carcinogenesis-2006-sartippour-2424-33

8/8/2019 Carcinogenesis-2006-Sartippour-2424-33

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The combination of green tea and tamoxifen is effective against breast cancer

Maryam R.Sartippour1,3

, Richard Pietras2

, DianaC.Marquez-Garban2, Hsiao-Wang Chen2, David Heber3,Susanne M.Henning

3, Guilan Sartippour

1,

Liping Zhang1, Ming Lu1, Olga Weinberg2,Jian Yu Rao

4and Mai N.Brooks

1,3,

1Department of Surgery, 2Department of Medicine, Division of Oncology,3Center for Human Nutrition and 4Department of Pathology,University of California, Los Angeles, CA 90095, USA

To whom correspondence should be addressed at: Department of Surgery,Division of Oncology, University of California, Box 951782, Los Angeles,CA 90095-1782, USA. Tel: +1 310 206 2215; Fax: +1 310 825 7575;Email: [email protected]

Epidemiologic data have suggested that green tea may

prevent breast cancer. Studies in our laboratory haveprovided evidence that green tea extract inhibits breastcancer growth by a direct anti-proliferative effect on thetumor cells, as well as by indirect suppressive effectson the tumor-associated endothelial cells. In this study, weasked whether concurrent administration of green teamay add to the anti-tumor effects of standard breastcancer therapy. We observed that green tea increased theinhibitory effect of tamoxifen on the proliferation of theER (estrogen receptor)-positive MCF-7, ZR75, T47Dhuman breast cancer cells in vitro. This combinationregimen was also more potent than either agent alone atincreasing cell apoptosis. In animal experiments, micetreated with both green tea and tamoxifen had the

smallest MCF-7 xenograft tumor size, and the highestlevels of apoptosis in tumor tissue, as compared witheither agent administered alone. Moreover, the suppres-sion of angiogenesis in vivo correlated with larger areas ofnecrosis and lower tumor blood vessel density in treatedxenografts. Green tea decreased levels of ER-a in tumorsboth in vitro and in vivo. We also observed that green teablocked ER-dependent transcription, as well as estradiol-induced phosphorylation and nuclear localization ofmitogen-activated protein kinase. To our knowledge, thisstudy is the first to show the interaction of green tea withthe ER pathway, as well as provide mechanistic evidencethat the combination of green tea and tamoxifen is morepotent than either agent alone in suppressing breast

cancer growth. These results may lead to future improve-ments in breast cancer treatment and prevention.

Introduction

Despite multiple advances in the treatment for breast cancer,the mortality remains high (1). Since there is no definitive

cure for advanced breast cancer, prevention strategies canpotentially save many lives. Current effective breast cancerprevention strategies for high-risk women and for adjuvanttherapy in estrogen receptor-a(ER-a) positive cancercases include tamoxifen (2). However, a 5-year course oftamoxifen may involve serious side effects. Thus, alternativeinterventions such as substances from natural herbal sourcesare needed to replace or to supplement current regimens.

Next to water, tea is the most widely consumed beveragein the world, and its ingestion in reasonable doses is con-sidered safe (3). There are reports linking the consumption ofgreen tea with an improved prognosis in breast cancer (4,5).EGCG (epigallocatechin-3-gallate), the major component

of green tea, was shown to suppress the growth of the breastcancer cell line Hs578t (6) and the ER-positive MCF-7 xenograft (7). Studies in our laboratory have providedevidence that green tea extract (GTE) inhibits breast cancergrowth by a direct anti-proliferative effect on tumor cells, aswell as by indirect suppressive effects on tumor-associatedendothelial cells (811). Our studies characterized anti-proliferative effects of individual catechin components ofgreen tea and determined their relative potency (8). It hasbeen generally reported that EGCG accounts for most, if notall, of the actions of green tea. We, on the other hand,observed that CG (catechin gallate), GC (gallocatechin), EC(epicatechin), EGC (epigallocatechin), ECG (epicatechin-3-gallate) and GCG (gallocatechin gallate) suppress both

ER-negative MDA-MB231 breast cancer cells and vascularendothelial cells. In fact, many of these catechin componentsare as potent as GTE or EGCG in our experiments. Thisexplains why GTE has a similar potency as EGCG dose perdose, although it contains only half as much EGCG.Globally, this observation supports our philosophy thatnatural products exert their positive effects based on thesum or synergy of their multiple mixed components.Therefore, it may not necessarily always be beneficial toisolate one single chemical compound and concentrate it inthe form of a pure drug.

The above results led to our interest in the questionwhether concurrent administration of green tea has anyadditive or synergistic effects on the widely used breast

cancer prevention drug tamoxifen. Thus, we set out to det-ermine if the combination of green tea and tamoxifen is morepotent than either agent administered alone, and to elucidateany mechanistic interaction(s) between green tea, estrogenand tamoxifen. Our findings suggest that green tea may havea supplemental role, with or without tamoxifen, in breastcancer treatment and prevention.

Materials and methods

Reagents and cells

GTE was obtained from Pharmanex (batch no. 990222; Brisbane, CA) byproprietary methods (8). GTE concentration was expressed as mg/ml media

Abbreviations: CAT, chloramphenicol acetyltransferase; CI, combinationindex; CG, catechin gallate; EC, epicatechin; EGC, epigallocatechin; EGCG,epigallocatechin-3-gallate; ER, estrogen receptor; ERE, estrogen responseelement; GTE, green tea extract; GC, gallocatechin; GCG, gallocatechingallate.

Carcinogenesis vol.27 no.12 pp.24242433, 2006doi:10.1093/carcin/bgl066

Advance Access publication June 19, 2006

# The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 2424


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for in vitro experiments, and as g/l drinking water for animal experiments.EGCG, EC, ECG, b-D-glucuronidase type X-A from Escherichia coli,arylsulfatase type VIII from abalone entrails, estradiol-17b and tamoxifenwere obtained from Sigma (St Louis, MO).

The human breast cancer cell lines MCF-7, ZR75, T47D and SKBR3were obtained from American Type Tissue Culture Collection (ATCC,Rockville, MD). Cells were routinely maintained in RPMI medium 1640(Life Technologies, Grand Island, NY) with 10% heat-inactivated fetalbovine serum (FBS; Omega Scientific, Tarzana, CA), 100 U/ml penicillinand 100 mg/ml streptomycin (Life Technologies). For estrogen-free

experiments, medium was changed to phenol red-free RPMI 1640 with0.5% dextran-coated charcoal (DCC) stripped FBS (Gemini Bio-Products,Woodland, CA). After 48 h, medium was changed to the same RPMIDCCFBS above, but containing estradiol-17b (1 nM) for 30 min before treatmentwith GTE and/or tamoxifen.

In vitro assay of proliferation

Cells were plated on to 48-well culture plates at 10000 cells/well andincubated at 37C in 5% CO2 for 24 h in RPMI with 10% FBS media. Thetest sample was applied the following day. In the negative control wells, thegrowth media were changed to RPMI without FBS. On the third day, 1 mCiof [methyl-

3H]thymidine (Amersham, Piscataway, NJ) was added to each

well. At 1215 h later, plates were washed with phosphate-buffered saline(PBS). The cells were fixed with trichloroacetic acid, washed with ethylalcohol and lysed with sodium hydroxide, using methods as describedpreviously (8). After adding glacial acetic acid, the radioactivity of celllysates was counted in scintillation solution (ScintiVerse, Fisher, Pittsburgh,

PA). In vitro assays were performed in triplicates.Mouse tissue catechin measurement

Nude mice were bred in a pathogen-free colony at our institution and6-week-old female mice were used for this study. All mice were housed fourper cage, and were fed ad libitum with sterilized food pellets and sterilewater. Each group of four mice was assigned to (i) water, (ii) GTE 0.625 g/lwater, (iii) GTE 1.25 g/l and (iv) GTE 2.5 g/l. After 4 days of treatment, themice were killed by exposure to a nitrogen gas environment, and themammary pads were removed.

Tissue polyphenol analysis was performed using a modification of themethod of Lee et al. (12). About 100200 mg of mammary tissue washomogenized in 0.8 ml of buffer A (40 mM NaH2PO4/0.1% EDTA, pH 5.8),mixed with 30 ml of buffer B (10% ascorbic acid40 mM NaH2PO4/0.1%EDTA), 1500 U b-D-glucuronidase, 24 U sulfatase and 20 ml of 2.3 mM CGinternal standard. The mixture was incubated at 37C for 45 min. Mammaryhomogenates were extracted three times with 2 ml ethylacetate each. Aliquotof 10 ml of 0.2% ascorbic acid/0.005% EDTA was added to the pooledsupernatant followed by vacuum concentration for 2 h at low heat using aSavant SC-100 Speed-Vac system (Savant Instruments, Farmingdale, NY).Samples were reconstituted in 200 ml of mobile phase A (75 mM citric acidand 25 mM ammonium acetate), and 20 ml aliquots were injected into theHPLC column. The column was eluted at room temperature with a lineargradient from 100% mobile phase A to 90% mobile phase A and 10% mobilephase B [75 mM citric acid25 mM ammonium acetate/acetonitrile (50:50)]in 4 min at a flow rate of 1 ml/min. The gradient was linearly changedfurther to 70% A/30% B (412 min), 66%A/34%B (1217 min), 63% A/37%B (1720 min), 57% A/43% B (2029 min), 100% B (2933 min), 100% A(3335 min) and maintained there until analysis of the next sample.

Mouse breast cancer xenografts

Six-week-old ovariectomized nude mice were purchased from HarlanSprague Dawley (Indianapolis, IN). All mice were housed four per cage,and were fed ad libitum with sterilized food pellets and sterile water.Mice were inoculated with estradiol-17b (1.7 mg/pellet; Innovative Researchof America, Sarasota, FL) before the injection of 5 107 MCF-7 cellssubcutaneously into the mid-back region. The tumor size was measured inthree dimensions with calipers twice weekly starting at Day 7. Mice wereobserved for any change in behavior, appearance or weight. When tumorsreached 3050 mm3, six animals were randomly assigned to each treatmentgroup. Control mice drank water, and experimental mice drank GTE atthe concentrations of 2.5 g/l and/or were inoculated subcutaneously withtamoxifen (20 mg/pellet; Innovative Research of America). At the end ofthe experiment at Day 64, the mice were killed and xenograft specimenswere harvested for further analyses.

Immunohistochemistry

Paraffin-embedded tumor specimens were prepared as described previously(8). We used antigen retrieval with 0.01 M sodium citrate (pH 6.0) in a95C water bath for 20 min, and 1 mg/ml trypsin (Sigma) at room

temperature for 3 min. Immunostaining was performed with the DAKOEnvision peroxidase rabbit ready-to-use system. The slides were sequentiallyincubated at room temperature as follows: (i) in DAKO antigen blockreagent to block non-specific antibody binding; (ii) with the rabbit anti-vonWillebrand factor (vWf; Dako, Carpinteria, CA) primary antibody at 1:250dilution for 1 h; (iii) with the DAKO secondary antibody to rabbit for30 min; and (iv) developed with DAKO DAB (diaminobenzidine) solution.Positively stained vessels were counted in five separate fields for eachspecimen using an Olympus model BH2 microscope. Following themethod established by Weidner (11), fields containing the highest density

of vWf-positive vessels, i.e. bursts, were identified at scanning power andthen counted at 400 magnification. The numbers for the five fields wereaveraged.

ER immunohistochemistry was performed as above, without trypsindigestion. The primary antibody was used at 1:50 dilution (Biocare Medical,Walnut Creek, CA). The secondary anti-mouse antibody was from DAKO.The histological slides were reviewed by a Board-certified pathologist(J.Y.R.) and graded from 0 to 2+, using standard clinical criteria.

Apoptosis assay

Apoptosis was measured by the TUNEL (TdT-dUTP terminal nick-endlabelling) assay. Cells were plated in the 2-well glass slides (Nalgen Nunc,Naperville, IL). After 48 h of incubation with GTE (5 or 10 mg/ml) and/ortamoxifen (1 or 10 nM), the ApopTag peroxidase kit (Intergen, Purchase,NY) was used to detect in situ apoptosis according to the manufacturersprotocol. Briefly, cells were fixed in paraformaldehyde, and post-fixed inice-cold ethanol/acetic acid (2:1 v/v). After applying the equilibration buffer,

the TdT (terminal deoxy-nucleotidyl transferase) was added, followed byanti-dioxigenin conjugate. Slides were stained with peroxidase substrate,counterstained by methyl green, dehydrated by xylene, and mounted under aglass coverslip to be viewed under the microscope at (400 magnification.

Paraffin-embedded specimens were cut into 5 mm sections, deparaffinizedand then pretreated with proteinase K using the above kit.

Western blot analysis

After being maintained in estrogen-free conditions for 48 h, and with 1 nMestradiol-17b for 30 min, cells were treated with GTE and/or tamoxifenfor 48 h. Cell lysates were centrifuged at 12 000 g for 10 min, and thesupernatant was retained. Protein concentration was measured by theBradford assay (Bio-Rad Laboratories, Hercules, CA). Approximately 50 mgof protein was separated by a 7.5% SDSPAGE, and transferred on to anitrocellulose membrane by electrophoretic blotting. The membrane wasblocked for 1 h with 5% non-fat dry milk in Tris-buffered saline (TBS), andthen incubated for 1 h with a 1:500 dilution of anti human ER-a affinity

purified rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz,CA). The blots were then washed three times over 1 h in TBST (TBS with0.1% Tween-20), and incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-rabbit IgG (1:2000), and thenwashed in TBST as before. The membranes were developed using theSupersignal West Pico Chemiluminescent Western blotting detectionsystem according to the manufacturers instructions (Pierce, ArlingtonHeights, IL).

Analysis of estradiol binding

ER-a binding was determined by ligand binding with [3H]estradiol-17b.Previous studies have shown that MCF-7 cells do not contain any significantlevels of ER-b (13). Effects of GTE on specific estradiol binding in MCF-7 cells were determined as before (14). Briefly, MCF-7 breast cancer cells(1 106 cells/condition) were incubated with 2 nM [3H]estradiol-17b (NEN,Boston, MA) and increasing concentrations of GTE (0.280 mg/ml). A 100-fold molar excess of unlabeled estradiol-17b was present with [3H]estradiol-17b in paired samples for the determination of displaceable binding. Sampleswere incubated for 1 h at room temperature with vortex every 15 min. Tostop the reaction, cells were placed on ice. Unbound steroids were removedwith DCC, and counts per minute (c.p.m.) were determined by liquidscintillation counting.

ER transcriptional activation

ER transcriptional activity was assessed using an ERECAT (estrogenresponse elementchloramphenicol acetyltransferase) reporter gene. Tran-sient transfections were performed in MCF-7 breast cancer cells withmethods as before using the pERE-BLCAT reporter vector (14,15). In brief,cells in 60 mm Petri dishes were transfected using 2.5 mg of pERE-BLCATvector and 0.5 mg of pCMV-b-galactosidase for transfection efficiency. Then12 ml of Fugene 6 (ROCHE Applied Science, Indianapolis, IN) was addedper plate. Transfection was performed for 24 h in the presence of 1% DCCFBS in phenol red-free RPMI. At 24 h after transfection, cells were treated

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with vehicle alone, 1 nM estradiol-17b, 5 or 10 mg/ml of GTE, orcombinations of these agents with tamoxifen. CAT reporter gene assay wasperformed after 18 h of treatment using the CAT-ELISA kit from RocheApplied Science. b-Galactosidase activity was assessed by using the b-GalELISA kit from Roche Applied Science for transfection efficiency.

p44/42 MAPK activity

Cells were maintained in estrogen-free conditions for 4872 h beforeexperiments, then treated with 2 nM estradiol in combination with selectedinhibitors, such as 20 nM ICI 182,780 (an anti-estrogen agent; Astra Zeneca,

Neward, DL), tamoxifen (10 nM) or GTE (40 mg/ml). Rapid effectsof estradiol (and selected inhibitors) on phosphorylation and nuclearlocalization of MAPK (mitogen-activated protein kinase) were tested usingconfocal microscopy with breast tumor cells (16). For immunofluorescencestudies with confocal microscopy, breast cells were grown on coverslipsand then fixed with methanol as described previously (17). Cells werethen immersed in primary phosphor-MAPK polyclonal antibody (anti-phospho-p44/p42 MAPK, Thr202/Tyr204, polyclonal antibody; New EnglandBiolabs, Beverly, MA), and a fluorescent-labeled secondary anti-rabbitantibody.

Cell hybrid DNA was stained using Hochest 33258 dye at 0.5 mg/ml.After repeated washes with PBS, coverslips were mounted on glassmicroscope slides using mounting medium Fluoroguard Antifade Reagent(Bio-Rad) and viewed using a Leica TCS SP MP Inverted ConfocalMicroscope (Leica, Bannockburn, IL).

Aromatase activity

Aromatase activity in lung cancer cell lines was determined using the[3H]H2O release method reported by Kinoshita and Chen (18). Breast cancer

cell line SKBR3 was plated and grown to confluency in 6-well cell cultureplates. Once confluent, cells were washed twice with PBS. To each well,1 ml of serum-free medium containing substrate, 100 nM [1b-

3H]Androst-4-

ene-3,17-dione (PerkinElmer, Boston, MA) and 500 nM progesterone (toinhibit 5a-reductase in cells) was added. The reaction was incubated for3 h at 37C. The reaction mixture was then removed and extracted with 1 mlof chloroform, vortexed and centrifuged for 10 min at a maximum speed of10 000 g. The upper aqueous layer was carefully removed and treated with1 ml of dextran-treated charcoal. After centrifugation at maximum speed,supernatant containing the product, [3H]H2O, was counted in a liquidscintillation counter. Protein concentration was determined using theBradford method, after dissolving cells in 0.5 M NaOH.

Statistical analysis

Descriptive statistics, such as mean and standard error, were used to

summarize the results. The Studentst-test was used for univariate analysis,and the ANOVA test was performed for comparison among the various

groups. Statistical significance was defined by P < 0.05.For analysis to detect additive, synergistic or antagonistic effects, the

multiple drug effect analysis method was used (19). Briefly, a combinationindex (CI) value was calculated to assess synergism or antagonism accordingto the following equation which assumes an independent mechanism of drugaction (mutual exclusivity): CI (D)1/(Dx)1 + (D)2/(Dx)2 + (D)1(D)2/(D

x)1(Dx)2, where (D)1 and (D)2 are the concentrations of Drug 1 and Drug 2

which combined produce x% inhibition, and (Dx

)1 and (Dx)2 are theconcentrations of each drug which alone produce x% inhibition. CI 1indicates an additive interaction, CI < 1 indicates synergy and CI > 1antagonism.

Results

In vitro proliferation studiesThe in vitro assays showed that individually, green tea ortamoxifen inhibited proliferation of human ER-positiveMCF-7 breast cancer cells (Figure 1A and B). The inhibitoryeffects of these compounds were dose-dependent. The ID50were as follows: 16.6 mg/ml of green tea and 1.2 mm oftamoxifen. We then tested the combination of green tea andtamoxifen. We used tamoxifen at a lower dose (nm) than thestandard in vitro dose (mm), in order to better detect potentialsynergy. The combination of green tea and tamoxifen wasmore effective than either agent given alone (Figure 1C, P

carcinogenesis-2006-sartippour-2424-33

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