Carcinogenesis vol.29 no.1 pp.129–138, 2008doi:10.1093/carcin/bgm233Advance Access publication November 4, 2007

Molecular mechanism of black tea polyphenols induced apoptosis in human skin cancercells: involvement of Bax translocation and mitochondria mediated death cascade

Babli Halder, Udayan Bhattacharya, SibabrataMukhopadhyay1 and Ashok K.Giri�

Molecular and Human Genetics Division and 1Department of Chemistry,Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur,Kolkata 700 032, India

�To whom correspondence should be addressed. Tel: þ91 33 2473 3491/0492/6793; Fax: þ91 33 2473 5197;Email: akgiri15@yahoo.com

Theaflavins (TF) and thearubigins (TR) are the most exclusivepolyphenols of black tea. Even though few previous reportsshowed the anticancer effects of TF through apoptosis, the poten-tial effect of TR has not been appraised. This study investigatedthe induction of apoptosis in human skin cancer cells after treat-ment of TF and TR. We report that both TF and TR could exertinhibition of A431 (human epidermoid carcinoma) and A375(human malignant melanoma) cell proliferation without adverselyaffecting normal human epidermal keratinocyte cells. Growth in-hibition of A375 cells occurred through apoptosis, as evident fromcell cycle arrest at G0/G1 phase, increase in early apoptotic cells,externalization of phosphatidylserine and DNA fragmentation. Inour pursuit to dissect the molecular mechanism of TF- and TR-induced apoptosis in A375 cells, we investigated whether celldeath is being mediated by mitochondria. In our system, Baxtranslocation to mitochondria persuaded depolarization of mito-chondrial membrane potential, cytochrome c release in cytosoland induced activation of caspase-9, caspase-3 and poly (ADP-ribose) polymerase cleavage. Our intricate investigations on apo-ptosis also explained that TF and TR augmented Bax:Bcl2 ratio,up-regulated the expression of p53 as well as p21 and inhibitedphosphorylation of the cell survival protein Akt. Furthermore, TFand TR elicited intracellular reactive oxygen species generation inA375 cells. These observations raise speculations that TF as wellas TR might exert chemopreventive effect through cell cyclearrest and induction of apoptogenic signals via mitochondrialdeath cascade in human skin cancer cells.

Introduction

Polyphenols present in food have been demonstrated to decrease var-ious types of experimental carcinogenesis. In recent years, identifica-tion of effective chemopreventive polyphenols in diets or dietarysupplements for human use is of much interest. Treatments with suchpolyphenols result in cell cycle arrest (1), thereby reducing the growthand proliferation of cancerous cells through apoptosis or programmedcell death (2). Tea (Camellia sinensis) polyphenols exert their potentanticancer activity and appear to be the ideal agents for chemopre-vention. During the fermentation process, tea catechins are polymer-ized by polyphenol oxidase (released from crushed tea leaves) to formtheaflavins (TF) and thearubigins (TR). TF and TR account for 3–6%and 12–18% of dry weight of black tea, respectively (3). Black tea hasbeen shown to be potent in inhibiting tumorigenesis in animal sys-tems, including lung (4), colon (5) and skin (6). Some reports provideevidence that black tea significantly inhibits proliferation and enhan-ces apoptosis in established mouse skin tumor (7). Black teapolyphenols, especially TF exert cancer chemopreventive activity

by inducing apoptotic signals (7,8). However, besides some sporadicreports on the protective role of black tea and its derivatives, thedetailed molecular mechanisms underlying their anticancerous effectare inconclusive.

Apoptosis triggered by various stimuli, is characterized by a seriesof distinct biochemical and morphological changes (9), including in-crease in reactive oxygen species (ROS) level (10), activation of cas-pases, cell shrinkage, chromatin condensation and nucleosomaldegradation (11). One of the most significant events in apoptosis ismitochondrial dysfunction. Loss of mitochondrial transmembrane po-tential (MTP) elicits the release of cytochrome c from mitochondria tocytosol (12). After release, it activates caspase-9 and subsequentlyactivates downstream caspase-3 (13). Activated caspase-3 cleaves in-tracellular protein poly (ADP-ribose) polymerase (PARP), which is animportant marker of apoptosis (14).

It has been evident that the proapoptotic protein Bax plays anessential role for the onset of MTP changes and induces cytochromec release, which is inhibited by the antiapoptotic protein Bcl2 (12).Studies have also shown that Bax:Bcl2 ratio increases during apopto-sis (15). Bax is a p53 target and is known to be transactivated ina number of systems during p53-mediated apoptosis (16). p53 isknown to mediate cell cycle arrest (17) and also acts as a transcriptionfactor that binds to a p53-specific DNA consensus sequence in re-sponsive genes such as p21 (18). Up-regulation of p21 functions as theinhibitor of cell cycle kinetics (19). Previous reports showed that Aktphosphorylation confers cell survival signals through up-regulation ofantiapoptotic protein Bcl2 and down-regulation of proapoptotic fac-tors, such as caspase-9 (20,21).

All these above reports indicate that the balance between the proa-poptotic and antiapoptotic proteins, dysfunction of mitochondria andactivation of caspases are the important factors deciding apoptotic celldeath of tumor cells.

Previously we have reported the antimutagenic and anticlastogeniceffects of TF and TR in vivo and in vitro in multiple test systems(22,23). Although the content of TR is much higher than that of TFin black tea, interestingly there are no such reports available on po-tential anticancerous effects of TR till date.

In the present study, we have investigated further and elucidated theputative pathways of TF- and TR-induced cancer cell death speciallyemphasizing on TR. The effect of different doses of TF and TR on thegrowth of A431 and A375 cells was examined and the efficacy ofthese polyphenols as a growth inhibitor on these cell lines was com-pared. A375 cells were selected because they were much more sus-ceptible than A431 cells toward the treatment of different doses of TFand TR. In the current study, we determined the role of the mitochon-drial death cascade in TF- and TR-induced apoptosis in A375 cells.Bax:Bcl2 ratio along with the tumor suppressor protein p53 and p21,which are important regulators of mitochondria-mediated apoptosis,was also evaluated. We also addressed the relevance of phosphory-lated Akt in apoptosis.

Materials and methods

Reagents

Primary antibodies (Bcl2, Bax, p21, p53, Akt-ser, PARP, cytochrome c,b-actin) and polyclonal secondary antibody were obtained from Santa Cruz Bio-technology, Inc. (Santa Cruz, CA). Cycle TEST PLUS DNA reagent kit andJC-1 kit from BD Biosciences (San Diego, CA), AnnexinV-PI Apoptosis De-tection Kit was obtained from BD PharMingen (San Diego, CA), Apo-directTUNEL ASSAY KIT, caspase protease assay kit, were purchased fromChemicon International Corporation. 4#, 6-diamidino-2-phenylindole (DAPI),FITC-AnnexinV, bromophenol blue, EGTA, NaF, Na3VO4, NP 40, PMSF,aprotinin, leupeptin, Pepstatin A, catalase, HEPES, 3[4-dimethylthiazol-2-71]-2-5-diphenyl tetrazolium bromide (MTT), H2O2, digitonin, molecular

Abbreviations: DAPI, 4#, 6-diamidino-2-phenylindole; MMP, mitochondrialmembrane potential; MTP, mitochondrial transmembrane potential; MTT,3[4-dimethylthiazol-2-71]-2-5-diphenyl tetrazolium bromide; NHEK, normalhuman epidermal keratinocytes; PARP, poly (ADP-ribose) polymerase; PI,propidium iodide; ROS, reactive oxygen species; TF, theaflavins; TR, thearubigins.

� The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 129

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grade BSA, Tween-20, Nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolylphospate, p-toluidine (NBT/BCIP), Tris–HCl, DMSO and pifithrin-a (p53 in-hibitor) were from Sigma-Aldrich (St Louis, MO). 2#, 7#-dichlorofluoresceindiacetate from Molecular Probes Inc. (Eugene, OR). Mitochondria/Cytosolfractionation kit from BioVision (Mountain View, CA). Caspases fluoremetricassay kit obtained from Chemicon International Corporation. All caspase in-hibitors Z-VAD-FMK (pan-caspase inhibitor), DEVD-CHO (caspase-3-specific inhibitor), LEHD-CHO (caspase-9-specific inhibitor), LY294002(PI3K/Akt inhibitor) were purchased from Calbiochem (La Jolla, CA). Bio-Rad Protein Assay Kit from Bio-Rad Labarotories, Hercules, CA.

Cell culture

A431 (human epidermoid carcinoma cells) and A375 (human malignant mela-noma cells) were purchased from National Centre for Cell Science, Pune, India,and were maintained in DMEM containing 10% FBS, 100 U/ml penicillin, 100lg/ml sterptomycin (all from Invitrogen Corporation, Grand Island, NY). Nor-mal human epidermal keratinocytes (NHEK) from Clonetics� and grown inKeratinocyte growth medium (KGMTM from Clonetics� Cambrex BioscienceWalkersville, Inc., Walkersville, MD). The cells were incubated at 37�C ina humidified atmosphere containing 5% CO2 inside a CO2 incubator.

Extraction of TF and TR from black tea

Both TF and TR were extracted from the black tea (Tata Tea Gold) as describedin previous papers (22,23). TF and TR were extracted according to the pro-cedure of Xie et al. (24). Extraction was carried out in the Medicinal ChemistryDepartment of our Institute. In brief, initially black tea (10 g) was extracted byboiling water (250 ml), then the aqueous fraction was further extracted withchloroform to remove caffeine and successively with ethyl acetate andn-butanol by liquid–liquid partition (24). Ethyl acetate fraction contains TF,whereas the n-butanol fraction contains TR (25). We have also estimated thetotal TF and TR content of our sample, which were calculated to be 2.3% and12.5%, respectively, of the dry weight of the sample.

MTT assay

Effect of TF and TR (0–125 lg/ml) on the viability of A375, A431 and NHEKcells was determined by MTT assay following the method of Mosmaan (26).Briefly �5000 of A431, A375 and NHEK cells per well were plated in 96-wellplates and treated with TF or TR (0, 15, 25, 50, 75, 100, 125 lg/ml) for 24 or48 h. At the end of stipulated time following TF and TR treatment, the mediumwas aspirated and MTT (50 ll of 5 mg/ml stock solution in PBS) was addedinto each well and incubated at 37�C for 2 h. After shaking, the purple-coloredprecipitate of formazan was dissolved in 150 ll of DMSO (Sigma-Aldrich).The color absorbance of each well was recorded at 540 nm with a Bio-Radmicroplate reader with a reference serving as blank. Then, IC50 value of TF andTR was calculated (27).

Detection of apoptosis by flow cytometry

A375 cells (1 � 106 in each case) were treated with different concentration ofTF or TR (25, 50, 75, 100 lg/ml) for 24 h. For the determination of cell cyclephase distribution, A375 cells were harvested, permeabilized and nuclear DNAwas labeled with propidium iodide (PI) using cycle TEST PLUS DNA reagentkit. Cell cycle phase distribution of nuclear DNA was determined on FACS(Becton Dickinson, San Diego, CA), fluorescence detector equipped with488 nm argon laser light source and 623 nm band-pass filter (linear scale)using CellQuest software (Becton Dickinson). A total of 10 000 events wereacquired and analyzed.

Apoptosis was measured using AnnexinV-PI Apoptosis Detection Kit. A375cells were harvested and PI and AnnexinV fluos were added directly to themedium and then analyzed on flow cytometer (equipped with 488 nm argonlaser light source, 515 nm band-pass filter for FITC fluorescence and 623 nmband-pass filter for PI fluorescence) using CellQuest software. A total of10 000 events were acquired and the cells were properly gated for analysis.

To confirm the nature of tumor killing by different concentrations of TF andTR, A375 cells were fixed, permeabilized and incubated with terminal deox-ynucleotidyl transferase enzyme and FITC-dUTP. Cells were washed, incu-bated with PI/RNAase solution and analyzed on FACS (Becton Dickinson).The fragmented DNA of apoptotic cells was labeled using Apo-direct TUNELASSAY KIT. The cells were then analyzed on FACS (equipped with 488 nmargon laser light source, 515 nm band-pass filter, FL1-H, and 623 nm band-passfilter, FL2-H) using CellQuest software (Becton Dickinson). A total of 10 000events were acquired and analyzed.

Detection of apoptosis by confocal microscopy

A375 cells were cultured over a glass coverslips in a 60-mm culture dish to�70% confluency and then treated with 0, 25, 50, 75, 100 lg/ml doses of TF orTR for 24 h. The cells were treated with DAPI followed by incubation withFITC-AnnexinV for labeling the nuclei and phosphatidylserine at outer mem-

brane of cell, respectively. Coverslips were mounted. Stained nuclei and pho-phatidylserine were visualized and photographed using a Leica TCS-SP2 laserscanning confocal microscope (Leica Microsystem, Heidelberg, Germany).DAPI (excitation at 372 nm and emission at 456 nm) and FITC (excitationat 490 nm and emission at 520 nm) were exited by the argon–krypton laser.Apoptosis was characterized by the morphological changes, namely, external-ization of phosphatidylserine, cytoplasmic and nuclear shrinkage, and chro-matin condensation.

Measurement of MMP

The loss of mitochondrial membrane potential (MMP) is a hallmark for apo-ptosis. It is an early event preceding phosphatidylserine externalization andcoinciding with caspase activation. MMP was measured using JC-1 kit follow-ing the protocol submitted by the supplier. The MMP detection kit usesa unique fluorescent cationic dye, JC-1 (5,5#6,6#-tetracholoro-1,1#,3,3#-tetraethylbenzimidazolylcarbocyanine iodide) (excitation at 488 nm andemission at 525 nm), to signal the loss of MMP.

Cells were harvested by trypsinization at 24 h of treatment with differentconcentrations (0, 25, 50, 75, 100 lg/ml) of TF or TR. Then, mitochondrialpermeability transition was determined by the staining of the cells with JC-1 asdescribed. Briefly, equal numbers of cells (1 � 106) were incubated with JC-1at 2.5 lg/ml in 1 ml PBS for 30 min at 37�C with moderate shaking. Cells werethen centrifuged at 300 g at 4�C for 5 min, washed twice with ice-cold PBS andfinally resuspended in 200 ll PBS. Mitochondrial permeability transition wassubsequently quantified on FACS (Becton Dickinson). Data are given in per-centage of cells with altered MMP.

Preparation of cytosolic and mitochondrial fractions

A375 cells were harvested after treatment with 0, 25, 50, 75, 100 lg/ml con-centrations of TF or TR for 24 h. Isolation of a highly enriched mitochondrialfraction and cytosolic fraction of cells was performed using a Mitochondria/Cytosol fractionation kit. Briefly, A375 cells (5 � 107) were centrifuged at600g for 5 min at 4�C, resuspended in ice-cold PBS and centrifuged at 600g for5 min at 4�C. Then the cells were resuspended in 1.0 ml of cytosol extractionbuffer mix containing DTT and protease inhibitors and incubated on ice for10 min. The cells were homogenized on ice. The homogenate was centrifugedat 700g for 10 min at 4�C and the supernatant was collected and centrifuged at10 000g for 30 min at 4�C. Then, the supernatant was collected as the cytosolicfraction. The pellet was resuspended in 0.1 ml mitochondrial extraction buffermix containing DTT and protease inhibitors, vortexed for 10 s, and saved as themitochondrial fraction. These isolated cytosolic and mitochondrial fractionswere subjected to western blot analysis for cytochrome c release assay and Baxtranslocation assay. Cytochrome c expression was analyzed with anti-cytochrome c polyclonal antibody and Bax expression was determined byanti-Bax polyclonal antibody. An alkaline phosphatase-conjugated goat anti-rabbit secondary antibody was used for this experiment.

Measurement of intracellular ROS level

A375 cells were seeded into 100-mm dishes (5 x 106 cells per dish) overnight.Cells were treated with 0, 25, 50, 75, 100 lg/ml of TF or TR for 24 h and thencollected by trypsinization; 200 ll of cell suspension containing 2 � 106 cells/mlwas added to 800 ll of PBS and was incubated with 2#, 7#-dichlorofluoresceindiacetate at 10 lM concentration for 15 min. H2O2 (25 lM) and TF or TR (0,25, 50, 75, 100 lg/ml) were added in the culture and the incubation wascontinued for an additional 20 min at 37�C. The production of intracellularH2O2 was measured using fluoremeter (excitation at 365 nm and emission at430 nm). Another set of experiment was performed simultaneously, with sametreatments and conditions, where catalase (50 U/ml) was added to the cellculture medium 5 min before the addition of different concentrations of TFand TR.

Activity of caspases

Caspase-3 and caspase-9 were assayed using caspases fluoremetric assay kitdesigned for each caspase submitted by the suppliers. A375 cells (1 � 105 perwell) were plated in triplicate in a 24-well tissue culture plate and treated withTF or TR (0, 25, 50, 75, 100 lg/ml) for 24 h. The activity of caspases wasdetermined fluoremetrically (excitation at 400 nm and emission at 505 nm).Different concentration of TF or TR treatment was done for 24 h either aloneor in combination with Z-VAD-FMK (pan-caspase inhibitor), DEVD-CHO(caspase-3-specific inhibitor), LEHD-CHO (caspase-9-specific inhibitor).

PARP cleavage

The intact PARP molecule (116 kDa) is activated by breaks in DNA and iscleaved by caspases to yield a p85 fragment that is a characteristic marker ofapoptosis. For the detection of PARP cleavage, A375 cells were treated withTF or TR (0, 25, 50, 75, 100 lg/ml) for 24 h and subjected to western blotting.

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An anti-PARP rabbit polyclonal antibody recognizes both 116-kDa and the85-kDa cleavage fragment. An alkaline phosphatase-conjugated goat anti-rabbitsecondary antibody was used for this experiment.

Western blot analysis

Western blot analysis was done to determine the expression of different proteins.A375 cells were treated with different concentrations of TF or TR (0, 25, 50, 75,100 lg/ml) for 24 h. Cells were harvested, washed with cold PBS (pH 7.4) andlysed with ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA,1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 1% NP 40, 1 mM PMSF, 10 lg/mlaprotinin and 10 lg/ml leupeptin, pH 7.4 for 30 min and centrifuged at 12 000gfor 30 min at 4�C. The protein concentration of the clear supernatant was col-lected by means of centrifugation and was evaluated using Bio-Rad ProteinAssay Kit. Aliquots of equal amounts of proteins from the cells were subjectedto SDS–PAGE. Thereafter, proteins were electrophoretically transferred to nitro-cellulose membrane and non-specific sites were blocked with 5% skimmed milkin 1% Tween-20 (Sigma-Aldrich) in 20 mM TBS (pH 7.5) and reacted witha primary polyclonal antibody, Bax, Bcl2, Akt, Akt-ser, p21, p53, cytochrome cPARP, cytochrome c and b-actin for 4 h at room temperature. After washing thetris-buffered saline containing 0.1% Tween-20, the membrane was then incu-bated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody.The protein bands were visualized using NBT–BCIP.

Statistical analyses

GraphPad Instat software was used for statistical analysis. All data are ex-pressed as the mean ± SD of three independent experiments. The differencesbetween the control and the treatment groups were determined by one-wayANOVA and posttests were done using Dunnett’s multiple comparison test todetermine the significant levels.

Results

TF and TR treatment inhibits cell viability in A431 and A375 cells

The cytotoxic effect of TF and TR on NHEK, A431 and A375 cellswas determined with varying concentrations of TF and TR (0, 15, 25,50, 75, 100, 125 lg/ml) for 24 h (Figure 1A I and A II) and 48 h(Figure 1B I and B II) by MTT assay. Treatment of lower doses of TFand TR did not exhibit any significant cytotoxic effects on the cells at24 h of incubation. Our study revealed that significant reduction in cellviability commenced at 75 and 100 lg/ml of TF treatment on A375and A431 cells, respectively (Figure 1A I), whereas significant dim-inution of viable cell count instigated at 100 and 125 lg/ml of TRtreatment on A375 and A431 cells, respectively (Figure 1A II). How-ever, during 48 h of incubation, inhibition of cell viability of A375 andA431 cells were increased in dose-dependent manner (Figure 1B I andB II), which were statistically significant (P , 0.05, P , 0.01).Treatment with TF and TR appeared to display inhibitory activitiesin A431 cells with estimated IC50 was 99.66 lg/ml, whereas in A375cells estimated IC50 was 50.02 lg/ml at 48 h of treatment. In case ofNHEK cells for both TF and TR, IC50 value (data not shown) was.125 lg/ml at 48 h of treatment. Treatment of ,25 lg/ml concen-trations of TF and TR did not produce any statistically significantreduction in cell proliferation. Based on cell viability assay in A431and A375 cells after treatment with different doses of TF and TR, wehave selected A375 cells and 0, 25, 50, 75, 100 lg/ml doses for ourfurther studies.

Fig. 1. Effects of TF and TR on the viability of NHEK, A431 and A375 cells in dose-dependent manner. Cells were treated with different concentrations (0, 15, 25,50, 75, 100 lg/ml) of TF or TR and grown in 96-well plate for (A I and A II) 24 h and (B I and B II) 48 h. Inhibitory effects of TF and TR were determined by MTTassay. Each value is expressed as mean ± SD (n 5 3). �P , 0.05 and ��P , 0.01 are the TF- or TR-treated groups compared with the control (0 lg/ml).

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Induction of apoptosis in A375 cells by TF and TR treatment

We thought that the inhibition of cell viability by TF and TR in A375cells might be mediated through the initiation of apoptotic cascade.Accordingly, we extended our study to examine the induction ofapoptosis and the arrest of cell cycle kinetics by TF and TR usingA375 cells. The cells were treated with different concentrations of TFor TR (0, 25, 50, 75, 100 lg/ml) for 24 h and analyzed for cell cycledistribution by means of flow cytometry. The flow cytometric datadepicted the effect of TF and TR on cell cycle phase distribution of theskin cancer cells. Compared with the vehicle-treated controls (0 lg/ml),the cultivation with different concentrations of TF and TR increasedthe population of cells in the G0/G1 phase with a reduction of cells inthe S phase. The TF treatment caused an arrest of 50% cells in G0/G1

phase of cell cycle at a 25 lg/ml dose and 56%, 65%, 78% arrestcaused at the higher doses of 50, 75, 100 lg/ml, respectively (Figure2A I), whereas in case of TR treatment the arrest of cell cycle kineticswas 47%, 57%, 68%, 75% at the doses of 25, 50, 75, 100 lg/ml,respectively (Figure 2A II). These results indicated that TF as wellas TR led to cell cycle arrest at the G0/G1 phase followed by apoptosisin dose-dependent manner. Then, we confirmed TF- and TR-induced

apoptosis by means of AnnexinV/PI double-staining method. In caseof TF, induction of apoptosis ranged from 8.7% to 59%, whereas incase of TR the range was 7.5–41% (Figure 2B II). We also examinedwhether the induction of apoptosis was specific for cancer cells ornormal cells following TF and TR incubation. Interestingly, any doseof both TF and TR did not induce significant apoptosis (0.02–4.7% forTF-treated cells and in case of TR treatment it was 0.02–2.7%) inNHEK cells (Figure 2B I), even at the highest dose (100 lg/ml) of thatused for carcinoma cell lines. This could be explained by the fact thatTF and TR induced externalization of phosphatidylserine at very earlyin apoptosis before any nuclear changes have occurred. All theseresults together indicated that the different dose of TF and TR (25,50, 75, 100 lg/ml) rapidly induced apoptosis in A375 cells.

Next, we quantified the extent of apoptosis by DNA fragmentationby means of flow cytometric analysis of the cells labeled with fluo-rescent-tagged dUTP. As is evident from Figure 2C I, significant DNAfragmentation augmented at higher dose that is 75 lg/ml of both TFand TR treatment for 24 h in A375 cells. However, in case of 48 h oftreatment statistically significant increment of DNA fragmentations inA375 cells initiated even at lower dose that is 25 lg/ml of both TF and

Fig. 2. Detection of TF- and TR-induced apoptosis in A375 cells. (A I and A II) Graphical representation of the flow cytometric analysis of A375 cell phasedistribution. A375 cells were seeded in 100-mm culture dishes and were treated with 0, 25, 50, 75, 100 lg/ml of (A I) TF or (A II) TR for 24 h. Nuclear DNA waslabeled by PI and determined by single-label flow cytometry. Each value is expressed as mean ± SD (n 5 3). �P, 0.05 and ��P, 0.01 are the TF- or TR-treatedgroup compared with the control (0 lg/ml). (B I and B II) Flow cytometric detection of apoptosis. NHEK and A375 cells were incubated with differentconcentrations of TF or TR (0, 25, 50, 75, 100 lg/ml) for 24 h. TF- or TR-induced apoptosis in (B I) NHEK and (B II) A375 cells was determined by flowcytometry using double-staining system. Control (untreated) and treated cells were labeled with PI and AnnexinV-tagged FITC and then fixed and analyzed ona flow cytometer. Dual parameter dot plot of FITC fluorescence (x-axis) versus PI fluorescence (y-axis) has been shown in logarithmic fluorescence intensity.Quadrants: lower left, live cells (�FITC); lower right, apoptotic cells (þFITC); upper right, necrotic cells or late phase of apoptotic cells (þPI, þFITC). (C I andC II) Detection of fragmented DNA by TUNEL assay. A375 cells were treated with 0, 25, 50, 75, 100 lg/ml of TF or TR for (C I) 24 h and (C II) 48 h and stainedwith FITC-conjugated dUTP and PI and analyzed by flow cytometry. Each value is expressed as mean ± SD (n 5 3). �P, 0.05 and ��P, 0.01 are the TF- or TR-treated groups compared with the control (0 lg/ml).

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TR (Figure 2C II). At the doses, 25–100 lg/ml of TF showed 17.67–57.98% of apoptotic cells, whereas 12.76–47.76% was observed duringthe treatment of TR at the dose ranged 25–100 lg/ml (Figure 2C II).

The induction of apoptosis by TF and TR was also evident from themorphologic alteration as shown by confocal microscopy after label-ing the cells with DAPI- and FITC-tagged AnnexinV. Even at thelower doses of TF and TR, several cells displayed early apoptoticmorphology (Figure 3).

Bax translocation to mitochondria upon the TF and TR treatment

It has been reported that translocation of proapoptotic protein Bax tomitochondria plays an important role in decreasing MMP and initiat-

ing mitochondrial death cascade. In our system, Bax translocationfrom cytosol to mitochondria was measured by western blot at 24 hof incubation in A375 cells. Our observation revealed that Bax level inmitochondria increased by 1.8- to 3.5-fold for TF and 1.6- to 3.5-foldfor TR in dose-dependent manner (Figure 4A). The above resultsraised the possibility of Bax-induced MMP loss in A375 cells asa result of TF and TR treatment.

Effect of TF and TR treatment on MMP

Loss of MTP is associated with mitochondrial dysfunction leading toapoptosis. Consequently, we next evaluated the effect of TF and TRon MMP. Our results showed that these two polyphenols significantly(P , 0.01) decreased MMP in A375 cells (Figure 4B).

Cytochrome c release in the cytosol by TF and TR treatment

To check the downstream events of mitochondrial death cascade inA375 cells, we investigated the release of cytochrome c from mito-chondria to cytosol. Western blot analysis showed that mitochondrialcytochrome c appeared in the cytosolic fractions after 24 h of incu-bation with TF or TR. Cytosolic cytochrome c increased by 2- to 4.6-fold for TF (25–100 lg/ml) and 1.8- to 3.5-fold for TR (25–100 lg/ml)(Figure 4C).

Effects of TF and TR on the generation of intracellular ROS

The prevailing notion implicates intracellular ROS as signaling inter-mediates that are involved in signal transduction pathways of apopto-sis. So, furthermore we investigated whether TF and TR treatmentresulted in the generation of ROS in the skin cancer cell. A statisti-cally significant increase (P , 0.01) of intracellular H2O2 level wasdetected after treatment with TF and TR, which was attenuated bypretreatment with catalase (Figure 4D). Our data also revealed that thepreincubation with the catalase prevented the TF- and TR-inducedapoptosis in A375 cells (Figure 4E).

Effect of TF and TR on caspases activity and PARP cleavage

Results of cytochrome c release and mitochondrial perturbation fur-ther led us to investigate the role of caspase cascade in TF- and TR-induced A375 cell apoptosis. Caspases are believed to play a centralrole in mediating various apoptotic responses. We examined whetherspecific caspases were involved in signaling pathway for the inductionof apoptosis in A375 cells after treatment with TF and TR for 24 h.Both the polyphenols induced statistically significant (P , 0.05 andP , 0.01) increase in the caspase-3 (Figure 5A) and caspase-9(Figure 5B) activation in A375 cells. We confirmed this result byusing respective caspase inhibitors to elucidate the functional roleof caspases in A375 cells. TF- and TR-induced apoptosis was com-pletely blocked by treatment with the respective blockers of caspase-9and caspase-3, which confirmed that TF- and TR-induced apoptosis isassociated with the activation of caspase-3 (Figure 5A) and caspase-9(Figure 5B). We have also studied the impact of catalase on TF- andTR-induced caspase-3 and caspase-9 activation. Exogenous catalaseinhibited TF- and TR-induced caspase-3 (Figure 5B) and caspase-9(Figure 5C) activation in A375 cells during the 24-h period. Then, weconducted experiments to investigate whether TF and TR inducedPARP cleavage as a hallmark sign of caspase-3 activation. Our resultsrevealed that the PARP cleavage was increased in dose-dependentmanner in A375 cells (Figure 5E).

Expression of apoptosis-associated proteins

After confirming that TF and TR induce apoptosis in A375 cells, wenext attempted to unveil further the mechanism of A375 killing. It iswell recognized that various proapoptotic and antiapoptotic proteinsplay a crucial role in mitochondria-mediated programmed cell death.So to examine the role of mitochondrial pathway in TF- and TR-treated apoptosis in A375 cells, we used the western blot analysis(Figure 6A I and A II) to measure the antiapoptotic (Bcl2) and proa-poptotic proteins (Bax, p53). We observed that different doses of TF

Fig. 3. Representative confocal pictures of TF- or TR-induced apoptoticcells (A375). The upper most row of the figure showing control cells (0 lg/ml).The doses of TF and TR (25 and 50 lg/ml) are mentioned at the extreme rightside of the figure. The first (extreme left) column showing only DAPI (blue)labeled cells, the second (middle) column showing only FITC-taggedAnnexinV (green) labeled cells and the third column showing merged (DAPIand FITC-AnnexinV) pictures. Arrows indicate apoptotic cells. Data shownhere are from a representative experiment repeated three times with similarresults.

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(Figure 6A I) or TR (Figure 6A II) up-regulated the expression of Baxand p53 and down-regulated the expression of Bcl2, thereby increas-ing Bax:Bcl2 ratio by 0.5- to 6-fold for TF (25–100 lg/ml) and 0.4- to5-fold for TR (25–100 lg/ml) (Figure 6B). Interestingly, the level ofp21 (cell cycle regulatory protein) was increased upon TF and TRtreatment. We further investigated the expression of the cell survivalprotein Akt. Expression of phosphorylated Akt (Akt-ser) was down-regulated upon TF and TR treatment. We made an effort to find outwhether expressions of p21 and Bax proteins depend on the p53 andalso if there was any relationship between up-regulation of Bcl2 pro-tein and the activation of Akt. In order to investigate the effect of TF(Figure 6C I) and TR (Figure 6C II) on the expression of p53, p21 andBax in A375 cells preincubated with p53 inhibitor, we decided toblock the activation of p53-responsive genes by pifithrin-a. Pifithrin-ahas been demonstrated to block p53-dependent transcriptionalactivation and apoptosis. In the absence of pifithrin-a, p53 expressionwas found to be dose dependent in A375 cells for TF (Figure 6A I)and TR (Figure 6A II) treatment. Upon addition of pifithrin-a, p53expression was reduced during TF (Figure 6C I) and TR (Figure 6C II)incubation when compared with the cells not treated with p53 blocker.

Similar to p53, the expression of p21 and Bax protein also substan-tially declined in TF-treated (Figure 6C I) and TR-treated (Figure 6CII) A375 cells, preincubated with pifithrin-a as compared with cellsnot treated with pifithrin-a. The inhibition suggests that pifithrin-aeffectively blocked the transactivation potential of p53. Next ourendeavor was to look into the effect of TF and TR on the expressionof Akt phosphhorylation and Bcl2 in A375 cells preincubated withPI3K/Akt inhibitor, LY294002. Akt phosphorylation was noticeablyinhibited in TF-treated (Figure 6D I) or TR-treated (Figure 6D II)A375 cells preincubated with LY294002 as compared with cells trea-ted with TF (Figure 6A I) or TR (Figure 6A II) alone. But the re-duction of Bcl2 expression was more about the same in case of TF-treated (Figure 6D I) and TR-treated (Figure 6D II) cells preincubatedwith LY294002 when compared with the cells treated with TF (Figure6A I) and TR (Figure 6A II) alone.

Discussion

Apoptosis plays a crucial role in eliminating the mutated hyperprolif-erating cells from the system. Thus, induction of apoptosis in tumor

Fig. 4. Effect of different concentrations of TF and TR on Bax translocation, MMP, cytochrome c release, ROS production in A375 cells. (A) Translocation of Baxfrom cytosol to mitochondria during TF- and TR-induced apoptosis. Cells were treated with different concentrations of TF or TR (0, 25, 50, 75, 100 lg/ml) for 24h. Relative density of bands obtained from western blot analysis was quantified and represented here as fold change. The Bax level in cytosol and mitochondria incontrol (0 lg/ml) was adjusted to 1-fold and the change of Bax level in the treated groups was represented as fold of control. Columns, mean of experiments donein triplicate; bars, SD. (B) Effect of TF and TR on MMP in A375 cells. Cells were treated with different concentrations (0, 25, 50, 75, 100 lg/ml) of TF or TR for24 h. TF- and TR-induced cells were labeled with JC-1 probe and the altered MMP was measured flow cytometrically. Columns, mean of experiments done intriplicate; bars, SD. ��P, 0.01 is the TF- or TR-treated group compared with the control (0 lg/ml). (C) Release of cytochrome c in cytosol from mitochondria inA375 cells after treatment with TF or TR in dose-dependent manner. Cells were treated with TF or TR (0, 25, 50, 75, 100 lg/ml) for 24 h. Cells were harvested andcytochrome c level in cytosolic and mitochondrial fractions was determined by western blot. Relative density of the bands of cytochrome c was quantified andrepresented here as fold change. The cytochrome c level in cytosol and mitochondria in control (0 lg/ml) was adjusted to 1-fold and the changes of cytochrome clevel in the treated groups were represented as fold of control. Columns, mean of experiments done in triplicate; bars, SD. (D) Estimation of H2O2 in A375 cellsafter treatment with different concentrations (0, 25, 50, 75, 100 lg/ml) of TF or TR for 24 h; 50 U/ml catalase can suppress H2O2 production in TF- and TR-treatedcells; 25 lM H2O2 was used as a positive control. H2O2-induced fluorescence was analyzed fluoremetrically. Each value is expressed as mean ± SD (n 5 3). ��P,

0.01 is the TF- or TR-treated groups compared with the control (0 lg/ml). (E) Effect of catalase on TF- and TR-induced apoptosis. Catalase (50 U/ml) was added tothe cell culture medium 5 min before the addition of different concentrations of TF and TR. Apoptosis was determined by flow cytometrically following 24 h of TFand TR exposure. Each value is expressed as mean ± SD (n 5 3). ��P , 0.01 is the TF- or TR-treated group compared with the control (0 lg/ml).

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cells may be considered as a protective mechanism against develop-ment and progression of cancer. The inhibitory activity of green teacatechins and black tea polyphenols on tumor growth has been re-ported, both in vitro and in vivo (7,28,29). However, information isvery limited on the antitumor activity of TF and there is no such reporton TR. Previously, we have reported the antimutagenic and anticlas-togenic effects of TF and TR in multiple test systems (22,23). So our

endeavor was to investigate the intrinsic molecular mechanism ofapoptosis in vitro and the probable signaling cascade of chemopre-vention induced by TF and TR in skin cancer cells, with specialemphasis on TR. Our study on growth inhibition demonstrated thatA375 cells were more susceptible than A431 cells toward the treat-ment of different concentrations of TF and TR. Our findings are inagreement with other studies demonstrating the growth inhibitory

Fig. 5. Activation of caspase-3 and caspase-9 after treatment of different concentrations of TF or TR in A375 cells. Cells were treated with TF or TR (0, 25, 50, 75,100 lg/ml) for 24 h and subjected to measurement of caspase activity by means of cleavage of color substrate. (A) DEVD-AFC for caspase-3 and (B) LEHD-AFCfor caspase-9. Effects of caspase-3 (A) and caspase-9 (B) inhibitors on TF- or TR-treated A375 cells were estimated in a coculture with caspase inhibitors. Cellswere incubated with each caspase inhibitor for 2 h before addition of TF or TR. Z-VAD-FMK, pan-caspase inhibitor, DEVD-CHO, caspase-3 inhibitors, LEHD-CHO caspase-9 inhibitor. Each value is expressed as mean ± SD (n 5 3). ��P , 0.01 is the TF- or TR-treated group compared with the control (0 lg/ml).(C and D) Effects of TF and TR on activity of caspase-3 and caspase-9 in A375 cells pretreated with catalase. Cells were pretreated with 50 U/ml exogenouscatalase for 5 min prior to addition of TF and TR at concentration indicated. (C) Caspase-3 and (D) capase-9 activity assay was performed immediately after 24 hincubation with TF or TR. (E) PARP cleavage was determined by western blotting using a rabbit polyclonal PARP antibody. A375 cells were treated with 0, 25, 50,75, 100 lg/ml of TF or TR for 24 h.

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Fig. 6. Western blot analysis of Bcl2, Bax, p21, p53 and Akt-ser and Akt protein levels upon treatment with TF and TR in different doses.(A I and A II) A375 cells were treated with 0, 25, 50, 75, 100 lg/ml of (A I) TF or (A II) TR for 24 h. Protein from the total cell lysate was subjected to SDS–PAGE and western blot using Bcl2, Bax, p21, p53, Akt-ser and b-actin antibody. Representative blot from three independent experiments was with identicalresults. Relative intensity of each band after normalization with the intensity of b-actin in a blot (below each western blot) was measured. (B) The ratio ofBax and Bcl2 protein expression was determined from three separate experiments by comparing the relative intensities of protein bands. Each value isexpressed as mean ± SD (n 5 3). (C I and C II) Western blot analysis of (C I) TF- and (C II) TR-induced p53, p21 and Bax protein expression in A375 cellsin the presence of p53-inhibitor, pifithrin-a (30 lmol/l). A375 cells were preincubated with pifithrin-a at a concentration of 30 lmol/l for 1.5 h before thetreatment of TF and TR. Then, the cells were treated with 0, 25, 50, 75, 100 lg/ml of TF or TR for 24 h. Protein from the total cell lysate was subjected toSDS–PAGE and western blot using p53, p21, Bax and b-actin antibody. Representative blot from three independent experiments was with identical results.Relative intensity of each band after normalization with the intensity of b-actin in a blot (below each western blot) was measured. (D I and D II) Western blotanalysis of (D I) TF- and (D II) TR-induced Akt, Akt-ser and Bcl2 protein expression in A375 cells in the presence of PI3k/Akt-inhibitor, LY294002.A375 cells were preincubated with LY294002. Then, the cells were treated with 0, 25, 50, 75, 100 lg/ml of TF (D I) or TR (D II) for 24 h. Protein from thetotal cell lysate was subjected to SDS–PAGE and western blot using Akt, Akt-ser and b-actin antibody. Representative blot from three independentexperiments was with identical results. Relative intensity of each band after normalization with the intensity of b-actin in a blot (below each western blot)was measured.

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effect of TF on various cancer cell lines such as H-ras-transformedmouse epidermal JB-6 cells (30) and mouse fibroblast NIH3T3 cells(31). Our observations indicate that TF exhibits more antiproliferativeactivity in comparison with that of TR in a dose-dependent manner.

Based on this investigation, next we decided to extend our series ofexperiments further in A375 cells to find out the induction of apopto-sis and its intrinsic mechanism. Previous studies have anticipated thatthe black tea TF were capable of inducing apoptosis in varieties ofhuman cancer cell lines (32). We observed that TF and TR treatmentresulted in augmentation of apoptosis in A375 cells, thus suggestingthat the inhibition of cell viability might be, in part, due to the in-duction of apoptosis. Nuclear DNA fragmentation, a classical featureof apoptotic cell death, was clearly shown in skin cancer cells withdifferent doses of TF and TR.

Eukaryotic cell cycle is regulated by signal transduction pathwaysmediated by a series of cell cycle regulators. One of those is p21 thathas been shown to induce tumor cell growth arrest and apoptosis(33,34). In this study, we determined the inhibition of A375 cellgrowth at different concentrations of TF and TR, accompanied witha G0/G1 cell cycle arrest.

Apoptosis is modulated by antiapoptotic and proapoptotic effectorsthat involve a large number of proteins. Therefore, to gain insight intomechanisms controlling apoptosis, we looked at the effect of TF andTR on antiapoptotic and proapoptotic proteins of the Bcl2 family.Bcl2 protein functions as a suppressor of apoptosis (35). Bax is a proa-poptotic protein and its predominance over Bcl2 promotes apoptosis(36). In the present study, we have found that treatment with differentconcentrations of TF and TR in A375 cells resulted in reduction ofBcl2 protein expression, whereas increased the expression of Bax,indicating that the increased ratio of Bax:Bcl2 proteins may be re-sponsible for the induction of apoptosis.

It is known that p53 may induce two sets of genes in response tostress signals thereby contributing to the decision-making growtharrest and apoptosis. This tumor suppressor protein may up-regulatethe proapoptotic protein Bax and is known to mediate growth arrestinvolving p21 as a major effector (37). In fact, p53-dependent induc-tion of p21 prevents entry of cells into S phase (38). In our present setof investigation, we have found increased expression of p53 and p21,suggesting that TF and TR treatment could inhibit cell cycle kineticsat G0/G1 phase thus inducing apoptogenic signal in A375 cells. Inorder to investigate whether the up-regulation of p21 and Bax iscorrelated with the elevation of p53 level in TF- and TR-treatedA375 cells, we decided to block p53 by pifithrin-a, a known p53inhibitor. Our results exhibited that abolition of p53 transactivationpotential by pifithrin-a reduced the expression level of p21 and Bax inA375 cells after treatment of TF and TR. So, it could be postulatedthat there might be a relationship between p53 activation and the up-regulation of p21 and Bax in TF- and TR-treated A375 cells. All theseelucidations corroborated the relationship between p53 status, p21induction and Bax:Bcl2 ratio in cell cycle deregulation and apoptosisin TF- and TR-treated A375 cells. However, the results of our studyalso illustrated that inhibition of Bcl2 expression was not changedsubstantially upon the treatment with PI3K/Akt inhibitor, LY294002,in TF- and TR-treated A375 cells as compared with the cells nottreated with Akt blocker. It could be hypothesized that Bcl2 expres-sion might not depend on the Akt phosphorylation in TF- and TR-treated A375 cells.

Translocation of Bax protein causes change in MTP that plays animportant role in the induction of apoptosis (12). The mitochondrialmembrane has only a limited permeability that is essential for gener-ation of the MTP (39). Permeabilization of this membrane allowsrelease of cytochrome c and activation of subsequent downstreamcaspases (12,13,40). Activated caspase-3 cleaves intracellular pro-teins such as PARP, which are vital to cell survival and growth, andPARP cleavage has been used as an important marker of apoptosis(14). In our present study, we have found that the treatment of TF andTR induced mitochondria-mediated apoptosis by translocation ofBax to mitochondria in A375 cells. This intracellular movement co-incided with the MTP change, release of cytochrome c into cytosol,

subsequent activation of downstream caspase-9, caspase-3 and PARPcleavage.

In a variety of cell types, the apoptosis-triggering effects of ROSwere noted in vitro and in vivo (41,42). TF and TR treatment elevatedintracellular ROS levels and also induced apoptosis in A375 cells.Furthermore, pretreatment of A375 cells with catalase could preventTF- and TR-induced intracellular ROS generation and apoptosis in-duction. This observation corroborated with the previous finding thatoxidative stress induces apoptotic pathways in the tumor cells (43). Inaddition, we noted a substantial prevention of TF- and TR-mediatedcaspase-3 and caspase-9 activation in A375 cells pretreated with ex-ogenous catalase. From these observations it could be suggested thatTF- and TR-derived intracellular ROS might have a role in caspase-3and caspase-9 activation and apoptosis induction in A375 cells.

In conclusion, our study indicates that both TF and TR induceapoptosis via mitochondria-mediated pathway and involvement ofproapoptotic and antiapoptotic proteins, thus providing a molecularbasis for understanding the chemopreventive effect of TF and TR thatmight be ideal candidates to induce effective apoptosis in human skincancer cells. However, presently the detailed mechanism underlyingthe antiproliferative role of TF and TR has been delineated in case ofA375 cells only. More work is required to find out the precise role ofTF and TR in different types of cancer cells to understand whether theaction of these polyphenols is cell line specific or via a general mech-anism. These observations will add new light in the field of develop-ing therapeutic strategies for cancer in near future. To our knowledge,this is the first report of the induction of apoptosis and its mechanismis induced by TR, an important polyphenol exclusively present inblack tea.

Acknowledgements

Authors are sincerely thankful to Mr Prasun Guha for his kind contribution inthis work. The authors are thankful to the Council of Scientific and IndustrialResearch, New Delhi, for providing Senior Research Fellowship to BabliHalder.

Conflict of Interest Statement: None declared.

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Received June 20, 2007; revised October 10, 2007; accepted October 18, 2007

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