Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea...

Mutation Research 523–524 (2003) 33–41

Anticlastogenic, antigenotoxic and apoptotic activity ofepigallocatechin gallate: a green tea polyphenol

Madhumita Roy, Sutapa Chakrabarty, Dona Sinha,Rathin Kumar Bhattacharya, Maqsood Siddiqi∗

Department of Environmental Carcinogenesis and Toxicology, Chittaranjan National Cancer Institute,37 S.P. Mukherjee Road, Kolkata 700 026, India

Received 23 April 2002; received in revised form 8 August 2002; accepted 15 August 2002

Abstract

Modulation of events characteristic of carcinogenesis or of cancer cells is being emphasized as a rational strategy tocontrol cancer. Green tea polyphenol epigallocatechin gallate (EGCG) has been shown to be highly active as a cancerchemopreventive agent. Certain cellular and molecular events relevant to carcinogenesis are also modified by EGCG. Thepresent investigation was carried out to examine the effects of EGCG on the cytogenetic change and DNA damage inducedby toxicant H2O2 and carcinogenN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster V-79 cells in culture.Cytogenetic change as evident by the formation of micronuclei and DNA damage in the form of comet tail length during singlecell gel electrophoresis was found to be significantly suppressed by EGCG in a dose dependent manner. Cells preincubatedwith EGCG were protected from subsequent damage by the genotoxic agents. Apoptosis, a highly organized physiologicalmechanism to eliminate injured or abnormal cells, is also implicated in multistage carcinogenesis. Initiated cells, cells atpromotional stage or fully transformed cells can be eliminated through apoptosis. It was observed that EGCG suppressedgrowth and proliferation of K-562 cells derived from human chronic myelogenic leukemia. Morphological features of treatedcells and characteristic DNA fragmentation revealed that the cytotoxicity was due to induction of apoptosis. This was mediatedby activation of caspase 3 and caspase 8. Results show that EGCG not only protects normal cells against genotoxic hazardbut also eliminate cancer cells through induction of apoptosis.© 2002 Elsevier Science B.V. All rights reserved.

Keywords: Micronuclei; DNA damage; Comet tail; Apoptosis; EGCG; V-79; K-562

1. Introduction

Modulation of events characteristic of carcino-genesis or of cancer cells is being emphasized as arational strategy to combat cancer. Changes in ploidy

∗ Corresponding author. Present address: Bose Institute, Cente-nary Building, P-1/12 C.I.T. Scheme VII-M, Kolkata 700 054,India. Tel.:+91-33-334-7434; fax:+91-33-334-3886.E-mail addresses: [email protected],[email protected] (M. Siddiqi).

of cells, non-random chromosome aberration andDNA damage are frequently associated with chemicaltoxicity and carcinogenesis. Prevention of manifesta-tion of these events may prevent induction of cancer.Apoptosis, a programmed cell death[1,2], also playsan essential role as a protective mechanism againstcarcinogenesis by eliminating genetically damagedcells, initiated cells or cells progressed to malignancy[3]. Induction of apoptosis thus is a highly desirablemode as a chemotherapeutic as well as a chemo-preventive strategy for cancer control. Indeed many

0027-5107/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.doi:10.1016/S0027-5107(02)00319-6

34 M. Roy et al. / Mutation Research 523–524 (2003) 33–41

chemopreventive agents act through the induction ofapoptosis as a mechanism to suppress carcinogenesis[4,5].

Epidemiological, in vitro cell culture and in vivoanimal studies have shown that green tea polyphenols,particularly the major polyphenol (−)-epigallocate-chin-3-gallate (EGCG), possess significant cancerchemopreventive activity[6,7]. It elicits a variety ofcellular and molecular responses[8] which includeantimutagenic activity[9], suppression of oxidativeDNA damage[10,11]and induction of apoptosis in tu-mor cells[12–15]. The present study was designed toexamine the effects of EGCG on cytokinesis-blockedmicronuclei formation and DNA damage in Chi-nese hamster V-79 cells induced by H2O2 andN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Therole of EGCG in the induction of apoptosis in humanleukemic cell line K-562 was also examined. Resultsshow that EGCG protects cells against genotoxic haz-ard as well as induces tumor cell death via apoptosis.

2. Materials and methods

2.1. Chemicals

MEM, RPMI 1640, fetal bovine serum (FBS),gentamycin, penicillin, streptomycin, trypan blue,agarose and Tris were purchased from GIBCO-BRLIndia Pvt. Ltd. (New Delhi, India). Cytochalasin B,EGCG, MNNG, Triton X-100, RNase A, proteinaseK, propidium iodide and ethidium bromide were ob-tained from Sigma–Aldrich (St. Louis, MO, USA).[Methyl-3H]thymidine (17.2 Ci/mmol) was obtainedfrom BRIT (Mumbai, India). Caspase 3 and caspase 8kits were purchased from Santa Cruz BiotechnologyInc. (Santa Cruz, CA, USA). Other reagents were ofanalytical grade and procured locally.

2.2. Cell cultures

Chinese hamster lung fibroblast cells (V-79) weremaintained in MEM supplemented with 10% heat in-activated FBS and gentamycin (40�g/ml), penicillin(100 units/ml) and streptomycin (10�g/ml). Chronicmyelogenic human leukemia cells (K-562) were main-tained in RPMI 1640 supplemented with 15% heat in-activated FBS and similar doses of antibiotics as used

for V-79. Cells were grown at 37◦C in a humidifiedatmosphere of 5% CO2/95% air.

2.3. Treatment

Exponentially growing V-79 cells were seeded in35 mm petridishes at a cell density of 5× 105 cellsper plate. After 24 h of growth, cells were treated for1 h with various concentrations of H2O2 or MNNG inthe absence or presence of various doses of EGCG. Inanother experiment, cells were pretreated with differ-ent doses of EGCG for 1 h and then washed with freshmedium and subsequently treated with a fixed con-centration of H2O2 or MNNG. Exponentially growingK-562 cells(1 × 104) were seeded in 96-well platesand after 48 h of growth treated with a series of con-centration of EGCG. Cells were allowed to grow for afurther period of 24 h before they were processed fordifferent analyses.

2.4. Scoring for micronuclei

The procedure provided by Fenech and Morley[16]was followed to detect micronucleus. V-79 cells weretreated with 3�g/ml cytochalasin B to block cytoki-nesis, and harvested 24 h later. Cells were then fixedin methanol:acetic acid (3:1) and stained with 5%Giemsa. Measurement of micronuclei in binucleatedcells was carried out under 100× magnification, and1000 cells were scored per slide.

2.5. Comet assay

DNA damage (single strand breaks) was measuredusing the alkaline single cell gel electrophoresis as-say. The method used was essentially that of Singhet al. [17]. Briefly, V-79 cells (1 × 103) were sus-pended in 0.6% (w/v) low-melting point agarose andlayered over a microscopic slide coated with a layer of0.75% normal-melting point agarose. The slides werethen immersed in a lysing solution of pH 10 for 1 hat 4◦C. The slides were presoaked in alkaline elec-trophoresis buffer (pH >13) for 15 min. Electrophore-sis was carried out for 20 min (300 mA, 25 mV). Theslides were washed thrice with a neutralizing solution,stained with 15�g/ml ethidium bromide and examinedunder a Nikon fluorescent microscope. The tail lengthof the comet was measured using an occulometer.

M. Roy et al. / Mutation Research 523–524 (2003) 33–41 35

2.6. Cell viability and proliferation

Growth inhibition of cells was determined by try-pan blue dye exclusion method. Cells (V-79 andK-562) were exposed to 0.2% trypan blue and countedin a hemocytometer. K-562 cells were treated with1�Ci [methyl-3H]thymidine and after 5 h cells wereharvested and washed with phosphate buffered saline(PBS) and spotted on Whatman 3 MM filter paperdisc. Amount of radioactivity was measured by liquidscintillation counting after processing of filter discsas described in our earlier report[18].

2.7. Microscopic examination

Treated K-562 cells were harvested, washed withPBS and to the cell pellet propidium iodide (50�g/ml)was added and incubated in the dark at room temper-ature for 10 min. Cells were spread on a slide and nu-clear morphology was examined under the fluorescentmicroscope.

2.8. DNA fragmentation

The isolation of fragmented DNA was carried outaccording to the procedure of Herrmann et al.[19].Treated K-562 cells were harvested and washed withPBS, and cell pellets were then treated for 10 s with100�l lysis buffer, pH 7.5, centrifuged for 5 min at1600× g and the supernatant was collected. To thiswas added SDS (1%) and the solution was treatedovernight with RNase A (5�g/ml) at 56◦C followedby digestion with proteinase K (2.5�g/ml) for 4 h at37◦C. After addition of 0.5 volume of 10 M ammo-nium acetate, DNA was precipitated with 2.5 volumeof cold ethanol. DNA was dissolved in a loading bufferand fragments were separated by electrophoresis on1.5% agarose gel and stained with ethidium bromide.

2.9. Caspase activity

The aspartic acid specific cysteine proteases caspase3 and caspase 8 were assayed by fluorimetric tech-nique using fluorescent tagged peptides. Cleavage ofsynthetic substrates DEVD-AFC and IETD-AFC bycaspase 3 and caspase 8 respectively releases AFC, andfree AFC emits a yellow fluorescence at 480–520 nm(peak at 505 nm) upon excitation at 400 nm. Cell lysate

was used to measure the activities following the pro-tocol submitted by the supplier.

3. Results

Both H2O2 and MNNG induced micronuclei for-mation in V-79 cells in a dose dependent fashion. Thefrequency of micronuclei induced by these agents wasconsiderably reduced when cells were simultaneouslytreated with EGCG. This reduction was dependent onthe dose of EGCG, and the effect was apparent at alllevels of each genotoxicant. These results are shownin Fig. 1. At a fixed concentration of H2O2 (15�g/ml)or MNNG (30�M) the IC50 of EGCG was calculatedto be 1.65�M for H2O2 and 1.5�M for MNNG.

Fig. 2 shows a typical comet formation denotingDNA damage after alkaline single cell gel elec-trophoresis of V-79 cells treated with MNNG. Comettail length, a measure of DNA damage, was foundto be increased with increasing dose of the H2O2or MNNG. The damage was considerably counter-acted by the increasing doses of EGCG in both cases(Fig. 3). At H2O2 concentration of 30�g/ml andMNNG concentration of 30�M the IC50 values ofEGCG were 74 and 50�M, respectively.

When cells were preincubated with different dosesof EGCG and then treated with H2O2 (15�g/ml) orMNNG (50�M) the frequency of micronuclei forma-tion was reduced in proportion to the pretreatment dose(Fig. 4). Similar observations have been made withrespect to comet tail length (Fig. 5), although H2O2concentration used to induce DNA damage was higher(45�g/ml). It is apparent that induction of DNA dam-age requires higher amount of H2O2 than that neededto induce micronuclei formation. It may also be notedthat it required higher dose of EGCG to neutralize theDNA damaging effect of the two genotoxicants whileconsiderably low dose was quite efficient in suppress-ing micronuclei formation (Figs. 1 and 3–5).

Treatment of leukemic cell line K-562 with EGCGshowed a dose dependent growth inhibition as evidentfrom viable cell counts by trypan blue dye exclusionmethod (Fig. 6a). At the concentrations tested EGCGdid not elicit any growth inhibition of lymphocytespurified from normal human blood (data not shown).In parallel with growth inhibition EGCG also sup-pressed cell proliferation as assessed by incorporation


Fig. 1. Induction of micronuclei formation in V-79 cells by H2O2

and MNNG as modified by different doses of EGCG.

Fig. 2. Single cell gel electrophoresis of V-79 cells showing comet (400×) after treatment with MNNG at dose: (a) 0�M, (b) 25�M, (c)50�M.

Fig. 3. Induction of DNA damage in V-79 cells by H2O2 andMNNG as assessed by comet assay and its modulation by differentdoses of EGCG.


Fig. 4. Effect of exposure of V-79 cells to different doses ofEGCG on the induction of micronuclei formation after subsequenttreatment with H2O2 and MNNG.

of [3H]thymidine in nuclear DNA. A decrease in theincorporation with increasing amounts of EGCG wasobserved. These data are depicted inFig. 6b.

When cells treated with EGCG were stained withpropidium iodide and examined under a fluorescentmicroscope alterations characteristic of nuclear mor-phology such as chromatin condensation and nuclearfragmentation were observed.Fig. 7 shows fluores-cence image of tumor cells treated with EGCG. Cellswere scored at random and classified into apoptotic

Fig. 5. Effect of exposure of V-79 cells to different doses of EGCGon the induction of DNA damage after subsequent treatment byH2O2 and MNNG as assessed by comet assay.

and non-apoptotic cells based on their nuclear mor-phology. Their ratio designated as apoptotic index wasseen to be increased with increasing concentrations ofEGCG (Fig. 8). Fragmented DNA from treated cellswas isolated free of genomic DNA and subjected toagarose gel electrophoresis. Examination revealed aladder formation characteristic of apoptosis.Fig. 9shows ladder formation at three doses of EGCG. Flu-orimetric assay of caspase 3 and caspase 8 in cellstreated with EGCG revealed that the activity of both


Fig. 6. (a) Growth inhibition of K-562 cells by different doses ofEGCG. (b) Effect of different doses of EGCG on the incorporationof [3H]thymidine into nuclear DNA of K-562 cells.

the proteases was elevated in proportion to the doseof EGCG. The activation of caspase 3 was more pro-nounced than that of caspase 8. The results are shownin Fig. 10.

4. Discussion

The micronucleus technique has been proposed asa useful tool for measurement of genotoxicity in in

Fig. 7. Fluorescent microscopic picture of K-562 cells (400×)treated with EGCG (250�M) showing fragmented nuclei.

vitro cultures. Micronucleus arise during cell divisioneither from chromosomes that are lagging in anaphaseor from chromosome fragments[16]. Micronucleustest also is used to assess the DNA damaging effectsof chemicals. Alkaline comet assay involving singlecell gel electrophoresis is a sensitive method to de-tect single strand breaks in DNA[17]. DNA suffersendogenous and exogenous oxidative damage fromfree radicals generated by a great number of geno-toxic agents. Both H2O2 and MNNG are known geno-toxic compounds. In the present study, both have been

Fig. 8. Apoptotic index in K-562 cells vs. dose of EGCG.


Fig. 9. Electrophoretic separation of fragmented DNA of K-562cells following treatment with different doses of EGCG.

shown to induce micronuclei formation as well asDNA single strand breaks in a dose dependent manner.It is also observed that EGCG, the major polypheno-lic constituent of green tea, significantly counteracts

Fig. 10. Activity of caspase 3 and caspase 8 in K-562 cells sub-sequent to treatment with different doses of EGCG. The proteaseactivity was determined fluorimetrically using fluorescent-labeledsynthetic substrates.

the genotoxic effects of H2O2 and MNNG. In the twotests employed here EGCG has displayed antigeno-toxic effect in a dose dependent manner.

The antigenotoxic role of tea polyphenols includ-ing EGCG has been previously assessed as theirantimutagenic activity against various mutagens andcarcinogens. Kuroda and Hara[9] have reviewed thesubject. Few studies have demonstrated the anticlasto-genic effects of tea polyphenols, particularly EGCG.The tea tannins have been shown to modify inducedsister-chromatid exchange and chromosome aberra-tions in several mutagen-treated cultured mammaliancells [20]. The green tea extract suppressed aflatoxinB1-induced chromosome aberration in bone marrowcells [21] and benzo[a]pyrene-induced micronucleiformation in lymphocytes[22]. In V-79 cells forwardgene mutation and cytokinesis-blocked micronucleiformation by mitomycin C was significantly inhib-ited by EGCG[23]. Recent studies have shown thatEGCG suppresses the level of 8-OH-dG, a marker foroxidative DNA damage, induced by tobacco-specificnitrosamine[10] and UV [24], provides protectionagainst the induction of DNA single strand breaksby tobacco-specific nitrosamine[11], free radicals[25,26]and by Glu-P-1, a food derived mutagen[27].None of these studies used comet assay. These ob-servations support our present findings with respectto amelioration by EGCG of damage sustained byDNA. Our observation further shows that cells previ-ously exposed to EGCG are resistant to DNA damagecaused by genotoxic agents, thus attributing a truechemopreventive role of EGCG.

It is evident from the present results that EGCGconcentration required to counteract micronuclei for-mation is much lower than that required for suppres-sion of DNA breaks. This is possibly due to the mildcytotoxic effect of EGCG on V-79 cells. In the mi-cronuclei assay cells are incubated for a long periodto induce cytokinesis, and cell killing was pronouncedafter such long exposure in the presence of high con-centration of EGCG. Since pretreatment at lower doseswas effective it is possible that effective dose to coun-teract genotoxicity is low. This may be also true forcomet assay, since concentration inside the cell is notexpected to be high.

In addition to its chemopreventive effect EGCG alsohas been shown to suppress cell growth and induceapoptosis in various cell systems[8,12–15]. We have


observed in the present study that EGCG is capableof suppressing the growth of K-562 cells derived fromhuman chronic myelogenic leukemia. Cell prolifera-tion as assessed by measuring DNA synthesis showsinhibition by EGCG, and this is found to be in parallelwith the suppression of cell viability. Morphologicalcharacterization of treated cells reveals that the modeof action of cell death induced by EGCG is mediatedthrough apoptosis. Thus, chromatin condensation andnuclear fragmentation of treated cells are clearly evi-dent and apoptotic index is positively correlated withthe treatment dose. Cleavage of DNA at the inter-nucleosomal linker sites yielding DNA fragments inmultiples of 180 base pairs is regarded as a biochem-ical hallmark of apoptosis[28]. Appearance of suchfragments resulting in a ladder formation is evidentwhen fragmented DNA free of genomic DNA is iso-lated from cells treated with EGCG and subjected toagarose gel electrophoresis.

Several mechanisms have been proposed to ex-plain EGCG-induced apoptosis. EGCG causes aG0/G1-phase cell cycle arrest[29] and apoptosis canbe mediated through inhibition of NF�B [29,30],inhibition of TNFα gene expression and its release[31], binding to Fas on the cell surface[32], mito-chondrial depolarization[14], inhibition of growthfactors’ signaling[13] and activation of caspase 8[32] and caspase 3[33]. Caspases, the cytoplasmicaspartate-specific cysteine proteases, play an impor-tant role in apoptosis[34]. Activation of caspasesappears to be directly responsible for many of themolecular and structural changes in apoptosis. Ourobservation on the elevation in the activities of cas-pase 3 and caspase 8 suggests that in K-562 cellsEGCG-induced apoptosis is mediated through activa-tion of these proteases, particularly caspase 3.

From the present results and available informationit is clearly evident that EGCG protects normal cellsfrom genotoxic or carcinogenic assault and is capableof eliminating tumor cells. This is strongly indicativeof a chemopreventive as well as a therapeutic role ofEGCG.

Acknowledgements

The authors acknowledge with thanks the encour-agement and interest of the Director, CNCI. We are

grateful to the authorities of the Institute for the awardof a senior research fellowship to SC and a junior re-search fellowship to DS.

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