In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells

Cell Biology International 31 (2007) 599e608 www.elsevier.com/locate/cellbi

In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells K.V.P. Chandra Mohan a, P. Gunasekaran b, E. Varalakshmi b, Y. Hara c, S. Nagini a,* a

Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608 002, Chidambaram, Tamil Nadu, India b Department of Virology, King Institute of Preventive Medicine, Chennai 600 032, Tamil Nadu, India c Mitsui Norin Co., Ltd, Shizuoka, Japan Received 11 September 2006; revised 7 November 2006; accepted 29 November 2006

Abstract We investigated the anticancer effects of green and black tea polyphenols alone and in combination with bovine milk lactoferrin (bLF) on human tongue squamous carcinoma (CAL-27) and normal human gingival fibroblast (HGF) cells. Both green (Polyphenon-E;P-E) and black tea polyphenols (Polyphenon-B;P-B) preferentially inhibit the growth of CAL-27 cells in a dose-dependent manner. Based on the IC50 values, P-E was found to be more effective than P-B and the combination of P-E and bLF (1:2 ratio) exhibited synergistic inhibition of CAL-27 cells. Analysis of the mechanism revealed nuclear fragmentation and condensation with appearance of the Ao peak indicative of apoptosis. Furthermore, tea polyphenols transduced the apoptosis signal via generation of reactive oxygen species and decrease in the Bcl-2/Bax ratio thereby inducing mitochondrial permeability transition with consequent activation of caspase-3. Overall, the potency of cytotoxic and apoptosis inducing effects of dietary agents on CAL-27 cells was in the order P-E and bLF combination (1:2 ratio) > P-E > P-B. These results suggest that a ‘‘designer’’ approach may be useful for oral cancer prevention strategies. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Anticancer; Apoptosis; Tea polyphenols; Lactoferrin

1. Introduction Chemoprevention by dietary agents has evolved as a promising approach to control the incidence of oral cancer, an important contributor to morbidity and mortality (Mignogna et al., 2004). Of late, chemoprevention by a combination of dietary phytochemicals with distinct molecular mechanisms has received growing consideration as a means to achieve higher efficacy and potency with reduced toxicity. In particular, combination regimens that use tea polyphenols as one of the constituents have been found to be potentially effective in Abbreviations: bLF, bovine lactoferrin; DCFH-DA, 20 ,70 -dichlorofluorescein diacetate; Djm, mitochondrial transmembrane potential; P-B, polyphenon-B; P-E, polyphenon-E; ROS, reactive oxygen species. * Corresponding author. Tel.: þ91 4144 239 842; fax: þ91 4144 238 145/ 238 080. E-mail addresses: [email protected], [email protected] (S. Nagini).

chemoprevention (Sakamoto, 2000; Suganuma et al., 2001; Ohigashi and Murakami, 2004; Zhou et al., 2004). In most parts of the world, tea is consumed together with milk. Both milk and tea are rich in bioactive compounds and nutraceuticals. Bovine milk lactoferrin (bLF), an 80 kDa iron-binding glycoprotein present in whey protein fraction of milk, and polyphenols in green and black tea are reported to exhibit a wide range of beneficial effects including chemopreventive activity by modulating multiple signal transduction pathways when administered as single agents (Yang et al., 2000; Tsuda et al., 2002; Moyers and Kumar, 2004). These findings suggest that a combination of bLF and tea polyphenols may have synergistic effects in inhibiting cancer development. However, it is important to establish the chemopreventive efficacies of agent combinations by evaluating cytotoxicity and apoptosis induction in cancer cell lines before embarking on whole animal bioassays or clinical trials.

1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.11.034

600

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

Apoptotic cell death is characterized by distinct morphological features such as cell shrinkage, chromatin condensation, nuclear DNA fragmentation, membrane blebbing and breakdown of the cell into apoptotic bodies. The two major pathways of apoptosis mediated by the mitochondria and death receptors result in activation of caspases that cleave a variety of cellular substrates eventually leading to cell death (Wang, 2001; Gupta, 2003). Both the pathways are regulated by Bcl-2 family proteins that may be antiapoptotic or proapoptotic. Evasion of apoptosis increases the likelihood of sustaining gene mutations required for malignant transformation. Induction of apoptosis is currently recognized as an active strategy to arrest proliferation of cancer cells (Wang, 2001; Gupta, 2003; Vermeulen et al., 2005). A large number of dietary constituents including tea polyphenols have been reported to induce apoptosis in malignant cells (Wang et al., 1999; Bhattacharyya et al., 2005; Nakazato et al., 2005a; Shimizu et al., 2006). Increasingly, the mitochondrion has become the focus of attention as a potential target for chemointervention (Wang, 2001). The present study was designed to evaluate the anticancer and apoptosis inducing effects of green and black tea polyphenols alone and in combination with bLF on human tongue squamous carcinoma (CAL-27) cells with special emphasis on the mitochondrial pathway of apoptosis. 2. Materials and methods 2.1. Chemicals bLF (lot No. 020119) of purity 96.2% was obtained from Morinaga Milk Industry Co., Ltd, Tokyo, Japan. The iron content of bLF was 18 mg/100 g. Green tea polyphenols (Polyphenon-E:P-E) and black tea polyphenols (Polyphenon-B:P-B) were kindly provided by Mitsui Norin Co., Ltd., Tokyo, Japan. The composition of Polyphenon-E and Polyphenon-B is same as described previously (Chandra Mohan et al., 2005, 2006). Polyphenon-E (P-E) is a mixture of epigallocatechin-3-gallate (64.6%), epigallocatechin (4.3%), epicatechin (9.4%), epicatechin-3-gallate (6.4%), gallocatechin-3-gallate (3.5%), catechin-3-gallate (0.2%), gallactocatechin (0.2%), catechins (1.1%) and caffeine (0.7%). Polyphenon-B (P-B) has the following composition: epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallocatechin-3-gallate (0.2%), free theaflavins (0.32%), theaflavinmonogallate-A (0.14%), theaflavinmonogallate-B (0.15%), theaflavindigallate (0.21%), tannin (35.6%) and caffeine (4.9%). All other reagents used were of analytical grade. Stock solutions of bLF and P-E were prepared in phosphate buffered saline (PBS). P-B was dissolved in PBS containing 0.5% dimethyl sulfoxide (DMSO). The stock solutions were then diluted with the medium prior to use to obtain the desired concentration. The final concentration of DMSO in the medium was less than 0.01 per cent that proved to have no detectable effect on cell growth. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO. Dithiothreitol (DTT), 3,3-diaminobenzidine tetrahydrochloride (DAB), 20 ,70 -dichlorofluorescein diacetate (DCFHDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide, proteinase K, phenylmethanesulfonyl fluoride (PMSF), rhodamine 123, and RNase A were purchased from Sigma Chemical Company, St. Louis, MO, USA.

University of Tennessee, College of Dentistry, Memphis, TN, USA were used in this study. The cells were grown in DMEM supplemented with 10% FBS, 50 U/ml penicillin G, and 50 mg/ml streptomycin sulphate. The cultures were maintained at 37  C in a humidified atmosphere of 5% CO2 in air. Exponentially growing cells were used for all the experiments.

2.3. Cytotoxicity assay Cytotoxicity was assessed by the MTT assay based on the reduction of MTT by mitochondrial dehydrogenases of viable cells to a purple formazon product (Mosmann, 1983). Briefly, cells were diluted in growth medium and seeded in 24-well plates (5  104 cells/well). After overnight growth, the growth medium was replaced with exposure medium (DMEM without FBS) containing indicated doses of bLF, P-E, P-B alone and a combination of bLF and P-E or P-B. After 24 h, the cells in each well were washed with 200 ml of PBS, and incubated with 100 ml of 500 mg/ml MTT in PBS at 37  C for 3 h. The MTT-formazon product dissolved in 200 ml of DMSO was estimated by measuring the absorbance at 570 nm in an ELISA plate reader. Cell survival was expressed as percentage of viable cells of treated samples to control samples. All the dietary agents were tested in triplicates and the experiments were repeated at least three times.

2.4. Nuclear morphology CAL-27 cells were plated at a density of 5  104 cells/well into 6-well chamber slides. After 80% confluence, CAL-27 cells were treated with dietary agents alone and in combination for 24 h. The cells were then washed with PBS, fixed in methanol: acetic acid (3:1, v/v) for 10 min and stained with 50 mg/ml propidium iodide for 20 min. Nuclear morphology of apoptotic cells with condensed/fragmented nuclei was examined under a fluorescent microscope and at least 1  103 cells were counted for assessing apoptotic cell death (Keum et al., 2002).

2.5. Cell cycle analysis Cell cycle distribution and measurement of the percentage of apoptotic cells were performed by flow cytometry (Tai et al., 2000). After treatment, floating cells in the medium were combined with attached cells harvested by trypsinisation. Cells were then washed with cold PBS and fixed in 80% ethanol in PBS at 20  C. After 12 h, fixed cells were pelleted and stained with propidium iodide (50 mg/ml) in the presence of RNase A (20 mg/ml) for 30 min at 37  C, and about 104 events were analysed on a Becton Dickinson FACScan flow cytometer. Cell cycle histograms were analysed using Cell Quest software. Apoptotic cells were distinguished form non-apoptotic intact cells by their decreased DNA content as determined by their lower propidium iodide staining intensity appearing in the area below subG0/G1 phase.

2.6. Determination of ROS generation To assess the generation of intracellular ROS, the oxidation-sensitive fluorescent probe DCFH-DA was used. Briefly, after treatment, CAL-27 cells were harvested and suspended in 0.5 ml PBS containing 10 mM DCFH-DA for 15 min at 37  C in the dark. DCFH-DA was taken up by the cells and deacetylated by cellular esterase to form a non-fluorescent product DCFH, which was converted to a green fluorescent product DCF by intracellular ROS produced by treated CAL-27 cells. The intensity of DCF fluorescence was measured by flow cytometry with excitation and emission settings of 488 and 530 nm respectively (Wang et al., 1999). A total of 104 events were counted and the histograms were analysed using Cell Quest software and compared with histograms of control untreated cells.

2.7. Determination of mitochondrial transmembrane potential 2.2. Cell lines and cell cultures Normal human gingival fibroblast (HGF) and human tongue squamous cell carcinoma (CAL-27) cell lines generously provided by Dr. D.A. Tipton,

The changes in mitochondrial transmembrane potential (Djm) were measured by uptake of the mitochondrial specific lipophilic cation dye rhodamine 123 (Scaduto and Grotyohann, 1999). After treatment, CAL-27 cells

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608 were pelleted by centrifugation for 10 min at room temperature and washed with PBS. The pelleted cells were incubated with 1 ml of exposure medium containing 10 mg/ml rhodamine 123 for 30 min at room temperature in dark, washed and resuspended in PBS. The samples (104 events) were then immediately subjected to flow cytometric analysis at an excitation wavelength of 488 nm and an emission wavelength of 545 nm. Histograms were analysed using Cell Quest software and compared with histograms of control untreated cells.

601

control cells)]  (O value of control cells). The data for nuclear morphology, cell cycle analysis, Bcl-2/Bax ratio and caspase 3 activity were analysed by Student’s t-test. The results were considered statistically significant if the p value was <0.05.

3. Results 3.1. Cytotoxicity assay

2.8. Immunofluorescence

2.9. Western blotting Following treatment with the dietary agents, CAL-27 cells were washed twice with ice-cold PBS, lysed in lysis buffer (50 mM TriseHCl, pH 8.0, 5 mM EDTA, 150 mM sodium chloride, 0.5% Nonidet P-40, 0.5 mM PMSF and 0.5 mM DTT) for 30 min at 4  C, and the supernatant was collected by centrifuging at 12,500  g for 20 min. About 50 mg of total protein from the supernatant as determined by Bradford’s protein estimation kit, was resolved on 10% SDS-PAGE, and then transferred to a nitrocellulose membrane using a semi-dry transfer system (BIORAD) (Towbin et al., 1979). The membranes were incubated in Tris buffered saline (TBS) (150 mM/L sodium chloride, 50 mM/L Tris, pH 7.4) containing 5% non-fat dry milk for 1h to block nonspecific binding sites. The blocks were then incubated with 1:1000 dilution of anti-Bcl-2, Bax (NeoMarkers, USA) and b-actin (Santa Cruz, CA, USA) overnight. The blocks were extensively washed with TBS containing 0.1% Tween-20 and the proteins were detected by incubating with corresponding horse-radish peroxidase-conjugated secondary antibodies (1:2000) for 60e 90 min at room temperature. After extensive washes in TBS containing 0.1% Tween 20, the transferred proteins were visualized using DAB. Densitometry was performed on an IISP flat-bed scanner and quantified with Total Lab 1.11 software.

We first examined the effects of different concentrations of P-E, P-B and bLF on the growth of CAL-27 and HGF cells (Fig. 1). Both P-E and P-B showed dose-dependent cytotoxic effects on CAL-27 cells with IC50 values of 20 and 40 mg/ml respectively. However, HGF cells were more resistant to the cytotoxic effects of P-E and P-B with IC50 values of 70 and 120 mg/ml respectively. Treatment with bLF did not induce any cytotoxic effects either in CAL-27 or in HGF cells. We next examined the cytotoxic effects of tea polyphenols in combination with bLF on CAL-27 and HGF cells. We chose the IC50 value of P-E and P-B on CAL-27 cells and tested this in combination with increasing concentration of bLF. Fig. 2A shows a U-shaped cytotoxicity curve induced by the combination of P-E with increasing concentration of bLF on CAL-27. Significant synergistic effects were observed with P-E (20 mg/ml) þ bLF (40 mg/ml) CAL-27 125 100

% of control

CAL-27 cells cultured to about 80% confluence in 6 well chamber slides were exposed for 24 h to dietary agents alone and in combination. Following treatment, cells were fixed in pre-chilled acetone at 4  C for 5 min. The fixed cells were permeabilised with 0.1% Triton X-100 in PBS and incubated with 1:1000 dilution of anti-Bcl-2 and Bax antibody (Dako, Carprinteria, CA, USA) at 4  C overnight. The proteins were then detected by incubating with fluorescein isothiocyanate (FITC)-conjugated secondary anti-mouse IgG antibody (Dako, Carprinteria, CA, USA) and visualized using a fluorescent microscope.

75 P-E

50

P-B bLF

25 0 0

2.10. Colorimetric estimation of caspase-3 activity

25

50

75

100

125

150

175

400

Treatment µg/ml, 24 h

2.11. Statistical analysis Cytotoxicity data are presented as mean percentages of control  S.D and linear regression analysis was used to calculate the IC50 values. Statistical analysis on the data for cytotoxicity of tea polyphenols alone and in combination with bLF on CAL-27 and HGF cells was done using analysis of variance (ANOVA). The nature of interaction between the combined effects of tea polyphenols and bLF was evaluated as described by Yokoyama et al. (2000). Synergism was calculated from the ratio of expected value (E)/observed value (O) of bLF and tea polyphenols combination treatment; a ratio of >1 indicates a synergistic effect; E value of bLF and tea polyphenols ¼ [(O value of bLF)/(O value of control cells)]  [(O value of tea polyphenols)/(O value of

HGF 125 100

% of control

Caspase-3 activity was assayed using CASP-3-C colorimetric kit (Sigma Chemical Company, St. Louis, MO, USA). After treatment, CAL-27 cells were lysed in lysis buffer containing 250 mM/L HEPES (pH 7.4), 25 mM/L CHAPS and 25 mM/L DTT. The supernatant was used as an enzyme source. The caspase-3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-nitroanilide (Ac-DEVD-pNA) by caspase-3 that results in release of p-nitroaniline (pNA) moiety. The concentration of pNA released from the substrate was calculated from the absorbance values at 405 nm or from a calibration curve prepared with defined pNA solutions.

75

P-E P-B

50

bLF

25 0 0

25

50

75

100

125

150

175

200

400

Treatment µg/ml, 24 h Fig. 1. Effects of polyphenon-E (P-E), Polyphenon-B (P-B) and bovine lactoferrin (bLF) on CAL-27 and HGF cell viability. Cell survival was measured by using MTT assay and expressed as percentage of viable cells of treated samples to control samples. Data are represented as mean  SD of two independent experiments each performed in triplicate. IC50 was calculated using linear regression analysis.

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

602

A

B

CAL-27

Cell viability (%)

Cell viability (%)

CAL-27 120

80 60 40 20 0

100

♦

♦

♦

40 120 1:3

40 160 1:4

♦

80 60 40 20 0

HGF

HGF 120

120

100

100

Cell viability (%)

Cell viability (%)

♦

80 60 40 20

0 20 P-E bLF (P-E+bLF ratio)

20 20 1:1

20 40 1:2

20 60 1:3

20 80 1:4

20 100 1:5

80 60 40 20 0 40 P-B bLF (P-B+bLF ratio)

40 40 1:1

40 80 1:2

40 200 1:5

Treatment µg/ml, 24h Fig. 2. Cytotoxicity of tea polyphenols alone and in combination with bLF on CAL-27 and HGF cells. A: CAL-27 and HGF cells were treated with bLF at the indicated concentration with P-E (20 mg/ml) for 24 h. B: CAL-27 and HGF cells were treated with bLF at the indicated concentration with P-B (40 mg/ml) for 24 h. Data are represented as means  SD of two independent experiments each performed in triplicate. ( e Cytotoxicity was significantly increased compared to P-E alone treated CAL-27 cells ( p < 0.001) (ANOVA followed by LSD). A e Cytotoxicity was significantly decreased compared to P-B alone treated CAL-27 cells ( p < 0.001) (ANOVA followed by LSD). ) e Synergistic effect (ratio of E/O value of a combination of P-E (20 mg/ml) and bLF (40 mg/ml) (1:2 ratio) is 1.69).

combination at a ratio of 1:2 (Table 1). HGF cells appeared to be less susceptible to the cytotoxic effect of P-E and bLF combination compared to CAL-27 cells. Fig. 2B illustrates the effect of treatment with a combination of P-B and bLF on the growth of CAL-27 and HGF cells. Cotreatment with P-B and bLF significantly reduced the cytotoxic effects of P-B on CAL-27 cells. These results suggest that bLF suppresses the anticancer effects of P-B. Overall, the results of MTT assay suggest that dietary agents showed preferential cytotoxic effects on CAL-27 cells compared to normal HGF cells and the order of their cytotoxic effects was P-E and bLF combination (1:2 ratio) > P-E > P-B. Since the

Table 1 Effects of bLF and P-E combination at a ratio of 1:2 on the cytotoxicity of CAL-27 cells Treatment

Untreated control bLF (40 mg/ml) P-E (20 mg/ml) bLF (40 mg/m) þ P-E (20 mg/ml)

Cell viability Observed %

Expected %a

Ratiob

100 100 49.38 29.18

49.38

1.69

Data are represented as means  SD of two independent experiments each performed in triplicate. a Expected value of bLF and P-E combination ¼ [(observed bLF treatment value)/(Control value)]  [(observed P-E treatment value)/(Control value)]  (Control value). b Ratio ¼ (expected value/observed value). A ratio of >1 indicates a synergistic effect, and a ratio of <1 indicates a less than additive effect.

dietary agents preferentially inhibited the growth of CAL27 cells and addition of bLF suppressed the anticancer effects of P-B, further studies were conducted only in CAL-27 cells by incubating with P-E (20 mg/ml), P-B (40 mg/ml) and bLF (40 mg/ml) alone and a combination of P-E (20 mg/ml) with bLF (40 mg/ml) to explore the mode of cell death. 3.2. Induction of apoptosis in CAL-27 cells by dietary agents To determine whether the cytotoxic effects induced by the dietary agents is caused by apoptosis, nuclear morphology was observed using the fluorescent DNA-binding agent propidium iodide. Incubation of CAL-27 cells with the dietary agents for 24 h significantly increased the number of apoptotic cells compared to control as evidenced by nuclear fragmentation and condensation (Fig. 3). Treatment of CAL-27 cells with P-E (20 mg/ml) and P-B (40 mg/ml) alone significantly increased the number of apoptotic cells compared to control ( p < 0.01 and p < 0.05 respectively). Although bLF alone did not induce apoptosis, cotreatment with P-E (20 mg/ml) and bLF (40 mg/ml) significantly increased the number of apoptotic nuclei to 60.24% ( p < 0.005 and p < 0.05 compared to control and P-E alone treated cells respectively). Specifically, 40 mg/ml bLF enhanced the apoptosis inducing potential of 20 mg/ml P-E by w1.9 fold. The order of apoptosis inducing potential of dietary agents was P-E and bLF (1:2) > P-E > P-B.

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

603

A

B

80

Apoptotic nuclei %

70 60 50 40 30 20 10 0

0

bLF 40

P-E 20

P-E+bLF 20:40

P-B 40

Treatment µg/ml, 24 h Fig. 3. Morphological changes (A) and the number of apoptotic nuclei (B) formed after treatment with dietary agents for 24 h. Error bar represents SD between counts of three independent experiments. ( e significantly increased compared to control ( p < 0.05) by Student’s t-test. (( e significantly increased compared to control ( p < 0.01) by Student’s t-test. ((( e significantly increased compared to control ( p < 0.005) by Student’s t-test. ) e significantly increased compared to P-E alone ( p < 0.05) by Student’s t-test.

3.3. Effect of dietary agents on the cell cycle control of CAL-27 cells To investigate whether the dietary agents have a role on cell cycle regulation, we analyzed the changes in cell cycle profiles by using fluorescence activated cell sorter. As shown in Fig. 4, incubation of CAL-27 cells with dietary agents for 24 h, significantly increased the proportion of cells with a reduced DNA content (subG0/G1 or A0 peak) indicative of apoptosis with loss of cells in G1 phase. Loss of DNA, a hallmark of apoptosis, occurs as a result of diffusion of degraded DNA out of the cells after endonuclease cleavage, and after staining with propidium iodide, these cells would have taken up less stain and appear in subG0/G1 or A0 peak i.e. to the left of the G0/G1 peak. Incubation

of CAL-27 cells with P-E and P-B alone significantly increased the proportion of cells with a reduced DNA content from 8.36 per cent (control) to 35.99 per cent (P-E) and 24.01 per cent (P-B). Although bLF alone did not significantly alter the percentage of subG0/G1 cells, cotreatment with 20 mg/ml P-E and 40 mg/ml bLF significantly increased the percentage of subG0/ G1 cells to 72.86% ( p < 0.005 and p < 0.01 compared to control and P-E alone treated cells respectively). Specifically, 40 mg/ml bLF enhanced the apoptosis inducing potential of 20 mg/ml P-E by w2 fold. The increase in subG0/G1 peak although indicative of cell death does not confirm apoptosis suggested by changes in nuclear morphology. In order to ascertain apoptotic cell death, we analyzed ROS generation, Djm, and expression of apoptosis-associated proteins.

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

604

A

40 μg/ml bLF

Control

20 μg/ml P-E+40 μg/ml bLF

B

20 μg/ml P-E

40 μg/ml P-B

90

% Sub G0/G1 cells

80 70 60 50 40 30 20 10 0

Control

bLF 40

P-E 20

P-E+bLF 20:40

P-B 40

Treatment µg/ml, 24 h Fig. 4. Cell cycle analysis of CAL-27 cells treated with dietary agents. (A) Representative histograms demonstrate cell population according to DNA content determined by propidium iodide staining. (B) Quantitative analysis of apoptotic cell population. Error bar represents SD of three independent experiments. ( e significantly increased compared to control ( p < 0.01) by Student’s t-test. (( e significantly increased compared to control ( p < 0.005) by Student’s t-test. ) e significantly increased compared to P-E alone treated cells ( p < 0.01) by Student’s t-test.

3.4. Effect of dietary agents on intracellular ROS generation

3.5. Effect of dietary agents on mitochondrial transmembrane potential (Djm)

To investigate whether ROS is involved in mediating apoptosis induced by the dietary agents we measured the intracellular generation of ROS using the fluorescent probe DCFH-DA. Treatment with dietary agents significantly increased ROS generation in CAL-27 treated cells compared to untreated control, and the order of ROS generation was PE þ bLF > P-E > P-B (Fig. 5A). The mean of DCF fluorescence increased from Ma ¼ 381 (control) to Mb ¼ 938 (P-E treated), Mc ¼ 1229 (P-E and bLF, 1:2 ratio) and Md ¼ 649 (P-B treated). Exposure of CAL-27 cells to bLF alone did not induce ROS generation (data not shown).

The increasing evidence that changes in Djm are linked to apoptosis led us to examine the influence of dietary agents alone and in combination on Djm of CAL-27 cells by flow cytometry using the fluorescent probe rhodamine 123. Rhodamine 123, a cationic dye, is selectively taken up by mitochondria to an extent that is directly proportional to the Djm. As illustrated in Fig. 5B, CAL-27 cells exposed to tea polyphenols for 24 h, showed a loss of Djm as revealed by reduced rhodamine 123 fluorescence intensity compared to control. The mean rhodamine 123 fluorescence declined from Ma ¼ 4636 (control) to Mb ¼ 2456.12 (P-E treated) and

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

A

605

B

ROS Ma = 381 Mb = 938

Ma = 4636.67 Mb = 2456.12

a

a

b

b

Ma = 381

Ma = 4636.67

Mc = 1229

Mc = 2098.75

a c c a

Ma = 381 Md = 649

Ma = 4636.67 Md = 2659.88

d a

a d

Fig. 5. Induction of ROS generation (A) and mitochondrial dysfunction (B) in CAL-27 cells by dietary agents. (a) Untreated control; (b) P-E (20 mg/ml); (c) P-E (20 mg/ml) þ bLF (40 mg/ml) (d) P-B (40 mg/ml).

Md ¼ 2659.88 (P-B treated). Although bLF alone did not show any significant alteration in Djm (data not shown) treatment with a combination of P-E and bLF showed a further sharp decline in rhodamine 123 fluorescence intensity to 2098.75 compared to control and P-E alone treated cells.

compared to untreated control and the order of Bcl-2/Bax decreasing potential of dietary agents was P-E þ bLF > P-E > P-B (Fig. 6A).

3.6. Effect of dietary agents on Bcl-2/Bax ratio

The assay of caspase 3 in CAL-27 cells treated with dietary agents revealed that apoptosis induction was mediated through activation of caspase 3 activity (Fig. 6B). Incubation of CAL27 cells with P-E, P-B, and a combination of P-E and bLF (1:2 ratio) significantly increased caspase 3 activity compared to control. While bLF did not exhibit any effect on caspase 3 activity, cotreatment of bLF with P-E increased enzyme activity by w1.46 fold.

We examined the effects of dietary agents on the expression of Bcl-2 and Bax that play a key role in regulating apoptosis by controlling Djm using immunofluorescence (data not shown) and Western blotting. Incubation of CAL-27 cells with P-E, P-B and a combination of P-E and bLF (1:2 ratio) significantly decreased the Bcl-2/Bax protein expression

3.7. Effect of dietary agents on caspase 3 activity

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

606

Bcl-2/Bax

A

B Bcl2 Bax

Caspase 3

β-actin 1.2

0.25 a 0.2

0.8

UA/ml

Bcl-2/Bax ratio

1

0.6

0.15 0.1

0.4 0.05

0.2 b 0

0

bLF 40

P-E 20

P-E+bLF 20:40

P-B 40

Treatment μg/ml, 24 h

0

0

bLF 40

P-E 20

P-E+bLF 20:40

P-B 40

Treatment μg/ml, 24 h

Fig. 6. The effect of dietary agents on Bcl-2/Bax ratio and caspase 3 activity in CAL-27 cells. Error bar represents SD of three independent experiments. b-actin was used as loading control. A- mmoles of p-nitroaniline formed/min.

4. Discussion The results of the present study demonstrate that tea polyphenols preferentially exert cytotoxic effects on human tongue squamous carcinoma (CAL-27) cells in a dose-dependent manner. The differential sensitivities of tumour and normal cells to cell death by tea polyphenols are in line with previous reports in literature (Weisburg et al., 2004; Park et al., 2005; Shimizu et al., 2005). The present investigation also provides evidence that P-E is more effective than P-B in inhibiting the growth of CAL-27 cells. Furthermore, a combination of P-E and bLF (1:2 ratio) exerts significant synergistic cytotoxic effect as reflected by a U-shaped growth curve, whereas cotreatment with bLF suppressed the anticarcinogenic effects of P-B. Overall, the order of the antiproliferative effects was P-E þ bLF(1:2) > P-E > P-B. The greater efficacy of P-E in inducing cytotoxic effects on CAL-27 cells apparently reflects its higher concentration of EGCG. These findings are substantiated by Weisburg et al. (2004), who demonstrated that tumour cells are more sensitive to the antiproliferative effects of green tea than black tea polyphenols. Tea polyphenols are known to mediate their anticancer properties by induction of apoptosis (Bhattacharyya et al., 2005; Kundu et al., 2005; Nakazato et al., 2005a,b; Qanungo et al., 2005). The present results also demonstrate that green and black tea polyphenols inhibit the growth of CAL-27 cells by inducing apoptosis as revealed by characteristic changes in nuclear morphology and Ao peak. There is increasing evidence that apoptosis induced by chemopreventive or chemotherapeutic agents is associated with perturbation of a specific phase of the cell cycle (Srivastava and Singh, 2004; Jo et al., 2005). As shown in Fig. 4, incubation of CAL-27 cells with tea polyphenols for 24 h resulted in loss of cells in Go/G1 phase with concomitant increase in appearance of apoptotic cells (Ao peak), suggesting that CAL-27 cells arrested in G0/G1 phase by tea polyphenols are preferentially undergoing apoptosis. While

bLF alone did not induce apoptosis of CAL-27 cells, cotreatment of P-E with bLF enhanced the apoptosis-inducing potential of P-E by w2 fold. The order of apoptosis inducing potential of dietary agents was same as that of the growth inhibitory effects i.e. P-E þ bLF(1:2) > P-E > P-B. Mitochondria, which play a pivotal role in apoptosis, are major sites of ROS generation. Excessive generation of ROS can lead to opening of the mitochondrial permeability transition pore with decline in Djm and consequent release of cytochrome c from the intermembrane space into the cytosol culminating in activation of the caspase cascade and apoptotic cell death (Wang, 2001; Chung et al., 2003). The results of the present study demonstrate that incubation of CAL-27 cells with P-E, P-B and P-E þ bLF (1:2 ratio) increased ROS generation, which in turn triggers apoptosis by disrupting mitochondrial function as revealed by decrease in Djm, and activation of caspase-3. EGCG has been reported to induce apoptosis in Jurkat cells by generation of ROS (Nakagawa et al., 2004). Several studies have provided evidence for the involvement of ROS, dissipation of Djm and caspase activity during apoptosis induced by EGCG and tea polyphenols. Moreover, addition of antioxidants such as glutathione, N-acetyl-L-cysteine and catalase was demonstrated to block ROS generation and apoptosis induced by tea polyphenols in cancer cells. Studies have also demonstrated that inhibition of caspase-3 inhibits the cytotoxic effects of tea polyphenols in various cancer cell lines (Bhattacharyya et al., 2005; Kundu et al., 2005; Nakazato et al., 2005a,b; Qanungo et al., 2005; Weisburg et al., 2004). Taken together, these results suggest that tea polyphenols induce apoptosis via ROS-induced mitochondrial perturbation and caspase 3-dependent pathway. ROS production, a key mediator of Djm collapse and mitochondrial mediated apoptosis is known to be influenced by expression of Bcl-2 (Mandavilli et al., 2005; Su et al., 2005; Yin et al., 2005). Overexpression of Bcl-2 reported in a wide range of malignancies prevents ROS production and therefore

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

apoptosis (Kirkin et al., 2004). Bcl-2 belongs to a family of proteins, the members of which may be proapoptotic or antiapoptotic. Bcl-2, the major antiapoptotic protein located primarily in the outer mitochondrial membrane, blocks apoptosis by preventing cytochrome c release from the mitochondria and inhibiting caspase-3 activity. Bax, the predominant proapoptotic member of the Bcl-2 family translocates from the cytosol to the mitochondria, interacts with outer membrane proteins to form pores thereby releasing cytochrome c and triggering caspasemediated apoptotic cell death. The ratio of Bcl-2/Bax appears to be critical in determining the overall propensity of a cell to undergo apoptosis and is a reliable indicator of the clinical response to cancer therapy (Lucken-Ardjomande and Martinou, 2005; Dejean et al., 2006). Tumours with elevated Bcl-2/Bax ratio exhibit poor response to chemotherapy and prognosis compared to tumours with low Bcl-2/Bax ratio. Bcl-2 and Bax have therefore become attractive targets for designing new anticancer drugs (Mackey et al., 1998; Lohmann et al., 2000). Our data shows a positive association between Bcl-2/Bax expression and mitochondrial events in the apoptotic process induced by tea polyphenols. The decrease in Bcl-2/Bax ratio observed in the present study can lead to a decrease in Djm with subsequent activation of caspase-3 activity probably via the cytochrome c/Apaf-1/caspase-9 pathway. These findings are consistent with previous reports on the apoptosis inducing effects of tea polyphenols on various cancer cell lines (Bhattacharyya et al., 2005; Kundu et al., 2005; Nakazato et al., 2005a,b; Qanungo et al., 2005). In a recent report, we have demonstrated that combined administration of tea polyphenols and bLF inhibits hamster buccal pouch carcinogenesis by inducing apoptosis through decreasing Bcl-2/Bax ratio and increasing caspase-3 expression (Chandra Mohan et al., 2006). Since a low ratio of Bcl-2/Bax appears to increase chemosensitivity, our results suggest that tea polyphenols and bLF combination may have therapeutic potential against oral cancer. In summary, the results of the present study demonstrate that tea polyphenols exert antiproliferative effects against CAL-27 cells that is mediated through apoptosis. The data indicate a key role for ROS and Bcl-2/Bax in mitochondrial mediated apoptosis by dissipating Djm and activating caspase-3. The study also emphasizes the greater efficacy of P-E both alone and in combination with bLF. The mechanism by which bLF enhances the anticarcinogenic effects of P-E and suppresses the anticarcinogenic effect of P-B is however unclear at present. Our results strengthen the observations of Suganuma et al. (1999) that green tea is a suitable candidate for use in combination with other cancer preventives. Since green tea polyphenols have already entered clinical trials in patients at high risk for liver and prostate cancers, it would be worthwhile to design similar trials in patients with oral premalignant lesions to evaluate the chemopreventive efficacy of P-E (Bettuzzi et al., 2006; Luo et al., 2006). Although additional studies on the effects of tea polyphenols on cell cycleassociated proteins, NFkB and Fas signaling are required, the results of the present study have opened potential avenues for targeting the mitochondria in apoptosis.

607

Acknowledgements This work was supported by a grant from the Mitsui Norin Co., Ltd., Tokyo, Japan. Financial support from the Indian Council of Medical Research, New Delhi, India in the form of a Senior Research Fellowship to Mr. K.V.P. Chandra Mohan is gratefully acknowledged. The authors are thankful to Dr. Usha K. Burra, Institute of Pathology, New Delhi, India for her help with immunofluorescence studies. References Bettuzzi S, Brausi M, Rizzi F, Castagnetti G, Peracchia G, Corti A. Chemoprevention of human prostate cancer by oral administration of green tea catechins in volunteers with high-grade prostate intraepithelial neoplasia: a preliminary report from a one-year proof-of-principle study. Cancer Res 2006;66:1234e40. Bhattacharyya A, Lahiry L, Mandal D, Sa G, Das T. Black tea induces tumor cell apoptosis by Bax translocation, loss in mitochondrial transmembrane potential, cytochrome c release and caspase activation. Int J Cancer 2005; 117:308e15. Chandra Mohan KVP, Hara Y, Abraham SK, Nagini S. Comparative evaluation of the chemopreventive efficacy of green and black tea polyphenols in the hamster buccal pouch carcinogenesis model. Clin Biochem 2005;38: 879e86. Chandra Mohan KVP, Devaraj H, Prathiba D, Hara Y, Nagini S. Antiproliferative and apoptosis inducing effect of lactoferrin and black tea polyphenol combination on hamster buccal pouch carcinogenesis. Biochim Biophys Acta 2006;1760:1536e44. Chung YM, Bae YS, Lee SY. Molecular ordering of ROS production, mitochondrial changes, and caspase activation during sodium salicylateinduced apoptosis. Free Radic Biol Med 2003;34:434e42. Dejean LM, Martinez-Caballero S, Manon S, Kinnally KW. Regulation of the mitochondrial apoptosis-induced channel, MAC, by Bcl-2 family proteins. Biochim Biophys Acta 2006;1762:191e201. Gupta S. Molecular signaling in death receptor and mitochondrial pathways of apoptosis (Review). Int J Oncol 2003;22:15e20. Jo EH, Kim SH, Ra JC, Kim SR, Cho SD, Jung JW, et al. Chemopreventive properties of the ethanol extract of Chinese licorice (Glycyrrhiza uralensis) root: induction of apoptosis and G1 cell cycle arrest in MCF-7 human breast cancer cells. Cancer Lett 2005;230:239e47. Keum YS, Kim J, Lee KH, Park KK, Surh YJ, Lee JM, et al. Induction of apoptosis and caspase-3 activation by chemopreventive [6]-paradol and structurally related compounds in KB cells. Cancer Lett 2002;177:41e7. Kirkin V, Joos S, Zornig M. The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 2004;1644:229e49. Kundu T, Dey S, Roy M, Siddiqi M, Bhattacharya RK. Induction of apoptosis in human leukemia cells by black tea and its polyphenol theaflavin. Cancer Lett 2005;230:111e21. Lohmann CM, League AA, Clark WS, Lawson D, DeRose PB, Cohen C. Bcl-2: Bax and Bcl-2:Bcl-x ratios by image cytometric quantitation of immunohistochemical expression on ovarian carcinoma: correlation with prognosis. Cytometry 2000;42:61e6. Lucken-Ardjomande S, Martinou JC. Regulation of Bcl-2 proteins and of the permeability of the outer mitochondrial membrane. C R Biol 2005;328: 616e31. Luo H, Tang L, Tang M, Billam M, Huang T, Yu J, et al. Phase IIa chemoprevention trial of green tea polyphenols in high-risk individuals of liver cancer: modulation of urinary excretion of green tea polyphenols and 8hydroxydeoxyguanosine. Carcinogenesis 2006;27:262e8. Mackey TJ, Borkowski A, Amin P, Jacobs SC, Kyprianou N. Bcl-2/Bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer. Urology 1998;52:1085e90. Mandavilli BS, Boldogh I, Van Houten B. 3-Nitropropionic acid-induced hydrogen peroxide, mitochondrial DNA damage, and cell death are

608

K.V.P.C. Mohan et al. / Cell Biology International 31 (2007) 599e608

attenuated by Bcl-2 overexpression in PC12 cells. Brain Res Mol Brain Res 2005;133:215e23. Mignogna MD, Fedele S, Lo Russo L. The World Cancer Report and the burden of oral cancer. Eur J Cancer Prev 2004;13:139e42. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55e63. Moyers SB, Kumar NB. Green tea polyphenols and cancer chemoprevention: multiple mechanisms and endpoints for phase II trials. Nutr Rev 2004;62: 204e11. Nakagawa H, Hasumi K, Woo JT, Nagai K, Wachi M. Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by ()-epigallocatechin gallate. Carcinogenesis 2004;25: 1567e74. Nakazato T, Ito K, Ikeda Y, Kizaki M. Green tea component, catechin, induces apoptosis of human malignant B cells via production of reactive oxygen species. Clin Cancer Res 2005a;11:6040e9. Nakazato T, Ito K, Miyakawa Y, Kinjo K, Yamada T, Hozumi N, et al. Catechin, a green tea component, rapidly induces apoptosis of myeloid leukemic cells via modulation of reactive oxygen species production in vitro and inhibits tumor growth in vivo. Haematologica 2005b;90: 317e25. Ohigashi H, Murakami A. Cancer prevention with food factors: alone and in combination. Biofactors 2004;22:49e55. Park HK, Han DW, Park YH, Park JC. Differential biological responses of green tea polyphenols in normal cells versus cancer cells. Curr Appl Phys 2005;5:449e52. Qanungo S, Das M, Haldar S, Basu A. Epigallocatechin-3-gallate induces mitochondrial membrane depolarization and caspase-dependent apoptosis in pancreatic cancer cells. Carcinogenesis 2005;26:958e67. Sakamoto K. Synergistic effects of thearubigin and genistein on human prostate tumor cell (PC-3) growth via cell cycle arrest. Cancer Lett 2000;151: 103e9. Scaduto Jr RC, Grotyohann LW. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 1999;76: 469e77. Shimizu M, Deguchi A, Lim JT, Moriwaki H, Kopelovich L, Weinstein IB. ()-Epigallocatechin gallate and polyphenon E inhibit growth and activation of the epidermal growth factor receptor and human epidermal growth factor receptor-2 signaling pathways in human colon cancer cells. Clin Cancer Res 2005;11:2735e46. Shimizu T, Nakazato T, Xian MJ, Sagawa M, Ikeda Y, Kizaki M. Resveratrol induces apoptosis of human malignant B cells by activation of caspase-3 and p38 MAP kinase pathways. Biochem Pharmacol 2006;71: 742e50.

Srivastava SK, Singh SV. Cell cycle arrest, apoptosis induction and inhibition of nuclear factor kappa B activation in anti-proliferative activity of benzyl isothiocyanate against human pancreatic cancer cells. Carcinogenesis 2004;25:1701e9. Su YT, Chang HL, Shyue SK, Hsu SL. Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Biochem Pharmacol 2005;70:229e41. Suganuma M, Okabe S, Kai Y, Sueoka N, Sueoka E, Fujiki H. Synergistic effects of ()-epigallocatechin gallate with ()-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9. Cancer Res 1999;59:44e7. Suganuma M, Ohkura Y, Okabe S, Fujiki H. Combination cancer chemoprevention with green tea extract and sulindac shown in intestinal tumor formation in Min mice. J Cancer Res Clin Oncol 2001;127:69e72. Tai KW, Chou MY, Hu CC, Yang JJ, Chang YC. Induction of apoptosis in KB cells by pingyangmycin. Oral Oncol 2000;36:242e7. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350e4. Tsuda H, Sekine K, Fujita K, Ligo M. Cancer prevention by bovine lactoferrin and underlying mechanisms e a review of experimental and clinical studies. Biochem Cell Biol 2002;80:131e6. Vermeulen K, Van Bockstaele DR, Berneman ZN. Apoptosis: mechanisms and relevance in cancer. Ann Hematol 2005;84:627e39. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001; 15:2922e33. Wang IK, Lin-Shiau SY, Lin JK. Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells. Eur J Cancer 1999;35: 1517e25. Weisburg JH, Weissman DB, Sedaghat T, Babich H. In vitro cytotoxicity of epigallocatechin gallate and tea extracts to cancerous and normal cells from the human oral cavity. Basic Clin Pharmacol Toxicol 2004;95: 191e200. Yang CS, Chung JY, Yang G, Chhabra SK, Lee MJ. Tea and tea polyphenols in cancer prevention. J Nutr 2000;130:472Se8. Yin HQ, Kim YH, Moon CK, Lee BH. Reactive oxygen species-mediated induction of apoptosis by a plant alkaloid 6-methoxydihydrosanguinarine in HepG2 cells. Biochem Pharmacol 2005;70:242e8. Yokoyama Y, Dhanabal M, Griffioen AW, Sukhatme VP, Ramakrishnan S. Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth. Cancer Res 2000;60:2190e6. Zhou JR, Yu L, Mai Z, Blackburn GL. Combined inhibition of estrogen-dependent human breast carcinoma by soy and tea bioactive components in mice. Int J Cancer 2004;108:8e14.