c and
especially its major polyphenolic component (À)-epigalloca- Smac/DIABLO. The role of ROS in mediating apoptosis
techin-3-gallate, is capable of inhibiting the growth of a variety in various cancer cells is well established (32, 33). The
of mouse and human cancer cells via the induction of apoptosis generation of ROS has been linked to the release of Smac or
in vitro (1, 16, 17). The mechanical studies of the effect of cytochrome c from mitochondria to the cytosol during
(À)-epigallocatechin-3-gallate on cell proliferation have shown apoptosis (34). Antioxidant, Mn-SOD, and catalase signifi-
the regulatory influence of (À)-epigallocatechin-3-gallate on cantly blocked ROS production, the loss of Dw m, caspase-3
the levels and activities of nuclear factor-nB, activator protein, activation, and (À)-epigallocatechin-3-gallate-induced apopto-
cyclin-dependent kinase inhibitor p21CIP1/WAF1 , phosphatidy- sis in myeloma cells. Previous studies have shown that both
linositol 3-kinase, and mitogen-activated protein kinases catalase and SOD abrogated ROS generation, and SOD
Clin Cancer Res 2005;11(16) August 15, 2005 6046 www.aacrjournals.org
Catechin Induces Apoptosis of Malignant B Cells
Fig. 6. (À)-Epigallocatechin-3-gallate (EGCG) potentiates As2O3-mediated apoptosis in myeloma cells. A , (À)-epigallocatechin-3-gallate-sensitive HS-sultan
(left) and (À)-epigallocatechin-3-gallate-less sensitive RPMI8226 (right) cells were cultured in the absence or the presence of low-dose As2O3 (2 Amol/L),
(À)-epigallocatechin-3-gallate (10 Amol/L), orAs2O3 + (À)-epigallocatechin-3-gallate for 24 hours. Cell viability was measured by trypan blue dye exclusion. Apoptotic cells
were counted byAnnexinV 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, (À)-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 Amol/L), (À)-epigallocatechin-3-gallate (10 Amol/L), or As2O3 + (À)-epigallocatechin-3-gallate for 3 hours. To determine the intracellular concentration of ROS
(OÀ, H2O2), HS-sultan and IM9 cells were stained with dehydroxyethidium (for OÀ) or dichlorodihydrofluorescein diacetate (for H2O2), and the fluorescence was measured by
2 2
flow cytometry. D, to assess the intracellular GSH level, HS-sultan (white column) and IM9 (black column) cells were stained with 20 Amol/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 Ag per lane) from HS-sultan cells were fractionated on 12.5% SDS-polyacrylamide gels and analyzed byWestern blotting with
antibodies against Mcl-1, Bcl-2, Procaspase-3, and h-actin proteins.
www.aacrjournals.org 6047 Clin Cancer Res 2005;11(16) August 15, 2005
Cancer Therapy: Preclinical
inhibited (À)-epigallocatechin-3-gallate-mediated H2O2 gener- As2O3 and (À)-epigallocatechin-3-gallate-induced apoptosis. It
ation (35, 36). In addition, it has been reported that ROS has been reported that GSH is an inhibitor of As2O3-induced
directly down-regulates the Bcl-2 and Mcl-1 levels (37). cell death either through conjugating As2O3 or sequestering
Therefore, catalase and SOD protected the down-regulation of ROS induced by As2O3 (9, 10). Some studies suggested that
Bcl-2 and Mcl-1 in (À)-epigallocatechin-3-gallate-treated mye- ascorbic acid decreases cellular GSH levels and potentiates
loma cells. These results suggest that ROS plays an upstream As2O3-mediated cell death in As2O3-resistant myeloma cells
important mediator during (À)-epigallocatechin-3-gallate- (8). The intracellular GSH levels after the treatment with
induced apoptosis in B-cell malignancies including myeloma cells. As2O3 plus (À)-epigallocatechin-3-gallate were considerably
Among all of the green tea phenolic compounds, (À)- decreased in HS-sultan and IM9 cells compared with those of
epigallocatechin-3-gallate is the most potent in terms of the the treatment with As2O3 or (À)-epigallocatechin-3-gallate
bioactivity, and (À)-epigallocatechin-3-gallate contains the alone. Previous study has shown that (À)-epigallocatechin-
most hydroxyl functional groups in its chemical structure. 3-gallate was oxidized by H2O2 to form a cytotoxic o-quinone
Previous studies on the antioxidative property of (À)-epigallo- and reacted with GSH to form glutathione conjugates (46).
catechin-3-gallate have shown both the trapping effect of ROS Therefore, it may be possible that oxidant and its components
as well as the inhibitory effect of lipid peroxidation (38). are depleting GSH in cells treated with (À)-epigallocatechin-
However, after neutralizing the peroxyl or other radicals, (À)- 3-gallate or As2O3. These findings and previous studies
epigallocatechin-3-gallate itself could be converted to phenoxyl indicate that the combination of As2O3 and (À)-epigalloca-
radical (39). In addition, under normal physiologic pH techin-3-gallate enhances apoptosis through decreased intra-
condition, (À)-epigallocatechin-3-gallate may undergo auto- cellular GSH levels and increased production of ROS in both
oxidation to form dimers, accompanying with the generation of cells. Our data suggest that (À)-epigallocatechin-3-gallate
ROS intermediates (40, 41). In the recent investigation, the increased the production of ROS and potentiated As2O3-
chemical property of (À)-epigallocatechin-3-gallate as a poten- induced cytotoxicity. Therefore, it is possible that the
tial pro-oxidant was highlighted by the blocking effects of GSH combination of (À)-epigallocatechin-3-gallate and ROS-
and NAC against (À)-epigallocatechin-3-gallate-induced apo- generating agents such as As2O3 or 2-methoxyestradiol
ptosis (42). It has also been reported that (À)-epigallocatechin- (known as a SOD inhibitor) would enhance therapeutic
3-gallate may induce the production of H2O2 in the culture activity and overcome drug resistance in myeloma cells.
media (43, 44). A component of green tea, catechin, is a natural compound
Oxidative damage has been suggested to be a key and seems more safe than popular chemotherapeutic agents. In
mechanism by which As2O3 causes cell death (45). As2O3- particular, it might be useful in older patients or in
induced apoptosis has been shown to be associated with the immunocompromised patients because of its safety and lack
generation of ROS in several experimental models. Antiox- of known toxicity. Because green tea extracts have already
idants and free radical scavengers are able to inhibit apoptosis entered phase I trials in patients with solid tumors in the
induced by As2O3 (8, 10). These observations suggest the United States (47), it would be useful to design similar clinical
possibility to develop new therapeutic strategies using the free trials with myeloma patients to evaluate its antimyeloma
radical-mediated mechanism of As2O3 to selectively kill cancer effects. Recent studies have indicated that green tea is an
cells. Based on the ability of both (À)-epigallocatechin- effective inhibitor of angiogenesis in vivo (48 – 50). Thus, (À)-
3-gallate and As2O3 to cause free radical generation, we epigallocatechin-3-gallate may also have the antiangiogenic
hypothesized that the combination of As2O3 and (À)- effect against multiple myeloma. Furthermore, the combination
epigallocatechin-3-gallate would enhance the cytotoxic activity of (À)-epigallocatechin-3-gallate and ROS-producing agents
in myeloma cells. The combination of As2O3 and (À)- may provide a new strategy to enhance therapeutic activity and
epigallocatechin-3-gallate resulted in a significant increase in overcome drug resistance. In conclusion, this component of
apoptosis compared with As2O3 or (À)-epigallocatechin- green tea may have potential as a novel therapeutic agent to
3-gallate treatment alone in all four investigated-malignant replace or augment the more cytotoxic agents currently used to
B cell lines. We also found that the combination of As2O3 and treat the myeloma patients.
(À)-epigallocatechin-3-gallate resulted in higher levels of ROS
than did of As2O3 or (À)-epigallocatechin-3-gallate alone. Acknowledgments
Furthermore, treatment of HS-sultan and IM9 cells with
catalase or Mn-SOD completely blocked the combination of We thank Kaori Saito for her excellent technical assistance.
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