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Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells
Biochemical and Biophysical Research Communications 362 (2007) 951–957 www.elsevier.com/locate/ybbrc
Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells Chiseko Noda a
c
a,b,*
, Jinsong He b, Tomoko Takano b, Chisato Tanaka c, Toshinori Kondo d, Kaoru Tohyama d, Hirohei Yamamura b,e, Yumi Tohyama c
Department of Nutrition Management, Faculty of Health Science, Hyogo University, Kakogawa, Hyogo 675-0101, Japan b Department of Genomic Science, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan Division of Biochemistry, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan d Department of Laboratory Medicine, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan e Hyogo Prefectural Institute of Public Health and Environmental Sciences, Kobe, Hyogo 650-0032, Japan Received 11 August 2007 Available online 24 August 2007
Abstract ()-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells. 2007 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Cancer-chemoprevention; Green tea; Epigallocahtechin-3-gallate; Reactive oxygen species; Etoposide; B cells
Green tea is a beverage that is commonly consumed in Asian countries. Data from epidemiological studies, although inconclusive, suggest that the consumption of green tea may reduce the incidence of several types of cancer [1,2]. Chemically, the water-extractable fraction of green tea contains several polyphenolic compounds including ()-epicatechin, ()-epigallocatechin, ()-epicatechin3-gallate, and ()-epigallocatechin-3-gallate (EGCG). As EGCG is the most abundant and possesses the most potent anti-tumor properties in the tea polyphenols [2], majority * Corresponding author. Address: Department of Nutrition Management, Faculty of Health Science, Hyogo University, Kakogawa, Hyogo 675-0101, Japan. Fax: +81 79 427 5112. E-mail address: [email protected] (C. Noda).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.079
of the mechanistic studies have focused on this compound. The chemopreventive and chemotherapeutic effects of EGCG have been reported with respect to different malignancies. EGCG has been shown to selectively inhibit cell growth and induce apoptosis in cancer cells without adversely affecting normal cells [3–6]. The anti-tumor effects of EGCG include cell cycle arrest and induction of apoptotic cell death [2,4–8]. Several mechanisms have been proposed concerning these cell biological effects of EGCG, including the direct inhibition of cyclin-dependent protein kinases, the induction of various negative regulators of cell cycle, and depolarization of mitochondrial membranes [9]. In the present study, we investigated the effect of EGCG on apoptotic cell death in human B lymphoblastoid Ramos cells, with particular focus on the involvement of the dys-
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function of mitochondria and generation of reactive oxygen species (ROS). In addition, to elucidate that EGCG has synergistic potential for cancer preventive activity with chemical cancer-preventive agents, we choose etopside, and investigate effect of co-treatment with EGCG and etoposide on apoptosis in B lymphoblastoid cells. Materials and methods Materials. ()-Epigallocatechin-3-gallate (EGCG) from green tea and ()-epicatechin, and diphenylene iodonium chloride (DPI), an inhibitor of the free radical producing NAD(P)H oxidase were purchased from Sigma (St. Louis, MO). Etoposide was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Mouse anti-cytochrome c monoclonal antibody and a rabbit anti-caspase-9 polyclonal antibody were from BD Pharmingen (San Diego, CA). RPMI-1640 medium supplemented with 2 mM glutamine and fetal bovine serum were purchased from Sigma (St. Louis, MO) and Invitorogen Corp. (Carlsbad, CA), respectively. Digitonin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cells and cell culture. The Ramos human lymphoblastoid cell line was maintained in RPMI-1640 medium supplemented with 10% fetal calf serum in 5% CO2 humidified air at 37 C. Detection of apoptosis by morphology and flow cytometry. To determine the proportion of apoptotic cells, cells were treated with several chemicals for indicated times. The cells were harvested for cytospin preparation and stained with May–Gruenwald–Giemsa solution for morphological evaluation. For flow cytometry the treated cells were analyzed with annexin V and propidium iodide (PI) staining using annexin V-FITC Vybrant Apoptosis Assay Kit (Molecular Probes, Eugene, OR) as described previously [10]. Analysis of mitochondrial transmembrane potential. Change of mitochondrial transmembrene potential was monitored by flow cytometry using Mito Tracker Orange CMTMRos (Molecular Probes, Inc., Eugene, OR) as described previously [10]. Briefly, cells were treated with EGCG, and then incubated with Mitotracker Orange for 30 min at 37 C. Fluorescence was measured immediately after the cells had been washed twice with PBS. Determination of ROS production. ROS production was monitored by flow cytometry using 5-(and 6-)-chloromethyl-2 0 ,7 0 -dichlorodihydrofluorescein diacetate, acetyl ester (CM–H2DCFDA) (Molecular Probes, Eugene, OR), a chloromethyl derivative of H2DCFDA. This dye readily diffuses into cells, its acetate groups are cleaved and subsequent oxidation yields a fluorescence adduct that is trapped inside the cells. Thus, the fluorescence intensity is proportional to the amount of peroxide produced by the cells. Cells were treated with EGCG, then incubated with CM–H2DCFDA for further 30 min at 37 C, and subjected to flow cytometry. For microscopic detection of ROS formation, cells were treated with EGCG and incubated with CM–H2DCFDA as described above. After the incubation the living cells were kept on ice and immediately observed with a confocal laser-scanning microscopy. DNA fragmentation assay. Cells were cultured for 16 h with or without the addition of various concentrations of EGCG. The cells were harvested, washed with PBS and lysed in 100 ll of hypotonic buffer containing 10 mM Tris–HCl (pH 7.4), 10 mM EDTA and 0.5 % Triton X-100 on ice for 10 min. Cell lysates were centrifuged at 12,000g for 20 min at 4 C. The supernatant which does not contain high molecular DNA was treated with RNase at 37 C for 1 h and then treated with proteinase K at 37 C for 1 h. DNA was precipitated by adding NaCl and isopropanol. The precipitated DNA was washed with 70% ethanol and analyzed by agarose gel electrophoresis. Western blot analysis. To examine cytosolic cytochrome c released from mitochondria, cells were treated with EGCG and a digitonin-permeabilization technique was used for preparation of cytosolic fraction [10,11]. The supernatants were collected as cytosolic extracts and subjected to Western blot analysis as described previously [10].
For immunoblot analysis of caspase-9, cells were treated with EGCG for 8 h, harvested and lysed in 100 ll of ice-cold lysis buffer (10 mM Tris– HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 2 mM PMSF and 10 lg/ml aprotinin). The lysates were clarified by centrifugation at 12,000g for 10 min at 4 C and subjected to Western blot analysis.
Results EGCG treatment induces apoptosis in human B lymphoblastoid Ramos cells Physiological cell death is characterized by apoptotic morphology, including chromatin condensation, internucleosomal degradation of DNA, and apoptotic body formation. In the initial experiments, we studied the dose- and time-dependency of EGCG-induced cell death in Ramos cells as determined by morphology. As shown in Fig. 1A, treatment with EGCG at the concentrations of more than 40 lM induced complete cell lysis (phase 2 of cell death) through apoptotic nuclear change (phase 1 of cell death) with time course, but treatment with ()-epicatechin even at 200 lM did not (data not shown). To confirm internucleosomal degradation of DNA, Ramos cells were treated with various concentrations of EGCG (20–100 lM) for 16 h, and DNA fragmentation analysis was performed. Clear DNA ladders were detected after treatment with 60–100 lM EGCG (Fig. 1B). High molecular weight DNA was observed in neither the non-treated nor the treated cells, because only fragmented DNA was extracted as described in Materials and methods. Next we examined another early apoptotic marker, translocation of phosphatidylserine from the internal to the external leaflet of the cell membrane by dual staining with fluorescenceconjugated annexin V and propidium iodide (PI). After treatment of cells with EGCG (40 and 60 lM) for 6 h, the percentage of annexin V-positive and PI-negative cells was almost identical to that in a population of non-treated cells, while after 20 h treatment with EGCG the percentage of cells undergoing apoptosis was increased from 1.98% to 24.6% at 40 lM and to 41.3% at 60 lM (Fig. 1C). EGCG treatment induces mitochondrial damage, cytochrome c release and caspase-9 cleavage There is increasing evidence that altered mitochondrial function is linked to apoptosis induced by various stimuli. Thus, to elucidate the possible involvement of mitochondrial damage in EGCG-induced apoptosis, we evaluated mitochondrial transmembrane potential by using MitoTracker Orange. After 2 h treatment with EGCG the incorporation of MitoTracker Orange was measured by flow cytometry. Relative to untreated control cells, cells exposed to EGCG had a sharp decline in MitoTracker Orange fluorescence and the fluorescence intensity shifted to left. The fluorescence intensities were 195.1 in non-treated cells, 50.8 in cells treated with 40 lM EGCG and 35.5 for 80 lM EGCG (Fig. 2A). The decline was maintained up
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Fig. 1. Induction of apoptosis in Ramos cells by EGCG. (A) Cells were cultured in the presence of 80 lM EGCG for 4 or 20 h, and the cells were harvested for cytospin preparation and stained with May–Gruenwald–Giemsa solution. The photos indicate untreated cells (intact cells), the cells after 4 h incubation (phase 1; apoptotic nuclear change) and the cells after 20 h incubation (phase 2; complete cell lysis). The original magnification is 400·. (B) Cells were treated with different concentrations of EGCG (20–100 lM) as indicated for 16 h, fragmented DNA was extracted and analyzed by electrophoresis in 2% agarose gel. The left lane (M) was marker DNA (uX174HaeIII digest). (C) Flow cytometric analysis of EGCG-induced apoptosis in Ramos cells. Cells were treated with EGCG (40 and 60 lM) for 6 and 20 h and then stained with fluorescence-conjugated annexin V and PI. Annexin Vpositve and PI-negative cells were considered apoptotic. The cells, which were negative for both annexin V and PI, were considered alive, and the cells, which were only PI-positive, were considered necrotic. The representative data from three independent experiments are shown.
to 4 h after treatment. These data indicate that the decrease in mitochondrial transmembrane potential occurs within 2 h after EGCG treatment. Next, we studied whether the loss of mitochondrial membrane potential results in the release of cytochrome c from mitochondria into the cytosol. With 50 lM EGCG, cytochrome c released into the cytosol was observed to a slight extent after 6 h. With 100 lM EGCG, a marked release of cytochrome c into the cytosol was detected even at 4 h, although cytochrome c release was negligible at 2 h (Fig. 2B). The release of cytochrome c from mitochondria may involve subsequent activation of caspase-9. Then, we examined the influence of EGCG on cleavage of the inactive pro-form (48 kDa) to the active form (37 kDa) of caspase-9 by immunoblot analysis. As shown in Fig. 2C, 37 kDa caspase-9 corre-
sponding to the active form was detected in EGCG-treated Ramos cells (60–100 lM for 8 h), but not in untreated cells. Appearance of the 37 kDa caspase was time-dependent: this form was detected after 4 h, and its level increased after 6–8 h (data not shown). Taken together, these data suggest that EGCG treatment induces mitochondrial dysfunction, the release of cytochrome c into the cytosol, and then activation of the cascade of caspases. EGCG treatment causes ROS generation and results in apoptosis EGCG not only functions as an antioxidant, but it also possesses the chemical properties of a pro-oxidant. Oxidative stress has been demonstrated to induce mito-
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Fig. 2. Induction of mitochondrial dysfunction and cleavage of pro-caspase-9 in Ramos cells by EGCG. (A) Loss of mitochondrial transmembrane potential. Cells were treated with EGCG (40 and 80 lM) for 2 and 4 h, further incubated with Mitotracker Orange (100 nM) for 30 min at 37 C, and the fluorescence intensity was analyzed by flow cytometry. (B) Release of cytochrome c from mitochondria. Ramos cells were treated with EGCG (50 and 100 lM) for 2, 4 and 6 h and cytosolic cytochrome c was detected by Western blotting. (C) Induction of cleavage of pro-caspase 9 (48 kDa) to 37 kDa form. Ramos cells were treated with different concentrations of EGCG (60–100 lM) as indicated for 8 h, and 48 and 37 kDa forms of caspase 9 were detected by Western blotting. The representative data from three independent experiments are shown.
chondrial permeability transition [12]. We therefore studied the effects of EGCG on the accumulation of intracellular ROS using the fluorescent probe CM–H2DCFDA. As this fluorochrome is taken up into cells, intracellular esterases cleave the diacetate group and CM–H2DCF is formed. CM–H2DCF is oxidized by ROS produced in cells, yielding the fluorescence product. Ramos cells were treated with EGCG and incubated with CM–H2DCFDA, and then the fluorescence was immediately observed using confocal laser-scanning microscopy or quantified by flow cytometry. Fluorescence was noted in almost all cells treated with 80 lM EGCG (Fig. 3A), indicating that treatment with EGCG up-regulated the intracellular level of ROS in Ramos cells. Fig. 3B shows that the level of intracellular ROS was increased by EGCG treatment. At a dose of 80 lM EGCG the intracellular ROS level was higher than the case of 40 lM after 0.5 h treatment, but ROS level was faintly declined after 4 h at 80 lM EGCG (data not shown). The data shown here indicate that the accumulation of ROS by EGCG treatment is an early event. We further examined the effect of DPI, an inhibitor of the free radical producing NAD(P)H oxidase [13], on apoptosis induction of EGCG. Fig. 3C indicates that pretreatment of DPI significantly suppressed the increase in EGCG-induced intracellular ROS level by CM– H2DCFDA assay. Fig. 3D shows that EGCG caused morphological apoptotic change via phase 1 to phase 2 (also shown in Fig. 1A) but pretreatment of DPI delayed the progression of apoptosis from phase 1 to phase 2.
EGCG and etoposide induce apoptotic cell death synergistically Today, several anti-cancer agents are available for clinical use. Most of these agents, however, can cause adverse effects. If EGCG can interact synergistically with anti-cancer agents to improve their cancer preventive activity, it might be possible to reduce the doses of these drugs and thus also reduce their adverse effects. To investigate a possible synergy between EGCG and a chemical cancer preventive agent, we chose the known cancer preventive agent etoposide, which is clinically used against several types of cancer including lymphoma [14]. To determine the threshold concentrations of etoposide and EGCG that induce apoptosis, Ramos cells (1 · 105 cells/ml) were cultured in the presence of various concentrations of etoposide or EGCG for 3–4 days, during which time the cells were not overgrowing, and their morphological changes were observed under a microscope. Apoptotic cells were noted after treatment with EGCG even at a low concentration of 10 lM or after treatment with 40 ng/ml etoposide (data not shown). Flow cytometric analyses revealed that treatment with either EGCG (7.5 lM) alone or etoposide (20 ng/ml) alone for 3 days did not significantly induce cell apoptosis. The percentages of apoptotic (annexin V-positive and PI-negative) cells were 3.9% (control), 4.1% (7.5 lM EGCG), and 7.6% (20 ng/ml etoposide). We found that co-treatment with etoposide and EGCG synergistically induced apoptosis in Ramos cells: the percentage of apoptotic cells was 24.2% (Fig. 4).
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Fig. 3. Induction of ROS generation by EGCG and its suppression by pretreatment of DPI. (A) Cells were treated with or without EGCG (80 lM) for 4 h, incubated for further 30 min with CM–H2DCFDA (1 lM) and then immediately observed with a confocal laser-scanning microscopy. (B) Cells were treated with or without EGCG (40 and 80 lM) for 0.5 h and after further incubation with CM–H2DCFDA (1 lM). The fluorescence in the cells was immediately assayed using flow cytometry. The representative data from three independent experiments are shown. (C) Cells were treated with or without EGCG (40 lM) for1 h, or pretreated with 1 lM DPI for 10 min before EGCG treatment. After further incubation with CM–H2DCFDA (1 lM, the fluorescence in the cells was immediately assayed using flow cytometry. The histogram shows the mean values with the standard deviation of the median fluorescence intensity from three independent experiments. Student’s t-test was performed and the data between ‘untreated’ and ‘EGCG’, and the data between ‘EGCG’ and ‘DPI+EGCG’ were both significantly different (p < 0.01). (D) Cells were treated with EGCG (40 or 80 lM) or pretreated with 1 lM DPI for 10 min before EGCG treatment. Another aliquot was treated only with DPI. After incubation for 20 h, the cells were harvested for cytospin preparation and stained with May–Gruenwald–Giemsa solution. The percentage of apoptotic cells was evaluated according to the morphology: phase 1 (apoptotic nuclear change) and phase 2 (complete cell lysis) as indicated in Fig. 1A.
Discussion
Fig. 4. Synergistic effects of EGCG with etoposide on induction of apoptosis in Ramos cells. Cells were treated with either EGCG 7.5 lM) or etoposide (0.02 lg/ml) alone, or with the co-existence for 3 days and then stained with fluorescence-conjugated annexin V and PI. Apoptosis was quantified by flow cytometry. The representative data from three independent experiments are shown.
Two central pathways have been shown to be involved in the process of apoptotic cell death: one is the direct involvement of caspase proteases (especially caspase-8), and the other is the mitochondrial pathway in which caspases are activated as a downstream event of apoptosis. In addition to these pathways, lysosomes are involved as the primary organelles in apoptotic cell death induced by some signals, such as B cell receptor-induced apoptosis, as reported in our laboratory [10]. The data presented here suggest that mitochondrial dysfunction is the main mechanism involved in apoptotic cell death induced by EGCG treatment in B lymphoblastoid Ramos cells. Furthermore, we have found that the loss of mitochondrial transmembrane potential and the increase of ROS generation are early events caused by EGCG treatment. These findings are comparable with previous findings
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from a study using lymphoma U937 cells treated with the oolong tea polyphenol theasinensin [15]. Although a few previous studies have demonstrated cytochrome c release into the cytosol after EGCG treatment [16,17], the signaling pathway by which EGCG influences the mitochondria is not well established. The following two possibilities are proposed: (i) EGCG increases ROS generation which leads to mitochondrial dysfunction; (ii) Mitochondrial dysfunction is caused by EGCG treatment in some fashion and this results in increase of ROS generation. While tea and other plant polyphenols are generally recognized as antioxidants [2], it is known that tea polyphenols also have pro-oxidant properties [18]. After neutralizing peroxyl and/or other radicals, EGCG itself could be converted to a phenoxyl radical [19]. In addition, under normal physiological conditions, EGCG may undergo auto-oxidation to form dimers, accompanying the generation of ROS intermediates [20]. In fact, recent studies have demonstrated that the cell-killing activity of tea polyphenols, at least in vitro, may be related to their pro-oxidant activity [7,17]. In the present study using Ramos cells, apoptosis induced by EGCG was at least partially prevented by an antioxidant DPI. These data will warrant further studies to clarify whether ROS generation caused by EGCG triggers intracellular apoptotic signaling. In the present study we also found that co-treatment with EGCG and etoposide enhanced apoptosis in Ramos cells (Fig. 4), although single use of etoposide did not cause any increase in the intracellular level of ROS by CM– H2DCFDA assay nor morphological apoptosis (data not shown). Etoposide is clinically used as a topoisomerase II inhibitor [21]. Most of the commonly used inhibitors of topoisomerase II cause severe side effects. Thus, it is noteworthy that co-treatment with EGCG and etoposide considerably lowered the effective concentrations of both substances compared with treatment with either alone. Etoposide has also been demonstrated to cause G2 phase arrest of the cell cycle by inhibiting p34cdc-2 kinase [22] and to activate caspases and other cysteine proteases [23]. The effect of EGCG on the cell cycle is different from that of etoposide: EGCG induces a G0-G1 phase arrest of the cell cycle [4,6], and acts as a topoisomerase I inhibitor in human colon cancer cells [24]. Thus, these differences in the biological activities of EGCG and etoposide may be relevant for their synergic interaction. Given these previous findings, together with the findings of the present study, EGCG could prove to be useful in the treatment of some types of cancer, although further study is needed to clarify the molecular mechanisms underlying the synergic effects of EGCG and cancer-preventive agents and the clinical applications of EGCG. Acknowledgment This study was supported in part by The Osaka Medical Research Foundation for Incurable Diseases.
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Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells Chiseko Noda a
c
a,b,*
, Jinsong He b, Tomoko Takano b, Chisato Tanaka c, Toshinori Kondo d, Kaoru Tohyama d, Hirohei Yamamura b,e, Yumi Tohyama c
Department of Nutrition Management, Faculty of Health Science, Hyogo University, Kakogawa, Hyogo 675-0101, Japan b Department of Genomic Science, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan Division of Biochemistry, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan d Department of Laboratory Medicine, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan e Hyogo Prefectural Institute of Public Health and Environmental Sciences, Kobe, Hyogo 650-0032, Japan Received 11 August 2007 Available online 24 August 2007
Abstract ()-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells. 2007 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Cancer-chemoprevention; Green tea; Epigallocahtechin-3-gallate; Reactive oxygen species; Etoposide; B cells
Green tea is a beverage that is commonly consumed in Asian countries. Data from epidemiological studies, although inconclusive, suggest that the consumption of green tea may reduce the incidence of several types of cancer [1,2]. Chemically, the water-extractable fraction of green tea contains several polyphenolic compounds including ()-epicatechin, ()-epigallocatechin, ()-epicatechin3-gallate, and ()-epigallocatechin-3-gallate (EGCG). As EGCG is the most abundant and possesses the most potent anti-tumor properties in the tea polyphenols [2], majority * Corresponding author. Address: Department of Nutrition Management, Faculty of Health Science, Hyogo University, Kakogawa, Hyogo 675-0101, Japan. Fax: +81 79 427 5112. E-mail address: [email protected] (C. Noda).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.079
of the mechanistic studies have focused on this compound. The chemopreventive and chemotherapeutic effects of EGCG have been reported with respect to different malignancies. EGCG has been shown to selectively inhibit cell growth and induce apoptosis in cancer cells without adversely affecting normal cells [3–6]. The anti-tumor effects of EGCG include cell cycle arrest and induction of apoptotic cell death [2,4–8]. Several mechanisms have been proposed concerning these cell biological effects of EGCG, including the direct inhibition of cyclin-dependent protein kinases, the induction of various negative regulators of cell cycle, and depolarization of mitochondrial membranes [9]. In the present study, we investigated the effect of EGCG on apoptotic cell death in human B lymphoblastoid Ramos cells, with particular focus on the involvement of the dys-
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function of mitochondria and generation of reactive oxygen species (ROS). In addition, to elucidate that EGCG has synergistic potential for cancer preventive activity with chemical cancer-preventive agents, we choose etopside, and investigate effect of co-treatment with EGCG and etoposide on apoptosis in B lymphoblastoid cells. Materials and methods Materials. ()-Epigallocatechin-3-gallate (EGCG) from green tea and ()-epicatechin, and diphenylene iodonium chloride (DPI), an inhibitor of the free radical producing NAD(P)H oxidase were purchased from Sigma (St. Louis, MO). Etoposide was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Mouse anti-cytochrome c monoclonal antibody and a rabbit anti-caspase-9 polyclonal antibody were from BD Pharmingen (San Diego, CA). RPMI-1640 medium supplemented with 2 mM glutamine and fetal bovine serum were purchased from Sigma (St. Louis, MO) and Invitorogen Corp. (Carlsbad, CA), respectively. Digitonin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cells and cell culture. The Ramos human lymphoblastoid cell line was maintained in RPMI-1640 medium supplemented with 10% fetal calf serum in 5% CO2 humidified air at 37 C. Detection of apoptosis by morphology and flow cytometry. To determine the proportion of apoptotic cells, cells were treated with several chemicals for indicated times. The cells were harvested for cytospin preparation and stained with May–Gruenwald–Giemsa solution for morphological evaluation. For flow cytometry the treated cells were analyzed with annexin V and propidium iodide (PI) staining using annexin V-FITC Vybrant Apoptosis Assay Kit (Molecular Probes, Eugene, OR) as described previously [10]. Analysis of mitochondrial transmembrane potential. Change of mitochondrial transmembrene potential was monitored by flow cytometry using Mito Tracker Orange CMTMRos (Molecular Probes, Inc., Eugene, OR) as described previously [10]. Briefly, cells were treated with EGCG, and then incubated with Mitotracker Orange for 30 min at 37 C. Fluorescence was measured immediately after the cells had been washed twice with PBS. Determination of ROS production. ROS production was monitored by flow cytometry using 5-(and 6-)-chloromethyl-2 0 ,7 0 -dichlorodihydrofluorescein diacetate, acetyl ester (CM–H2DCFDA) (Molecular Probes, Eugene, OR), a chloromethyl derivative of H2DCFDA. This dye readily diffuses into cells, its acetate groups are cleaved and subsequent oxidation yields a fluorescence adduct that is trapped inside the cells. Thus, the fluorescence intensity is proportional to the amount of peroxide produced by the cells. Cells were treated with EGCG, then incubated with CM–H2DCFDA for further 30 min at 37 C, and subjected to flow cytometry. For microscopic detection of ROS formation, cells were treated with EGCG and incubated with CM–H2DCFDA as described above. After the incubation the living cells were kept on ice and immediately observed with a confocal laser-scanning microscopy. DNA fragmentation assay. Cells were cultured for 16 h with or without the addition of various concentrations of EGCG. The cells were harvested, washed with PBS and lysed in 100 ll of hypotonic buffer containing 10 mM Tris–HCl (pH 7.4), 10 mM EDTA and 0.5 % Triton X-100 on ice for 10 min. Cell lysates were centrifuged at 12,000g for 20 min at 4 C. The supernatant which does not contain high molecular DNA was treated with RNase at 37 C for 1 h and then treated with proteinase K at 37 C for 1 h. DNA was precipitated by adding NaCl and isopropanol. The precipitated DNA was washed with 70% ethanol and analyzed by agarose gel electrophoresis. Western blot analysis. To examine cytosolic cytochrome c released from mitochondria, cells were treated with EGCG and a digitonin-permeabilization technique was used for preparation of cytosolic fraction [10,11]. The supernatants were collected as cytosolic extracts and subjected to Western blot analysis as described previously [10].
For immunoblot analysis of caspase-9, cells were treated with EGCG for 8 h, harvested and lysed in 100 ll of ice-cold lysis buffer (10 mM Tris– HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 2 mM PMSF and 10 lg/ml aprotinin). The lysates were clarified by centrifugation at 12,000g for 10 min at 4 C and subjected to Western blot analysis.
Results EGCG treatment induces apoptosis in human B lymphoblastoid Ramos cells Physiological cell death is characterized by apoptotic morphology, including chromatin condensation, internucleosomal degradation of DNA, and apoptotic body formation. In the initial experiments, we studied the dose- and time-dependency of EGCG-induced cell death in Ramos cells as determined by morphology. As shown in Fig. 1A, treatment with EGCG at the concentrations of more than 40 lM induced complete cell lysis (phase 2 of cell death) through apoptotic nuclear change (phase 1 of cell death) with time course, but treatment with ()-epicatechin even at 200 lM did not (data not shown). To confirm internucleosomal degradation of DNA, Ramos cells were treated with various concentrations of EGCG (20–100 lM) for 16 h, and DNA fragmentation analysis was performed. Clear DNA ladders were detected after treatment with 60–100 lM EGCG (Fig. 1B). High molecular weight DNA was observed in neither the non-treated nor the treated cells, because only fragmented DNA was extracted as described in Materials and methods. Next we examined another early apoptotic marker, translocation of phosphatidylserine from the internal to the external leaflet of the cell membrane by dual staining with fluorescenceconjugated annexin V and propidium iodide (PI). After treatment of cells with EGCG (40 and 60 lM) for 6 h, the percentage of annexin V-positive and PI-negative cells was almost identical to that in a population of non-treated cells, while after 20 h treatment with EGCG the percentage of cells undergoing apoptosis was increased from 1.98% to 24.6% at 40 lM and to 41.3% at 60 lM (Fig. 1C). EGCG treatment induces mitochondrial damage, cytochrome c release and caspase-9 cleavage There is increasing evidence that altered mitochondrial function is linked to apoptosis induced by various stimuli. Thus, to elucidate the possible involvement of mitochondrial damage in EGCG-induced apoptosis, we evaluated mitochondrial transmembrane potential by using MitoTracker Orange. After 2 h treatment with EGCG the incorporation of MitoTracker Orange was measured by flow cytometry. Relative to untreated control cells, cells exposed to EGCG had a sharp decline in MitoTracker Orange fluorescence and the fluorescence intensity shifted to left. The fluorescence intensities were 195.1 in non-treated cells, 50.8 in cells treated with 40 lM EGCG and 35.5 for 80 lM EGCG (Fig. 2A). The decline was maintained up
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Fig. 1. Induction of apoptosis in Ramos cells by EGCG. (A) Cells were cultured in the presence of 80 lM EGCG for 4 or 20 h, and the cells were harvested for cytospin preparation and stained with May–Gruenwald–Giemsa solution. The photos indicate untreated cells (intact cells), the cells after 4 h incubation (phase 1; apoptotic nuclear change) and the cells after 20 h incubation (phase 2; complete cell lysis). The original magnification is 400·. (B) Cells were treated with different concentrations of EGCG (20–100 lM) as indicated for 16 h, fragmented DNA was extracted and analyzed by electrophoresis in 2% agarose gel. The left lane (M) was marker DNA (uX174HaeIII digest). (C) Flow cytometric analysis of EGCG-induced apoptosis in Ramos cells. Cells were treated with EGCG (40 and 60 lM) for 6 and 20 h and then stained with fluorescence-conjugated annexin V and PI. Annexin Vpositve and PI-negative cells were considered apoptotic. The cells, which were negative for both annexin V and PI, were considered alive, and the cells, which were only PI-positive, were considered necrotic. The representative data from three independent experiments are shown.
to 4 h after treatment. These data indicate that the decrease in mitochondrial transmembrane potential occurs within 2 h after EGCG treatment. Next, we studied whether the loss of mitochondrial membrane potential results in the release of cytochrome c from mitochondria into the cytosol. With 50 lM EGCG, cytochrome c released into the cytosol was observed to a slight extent after 6 h. With 100 lM EGCG, a marked release of cytochrome c into the cytosol was detected even at 4 h, although cytochrome c release was negligible at 2 h (Fig. 2B). The release of cytochrome c from mitochondria may involve subsequent activation of caspase-9. Then, we examined the influence of EGCG on cleavage of the inactive pro-form (48 kDa) to the active form (37 kDa) of caspase-9 by immunoblot analysis. As shown in Fig. 2C, 37 kDa caspase-9 corre-
sponding to the active form was detected in EGCG-treated Ramos cells (60–100 lM for 8 h), but not in untreated cells. Appearance of the 37 kDa caspase was time-dependent: this form was detected after 4 h, and its level increased after 6–8 h (data not shown). Taken together, these data suggest that EGCG treatment induces mitochondrial dysfunction, the release of cytochrome c into the cytosol, and then activation of the cascade of caspases. EGCG treatment causes ROS generation and results in apoptosis EGCG not only functions as an antioxidant, but it also possesses the chemical properties of a pro-oxidant. Oxidative stress has been demonstrated to induce mito-
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Fig. 2. Induction of mitochondrial dysfunction and cleavage of pro-caspase-9 in Ramos cells by EGCG. (A) Loss of mitochondrial transmembrane potential. Cells were treated with EGCG (40 and 80 lM) for 2 and 4 h, further incubated with Mitotracker Orange (100 nM) for 30 min at 37 C, and the fluorescence intensity was analyzed by flow cytometry. (B) Release of cytochrome c from mitochondria. Ramos cells were treated with EGCG (50 and 100 lM) for 2, 4 and 6 h and cytosolic cytochrome c was detected by Western blotting. (C) Induction of cleavage of pro-caspase 9 (48 kDa) to 37 kDa form. Ramos cells were treated with different concentrations of EGCG (60–100 lM) as indicated for 8 h, and 48 and 37 kDa forms of caspase 9 were detected by Western blotting. The representative data from three independent experiments are shown.
chondrial permeability transition [12]. We therefore studied the effects of EGCG on the accumulation of intracellular ROS using the fluorescent probe CM–H2DCFDA. As this fluorochrome is taken up into cells, intracellular esterases cleave the diacetate group and CM–H2DCF is formed. CM–H2DCF is oxidized by ROS produced in cells, yielding the fluorescence product. Ramos cells were treated with EGCG and incubated with CM–H2DCFDA, and then the fluorescence was immediately observed using confocal laser-scanning microscopy or quantified by flow cytometry. Fluorescence was noted in almost all cells treated with 80 lM EGCG (Fig. 3A), indicating that treatment with EGCG up-regulated the intracellular level of ROS in Ramos cells. Fig. 3B shows that the level of intracellular ROS was increased by EGCG treatment. At a dose of 80 lM EGCG the intracellular ROS level was higher than the case of 40 lM after 0.5 h treatment, but ROS level was faintly declined after 4 h at 80 lM EGCG (data not shown). The data shown here indicate that the accumulation of ROS by EGCG treatment is an early event. We further examined the effect of DPI, an inhibitor of the free radical producing NAD(P)H oxidase [13], on apoptosis induction of EGCG. Fig. 3C indicates that pretreatment of DPI significantly suppressed the increase in EGCG-induced intracellular ROS level by CM– H2DCFDA assay. Fig. 3D shows that EGCG caused morphological apoptotic change via phase 1 to phase 2 (also shown in Fig. 1A) but pretreatment of DPI delayed the progression of apoptosis from phase 1 to phase 2.
EGCG and etoposide induce apoptotic cell death synergistically Today, several anti-cancer agents are available for clinical use. Most of these agents, however, can cause adverse effects. If EGCG can interact synergistically with anti-cancer agents to improve their cancer preventive activity, it might be possible to reduce the doses of these drugs and thus also reduce their adverse effects. To investigate a possible synergy between EGCG and a chemical cancer preventive agent, we chose the known cancer preventive agent etoposide, which is clinically used against several types of cancer including lymphoma [14]. To determine the threshold concentrations of etoposide and EGCG that induce apoptosis, Ramos cells (1 · 105 cells/ml) were cultured in the presence of various concentrations of etoposide or EGCG for 3–4 days, during which time the cells were not overgrowing, and their morphological changes were observed under a microscope. Apoptotic cells were noted after treatment with EGCG even at a low concentration of 10 lM or after treatment with 40 ng/ml etoposide (data not shown). Flow cytometric analyses revealed that treatment with either EGCG (7.5 lM) alone or etoposide (20 ng/ml) alone for 3 days did not significantly induce cell apoptosis. The percentages of apoptotic (annexin V-positive and PI-negative) cells were 3.9% (control), 4.1% (7.5 lM EGCG), and 7.6% (20 ng/ml etoposide). We found that co-treatment with etoposide and EGCG synergistically induced apoptosis in Ramos cells: the percentage of apoptotic cells was 24.2% (Fig. 4).
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Fig. 3. Induction of ROS generation by EGCG and its suppression by pretreatment of DPI. (A) Cells were treated with or without EGCG (80 lM) for 4 h, incubated for further 30 min with CM–H2DCFDA (1 lM) and then immediately observed with a confocal laser-scanning microscopy. (B) Cells were treated with or without EGCG (40 and 80 lM) for 0.5 h and after further incubation with CM–H2DCFDA (1 lM). The fluorescence in the cells was immediately assayed using flow cytometry. The representative data from three independent experiments are shown. (C) Cells were treated with or without EGCG (40 lM) for1 h, or pretreated with 1 lM DPI for 10 min before EGCG treatment. After further incubation with CM–H2DCFDA (1 lM, the fluorescence in the cells was immediately assayed using flow cytometry. The histogram shows the mean values with the standard deviation of the median fluorescence intensity from three independent experiments. Student’s t-test was performed and the data between ‘untreated’ and ‘EGCG’, and the data between ‘EGCG’ and ‘DPI+EGCG’ were both significantly different (p < 0.01). (D) Cells were treated with EGCG (40 or 80 lM) or pretreated with 1 lM DPI for 10 min before EGCG treatment. Another aliquot was treated only with DPI. After incubation for 20 h, the cells were harvested for cytospin preparation and stained with May–Gruenwald–Giemsa solution. The percentage of apoptotic cells was evaluated according to the morphology: phase 1 (apoptotic nuclear change) and phase 2 (complete cell lysis) as indicated in Fig. 1A.
Discussion
Fig. 4. Synergistic effects of EGCG with etoposide on induction of apoptosis in Ramos cells. Cells were treated with either EGCG 7.5 lM) or etoposide (0.02 lg/ml) alone, or with the co-existence for 3 days and then stained with fluorescence-conjugated annexin V and PI. Apoptosis was quantified by flow cytometry. The representative data from three independent experiments are shown.
Two central pathways have been shown to be involved in the process of apoptotic cell death: one is the direct involvement of caspase proteases (especially caspase-8), and the other is the mitochondrial pathway in which caspases are activated as a downstream event of apoptosis. In addition to these pathways, lysosomes are involved as the primary organelles in apoptotic cell death induced by some signals, such as B cell receptor-induced apoptosis, as reported in our laboratory [10]. The data presented here suggest that mitochondrial dysfunction is the main mechanism involved in apoptotic cell death induced by EGCG treatment in B lymphoblastoid Ramos cells. Furthermore, we have found that the loss of mitochondrial transmembrane potential and the increase of ROS generation are early events caused by EGCG treatment. These findings are comparable with previous findings
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from a study using lymphoma U937 cells treated with the oolong tea polyphenol theasinensin [15]. Although a few previous studies have demonstrated cytochrome c release into the cytosol after EGCG treatment [16,17], the signaling pathway by which EGCG influences the mitochondria is not well established. The following two possibilities are proposed: (i) EGCG increases ROS generation which leads to mitochondrial dysfunction; (ii) Mitochondrial dysfunction is caused by EGCG treatment in some fashion and this results in increase of ROS generation. While tea and other plant polyphenols are generally recognized as antioxidants [2], it is known that tea polyphenols also have pro-oxidant properties [18]. After neutralizing peroxyl and/or other radicals, EGCG itself could be converted to a phenoxyl radical [19]. In addition, under normal physiological conditions, EGCG may undergo auto-oxidation to form dimers, accompanying the generation of ROS intermediates [20]. In fact, recent studies have demonstrated that the cell-killing activity of tea polyphenols, at least in vitro, may be related to their pro-oxidant activity [7,17]. In the present study using Ramos cells, apoptosis induced by EGCG was at least partially prevented by an antioxidant DPI. These data will warrant further studies to clarify whether ROS generation caused by EGCG triggers intracellular apoptotic signaling. In the present study we also found that co-treatment with EGCG and etoposide enhanced apoptosis in Ramos cells (Fig. 4), although single use of etoposide did not cause any increase in the intracellular level of ROS by CM– H2DCFDA assay nor morphological apoptosis (data not shown). Etoposide is clinically used as a topoisomerase II inhibitor [21]. Most of the commonly used inhibitors of topoisomerase II cause severe side effects. Thus, it is noteworthy that co-treatment with EGCG and etoposide considerably lowered the effective concentrations of both substances compared with treatment with either alone. Etoposide has also been demonstrated to cause G2 phase arrest of the cell cycle by inhibiting p34cdc-2 kinase [22] and to activate caspases and other cysteine proteases [23]. The effect of EGCG on the cell cycle is different from that of etoposide: EGCG induces a G0-G1 phase arrest of the cell cycle [4,6], and acts as a topoisomerase I inhibitor in human colon cancer cells [24]. Thus, these differences in the biological activities of EGCG and etoposide may be relevant for their synergic interaction. Given these previous findings, together with the findings of the present study, EGCG could prove to be useful in the treatment of some types of cancer, although further study is needed to clarify the molecular mechanisms underlying the synergic effects of EGCG and cancer-preventive agents and the clinical applications of EGCG. Acknowledgment This study was supported in part by The Osaka Medical Research Foundation for Incurable Diseases.
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