Hops (Humulus Lupulus) Inhibits Oxidative Estrogen Metabolism and Estrogen-Induced Malignant Transformation in Human Mammary Epithelial Cells (MCF-10A)

Published OnlineFirst October 13, 2011; DOI: 10.1158/1940-6207.CAPR-11-0348

Cancer Prevention Research Article Research

Hops (Humulus lupulus) Inhibits Oxidative Estrogen Metabolism and Estrogen-Induced Malignant Transformation in Human Mammary Epithelial cells (MCF-10A)

L.P. Madhubhani P. Hemachandra, R. Esala P. Chandrasena, Shao-Nong Chen, Matthew Main, David C. Lankin, Robert A. Scism, Birgit M. Dietz, Guido F. Pauli, Gregory R.J. Thatcher, and Judy L. Bolton

Abstract Long-term exposure to estrogens including those in traditional hormone replacement therapy (HRT) increases the risk of developing hormone-dependent cancers. As a result, women are turning to over-the- counter (OTC) botanical dietary supplements, such as black cohosh (Cimicifuga racemosa) and hops (Humulus lupulus), as natural alternatives to HRT. The two major mechanisms which likely contribute to estrogen and/or HRT cancer risk are: the estrogen receptor–mediated hormonal pathway; and the chemical carcinogenesis pathway involving formation of estrogen quinones that damage DNA and proteins, hence initiating and promoting carcinogenesis. Because, OTC botanical HRT alternatives are in widespread use, they may have the potential for chemopreventive effects on estrogen carcinogenic pathways in vivo. Therefore, the effect of OTC botanicals on estrogen-induced malignant transformation of MCF-10A cells was studied. Cytochrome P450 catalyzed hydroxylation of estradiol at the 4-position leads to an o-quinone believed to act as the proximal carcinogen. Liquid chromatography/tandem mass spectrometry analysis of estradiol metabolites showed that 4-hydroxylation was inhibited by hops, whereas black cohosh was without effect. Estrogen-induced expression of CYP450 1B1 and CYP450 1A1 was attenuated by the hops extract. Two phenolic constituents of hops (xanthohumol, XH; 8-prenylnaringenin, 8-PN) were tested: 8-PN was a potent inhibitor, whereas XH had no effect. Finally, estrogen-induced malignant transformation of MCF-10A cells was observed to be signiﬁcantly inhibited by hops (5 mg/mL) and 8-PN (50 nmol/L). These data suggest that hops extracts possess cancer chemopreventive activity through attenuation of estrogen metabolism mediated by 8-PN. Cancer Prev Res; 5(1); 73–81. 2011 AACR.

Introduction positive cells (hormonal pathway) and the formation of reactive estrogen metabolites (chemical pathway, Fig. 1; Long-term exposure to estrogen resulting from a combi- ref. 5). Understanding these mechanisms can lead to strat- nation of early onset of menstruation, nulliparity or delayed egies for prevention of estrogen-dependent cancers which first child birth, short duration of breast feeding, late meno- can enhance the quality of life for women as well as pause, and use of hormone replacement therapy (HRT; significantly reduce the cost of health care. refs. 1, 2) increases the risk of hormone-dependent cancers Modulation of the hormonal mechanism has had some in women (3, 4). Two major mechanisms for estrogen success with selective estrogen receptor modulators and carcinogenesis have been proposed which include estro- aromatase inhibitors (6–9). Reduction in estrogen cancer gen-induced cell proliferation in estrogen receptor (ER)- risk could also be achieved through modulation of enzymes involved in generation of reactive estrogen metabolites (chemical pathway, Fig. 1). In breast epithelium, fi Authors' Af liation: Department of Medicinal Chemistry and Pharmacog- 17b-estradiol (E2)canbeconvertedtoits2-hydroxyes- nosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois tradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2)catechols catalyzed by CYP450 1A1/1A2 and CYP450 1B1, Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). respectively (Fig. 1). Both catechols are further oxidized to form reactive electrophilic o-quinones which have the Corresponding Author: Judy L. Bolton, Department of Medicinal Chem- istry and Pharmacognosy, College of Pharmacy, University of Illinois at potential to cause DNA and protein damage leading to Chicago, 833 S. Wood Street, Chicago, IL 60612-7231. Phone: 312-996- carcinogenesis (5). Several detoxification pathways can 5280; Fax: 312-996-7170; E-mail: [email protected] potentially neutralize catechols and quinones in cells; for doi: 10.1158/1940-6207.CAPR-11-0348 example, catechol O-methyl transferase (COMT) converts 2011 American Association for Cancer Research. catechols to methyl ether metabolites that cannot form

www.aacrjournals.org 73

Downloaded from cancerpreventionresearch.aacrjournals.org on September 28, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst October 13, 2011; DOI: 10.1158/1940-6207.CAPR-11-0348

Hemachandra et al.

R R R

P450 1B1 [O]

Hops HO HO O E (E ) 4-OHE (E ) Figure 1. Scheme of potential 1 2 OH 1 2 O 4-OHE1(E2)-Q NQO1 mechanisms by which hops can Hops modulate estrogen metabolism in P450 1A1 COMT DNA breast epithelial cells. Hops inhibits Hops E2-induced upregulation of R R CYP450 1A1 and CYP450 1B1 Genotoxicity resulting in reduced formation of HO catechol estrogens leading to less quinone production; hence, DNA HO HO damage and malignant 2-OHE1(E2) 4-MeOE1(E2) transformation is decreased. OCH3 Because COMT converts 2-OHE1 and 4-OHE1 into the corresponding COMT 2- and 4-methoxy metabolites, O these served as indices of catechol Estrone (E1): R, R OH estrogen formation. Estradiol (E2): R,

H3CO

HO 2-MeOE1(E2)

an o-quinone (10; Fig. 1). The expression of these ingredients, including xanthohumol (XH) that has shown enzymesdiffersfromonetissuetotheotherandcanbe chemopreventive activity (24–27). Black cohosh extracts altered when a normal cell is transformed into a cancer have been reported to inhibit breast cancer cell growth both cell, leading to an imbalance in estrogen metabolism (11, in ER-positive and ER-negative cell lines (28, 29). We 12). Agents that modulate enzyme activity through the hypothesized that hops and black cohosh can modulate inhibition or regulation of transcription are expected to estrogen metabolism and inhibit malignant transformation have chemopreventive properties, provided these agents in MCF-10A cells by altering enzyme expression in the attenuate estrogen quinone formation (3, 4). chemical carcinogenesis pathway (Fig. 1); this hypothesis The MCF-10A cell line is a nontumorigenic, immortalized was borne out for hops extracts in the present study. human breast epithelial cell line, which is classified as ERa negative because estrogens do not induce proliferation (13). Materials and Methods The latter attribute makes this a useful model system for study of chemical carcinogenesis as modulation of estrogen Chemicals and reagents metabolism can be clearly shown in this system without All the chemicals and reagents were obtained from Sigma interference from hormonal carcinogenic pathways. In or Invitrogen unless stated otherwise. All the standard addition, MCF-10A cells can be transformed into a malig- compounds of estrogen metabolites were obtained from nant phenotype by estrogenic compounds including E2 and Steraloides Inc. Antibodies were obtained from Santa Cruz equine estrogens (14, 15). More importantly, it has been Biotechnology and Cell Signaling Technology. Estrone- reported that over-the-counter (OTC) botanical dietary 2,4,16,16-d4, 4-hydroxyestrone-1,2,16,16-d4, and 2-meth- supplements, such as resveratrol, can modulate the estro- oxyestrone-1,4,16,16-d4 were obtained from CDN Isotopes gen-induced malignant transformation and CYP450 Inc. and used as internal standards in estrogen metabolism enzyme expression in MCF-10A cells (16) and in the related experiments. MCF-10F cell line (17). Black cohosh [Cimicifuga racemosa (L.) Nutt.; syn Actaea Plant materials and phenolic compounds racemosa (Nutt.) L] and hops (Humulus lupulus L.) are Authentic C. racemosa (L.) Nutt. (syn. Actaea racemosa L.; currently popular remedies for postmenopausal women as black cohosh) rhizomes/roots (BC #192) were acquired natural alternatives to HRT (18, 19). Their mechanisms of through our industrial partner, Naturex, formerly Pure action are not completely known; however, estrogenic World Botanicals and were botanically verified and char- activity for hops (20–22) and serotonergic effects for black acterized by the UIC/NIH Center for Botanical Dietary cohosh (23) have been reported. Hops, commonly used Supplements (30). The hops (Humulus lupulus) extract used as a flavoring agent in beer, has been used as a dietary for the experiments was an ethanol extract of spent hops supplement for sleep disturbances and in the treatment dispersed in kieselguhr (plant materials were extracted of mood disorders (24). Hops contain numerous active with ethanol after supercritical CO2 extraction of

74 Cancer Prev Res; 5(1) January 2012 Cancer Prevention Research

Hops Modulates Estrogen Carcinogenesis

pelletized strobiles of Humulus lupulus cv. Nugget), which dansyl chloride (1 mg/mL in acetone). The reaction mixture was obtained from Hopsteiner (Mainburg, Germany/ was incubated at 60C for 10 minutes to complete the New York). The kieselguhr was removed by methanol derivatization. Samples (10 mL) were analyzed by liquid filtration. Quantitative liquid chromatography/mass spec- chromatography/tandem mass spectrometry (LC/MS-MS) trometric (LC/MS) analysis using authentic reference com- as described later. pounds as calibrants revealed that this hops extract con- tained 5.4% XH and 0.084% 8-prenylnaringenin (8-PN). LC/MS-MS method XN was isolated and purified (>99.5% purity both by All of the metabolism experiments were carried out quantitative HNMR and LC/MS) as described previously by positive ion electrospray tandem mass spectrometric (26). 8-PN was synthesized and purified (95.0% purity by methodology on an API 3000 (Applied Biosystem) triple quantitative HNMR) using the modified literature proce- quadruple mass spectrometer equipped with Agilent dure as previously reported (21). 1200 HPLC (Agilent Technologies). Liquid chromatography was carried out on a 150 mm 3 mm i.d. column Cell lines and cell culture conditions packed with 3.5-mm particles, XBridge C-18 column MCF-10A cells were obtained from American Type (Waters). The mobile phase, operating at the flow rate of Culture Collection and maintained in Dulbecco’s Modi- 300 mL per minute consisted of water with 0.1% (v/v) formic fied Eagle’s Medium and F12 medium (DMEM/Ham’s acid as solvent A and 0.1% (v/v) formic acid in methanol F-12) supplemented with 1% penicillin–streptomycin, as solvent B. Initial conditions for the 30-minute run were 5% FBS, cholera toxin (0.1 mg/mL), epidermal growth set at 80% solvent B. The chromatographic gradient was factor (20 ng/mL), hydrocortisone (0.5 mg/mL), insulin held at the initial conditions for 5 minutes followed by a (10 mg/mL), and 5% CO2 at 37 C as described previously linear gradient of B from 80% to 95% over 20 minutes and (15). Estrogen-free medium for MCF-10A cells were pre- held at 95% solvent B for 5 minutes. The mass spectrometer pared supplementing charcoal–dextran-treated FBS to parameters were optimized as follows: the ion-spray phenol red–free DMEM/Ham’s F-12, whereas other com- voltage was 4.5 kV, the source temperature was 350C, the ponents remain the same. MCF-10A cells were authenti- nebulizer gas was 12 instrument units, the curtain gas was cated by single tandem repeat analysis. 8 units, and the collision gas was 5 units. The focusing potential was 370 V, and the declustering potential was 81 Analysis of estrogen metabolites in MCF-10A cells V. The collision energy for 2-methoxyestrone (2-MeOE1), MCF-10A cells were seeded in 6-well plates at a density 4-methoxyestrone (4-MeOE1), and 2-MeOE1-d4 was 59 V, of 20,000 cells per well. Cells were incubated with E2 whereas for 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (1 mmol/L) in the presence or absence of hops (5 mg/mL) (4-OHE1) and 4-OHE1-d4, it was 51 V. Collision energy for and black cohosh (20 mg/mL) for 6 days. Because E2,E1, and E1-d4 was 57 V. Multiple reactions monitoring 20 mg/mL of hops showed toxicity in MCF-10A cells, (MRM) channel of 504 ! 171 was set to detect E1, whereas lower concentrations of hops (5 mg/mL) were used for 506 ! 171 was set to detect E2. MRM channel of 534 ! 171 all the experiments. Because, hops showed a significant was set to detect both 4-MeOE1 and 2-MeOE1, whereas inhibition of estrogen metabolism at 5 mg/mL in MCF- 757 ! 170 and 538 ! 171 were set to detect 4-OHE1-d4 and 10A cells, a dose response was carried out. Different 2-MeOE1-d4, respectively. Estrogen metabolites were quan- concentrationsofhops(1,2.5,5,7.5and10mg/mL) tified with Analyst software (Applied Biosystems). Peak were tested in the presence of E2 (1 mmol/L). The effect of areas of 2-MeOE1 and 4-MeOE1 were normalized against the hops compounds XH and 8-PN on estrogen metab- 2-MeOE1-d4 internal standard and represented as relative olism were also studied. The dose-dependent effect of XH peak area. 2-MeOE1 and 4-MeOE1 relative peak areas in (0.1–2.5 mmol/L) and 8-PN (0.5–2.5 mmol/L) was tested E2-treated sample were considered as 100%, and all the inthepresenceofE2 (1 mmol/L). other samples were normalized against that and represen- Treatments were renewed every 3 days. Cell media were ted as relative peak area ratio in the graphs. collected (5 mL/well) after 3 days of treatment and stored at 20C. At the end of the treatment, cell media were Cytotoxicity collected and pooled with the third-day cell media Cytotoxicity assays were conducted in parallel to all the (10 mL/sample total volume). Ascorbic acid (2 mmol/L) metabolism experiments. MTT assay was conducted to and 5 nmol/L of each internal standard (E1-d4, 4-OHE1-d4, measure cell viability as described previously (32). and 2-MeOE1-d4) were added into each sample. The derivatization method of Xu and colleagues was used with minor Inhibition of human recombinant CYP450 1B1 modification (31) for the sample preparation and analysis. Human recombinant CYP450 1B1 isozymes with Briefly, the samples were lyophilized to approximately 2-mL CYP450 reductase were purchased from Sigma. Reaction aqueous solution, and the estrogen metabolites were mixture (1 mL) containing recombinant CYP450 1B1 extracted twice with dichloromethane (4 mL). Dichloro- (10 pmol/L), E2 (5 mmol/L), potassium phosphate buffer methane was evaporated under a stream of nitrogen gas (50 mmol/L, pH ¼ 7.4), and either hops (20 mg/mL) or and reconstituted with 200 mL of 0.1 mol/L sodium bicar- 8-PN (1 mmol/L) was preincubated at 37C for 5 minutes. bonate buffer (pH ¼ 9) and 200 mL of freshly prepared The reaction was initiated by adding NADPH (1 mmol/L)

www.aacrjournals.org Cancer Prev Res; 5(1) January 2012 75

Hemachandra et al.

into each reaction mixture and incubated for 1 hour at 37C. was used as the positive control. Cells were passaged once a The reaction was quenched by addition of 100 mLof week before confluency was achieved. At the end of the acetonitrile at 0C, and protein was removed by centrifu- treatment, cells were seeded on soft agar (0.3% agar) at a gation (10,000 rpm for 10 minutes). Ascorbic acid density of 5 104 cells per well in 12-well plates precoated (2 mmol/L) was added into each sample before 4-OHE1- with 0.6% agar base medium. Estrogen-free media were d4 (5 nmol/L) was added as the internal standard. Estrogen added as the feeding media on top of the soft agar layer. metabolites were extracted with dichloromethane (22 Cells were maintained in soft agar for 3 weeks, and media mL) and derivatized with dansyl chloride and analyzed by were refreshed every 3 days. After 3 weeks, colonies were LC/MS-MS as described earlier for the metabolism experi- stained with crystal violet (0.05%) and analyzed by an ments. Inhibition of CYP450 1B1 activity was further con- Olympus inverted microscope. Spherical formation of more firmed by the ethoxyresorufin-O-dealkylase (EROD) assay than 50 cells was taken as a colony. The number of colonies (Supplementary Fig. S1B) as described previously (33) with formed in each well were counted and represented as an minor modifications. Briefly, recombinant CYP450 1B1 average of triplicates SD. was incubated with E2 (1 mmol/L) and NADPH (1 mmol/L) in potassium phosphate buffer (50 mmol/L, Statistical analysis pH ¼ 7.4) in the presence and absence of different con- All of the metabolism experiments and Western blot centrations of hops (1–40 mg/mL). Activity of CYP450 1B1 analysis were carried out in triplicate. All data were was measured after incubating the reaction mixture at 37C expressed as the average SD. The statistical analysis of for 10 minutes and determined as percentage CYP450 1B1 these results consisted of t test or ANOVA by GraphPad activity calculated from the resorufin standard curve. Prism version 5 for Windows.

Immunoblotting Results Protein expression of CYP450 1B1 and CYP450 1A1 was analyzed by Western blot experiments as previously Analysis of estrogen metabolism in MCF-10A cells described (34) with minor modifications. Briefly, MCF- Oxidative estrogen metabolism was assessed by LC/MS- 10A cells were treated with E2 (1 mmol/L) in the presence MS, and MeOE1 metabolites were measured in MCF-10A and absence of hops (5 mg/mL) for 1, 3, and 6 days. Cells cell culture supernatants (Fig. 2, Supplementary Fig. S2). were harvested and protein lysates were prepared. Protein Because COMT converts 2-OHE1 and 4-OHE1 into the concentration was determined by bicinchoninic acid (BCA) assay. Equal aliquots of total protein samples (30 mg per b lane) were electrophoresed to separate proteins. -Actin A B level was measured as a gel loading and transferring control. 5 5 4-MeOE1 4-MeOE Anti-CYP450 1B1, anti-CYP450 1A1, anti-COMT, and anti- 1 b-actin antibodies were used as primary antibodies in 1:200, 2-MeOE 2-MeOE1 1:1,000, 1:1,000, and 1:2,000 dilution, respectively. Anti- 1 bodies were diluted in blocking solution (5% nonfat milk in ) ) 3 3 TBS with 0.1% Tween 20). Blots were incubated with 3 3 primary antibody overnight at 4C and with secondary antibody for 1 hour at room temperature. Blots were visu- alized with chemiluminescence substrate (Thermo Scien- Intensity (×10 tific). Imaging and analysis was done with FluoroChem Intensity (×10 software (Cell Biosciences). Each protein band density was 1 1 normalized to the respective b-actin band density and was represented as the relative protein expression. Three independent experiments were done to get the average, and the 17 18 19 20 17 18 19 20 results were represented as average SD. Time (min) Time (min)

Anchorage-independent growth assay Figure 2. Effect of black cohosh and hops on oxidative estrogen To determine the effects of hops and its flavonoids on metabolism in MCF-10A cells. Positive ion-electrospray selective estrogen-induced malignant transformation, anchorage- reaction monitoring (SRM) chromatograms of 2-MeOE1 and 4-MeOE1.A, fi independent growth assay was conducted as previously black cohosh had no signi cant effect on 2-MeOE1 and 4-MeOE1 formation, whereas (B) hops significantly inhibited the formation of 2- described (15) with minor modifications. Briefly, cells were MeOE1 and 4-MeOE1. MCF-10A cells were treated with and without black 6 plated in T-75 flasks at a density of 0.5 10 cells per flask cohosh (20 mg/mL) or hops (5 mg/mL) in the presence of E2 (1 mmol/L) for 6 and treated with hops and its active compounds. Treatment days. Cell media were collected, and the metabolites were extracted into was done with hops (5 mg/mL) or 8-PN (50 nmol/L) in the dichloromethane followed by the derivatization with dansyl chloride. m Samples were analyzed by LC/MS-MS in MRM mode as described in presence and absence of E2 (1 mol/L) for 3 weeks, twice a Materials and Methods. Overlaid SRM chromatograms represent the

week. Dimethyl sulfoxide (DMSO; 0.01%) was used as the formation of 2-MeOE1 and 4-MeOE1 in E2 treated (line) and E2 and (A) vehicle control in the experiments, whereas E2 (1 mmol/L) black cohosh (dashed line) or (B) hops (dotted line)-treated samples.

76 Cancer Prev Res; 5(1) January 2012 Cancer Prevention Research

Hops Modulates Estrogen Carcinogenesis

corresponding 2- and 4-methoxy metabolites (Fig. 1), these necessary before any significant inhibition of estradiol served as indices of catechol estrogen formation (35). When metabolism or ethoxyresorufin O-dealkylation activity was the cells were treated with E2 (1 mmol/L) for 6 days, and observed (Supplementary Fig. S1A and S1B). To determine the cell media were analyzed for metabolites, the majority the effect of hops on E2-induced P450 enzymes, MCF-10A of the metabolites detected were estrone derivatives, cells were treated with E2 (1 mmol/L) in the presence or whereas E2 metabolites were much less abundant (Supple- absence of hops (5 mg/mL) for 1, 3, and 6 days, and protein mentary Fig. S2). As a result, the methoxy estrone metabo- expression was analyzed by immunoblotting (Fig. 4). There lites (MeOE1) were used as biomarkers for metabolism, as was a significant time-dependent induction in CYP450 1B1 they were stable in cell media compared with the catechol and CYP450 1A1 with E2 treatment with maximum induc- estrogens and could be consistently and reproducibly tion at day 6 (Fig. 4). Cotreatment with hops (5 mg/mL) measured. significantly inhibited the induction of both CYP450 1B1 (Fig. 4A) and CYP450 1A1 (Fig. 4B). There was no signif- Hops inhibits estrogen metabolism in MCF-10A cells icant effect on COMT by either E2 or hops (Fig. 4C). whereas black cohosh had no significant effect To determine whether hops and black cohosh modulated Effect of xanthohumol and 8-prenylnaringenin on estrogen metabolism, MCF-10A cells were treated with E2 (1 estrogen metabolism m m mol/L) in the presence or absence of either hops (5 g/mL) XH exhibits chemopreventive activity (26), whereas 8-PN m or black cohosh extracts (20 g/mL) for 6 days. Because cell shows estrogenic activity (21). Because pure standards of m viability experiments indicated that 20 g/mL of hops were these compounds were available, they were tested for their ¼ toxic to MCF-10A cells [lethal concentration 50 (LC50) 11 effect on estrogen metabolism. No significant reduction in m 0.5 g/mL], hops extract was used at lower concentrations the formation of 2-MeOE and 4-MeOE was observed with m 1 1 (5 g/mL) in the metabolism experiments. Black cohosh cotreatment of different concentrations of XH in MCF-10A was not toxic and did not show any significant effect on cells (Fig. 5). In contrast, there was a significant reduction estrogen metabolism in MCF-10A cells (Fig. 2A). In con- (P < 0.001) in the formation of 2-MeOE and 4-MeOE in m 1 1 trast, cotreatment with hops (5 g/mL) significantly the presence of nanomolar amounts of 8-PN (Fig. 5). reduced (P < 0.005) the formation of both 2- and 4-estrone methylethers (Fig. 2B). Hops extract reduced estrogen Anchorage-independent growth inhibition by hops metabolism in a dose-dependent manner in MCF-10A cells and active compounds (Fig. 3). The ability to form anchorage-independent colonies in soft agar is considered an important hallmark of malignant Hops modulates estrogen-induced CYP450 enzymes in transformation (14). When MCF-10A cells were treated for MCF-10A cells 3 weeks and plated on soft agar for 3 weeks, there was a There are several important CYP450 enzymes involved in significant increase in anchorage-independent colony for- the estrogen chemical carcinogenesis pathway (Fig. 1). In mation in the E -treated sample (1 mmol/L) compared with breast epithelial cells, the CYP 1 family is mainly involved in 2 the negative control (0.01% DMSO; Fig. 6). Cotreatment phase I metabolism of E and E (5, 12). Direct inhibition of 1 2 with hops (5 mg/mL) significantly inhibited (P < 0.005) E - CYP450 1B1 metabolism by hops extracts was analyzed, 2 induced colony formation in soft agar. Finally, there was and these data showed that toxic concentrations were also a significant reduction (P < 0.0001) in colony formation with cotreatment with 8-PN (50 nmol/L).

100 Discussion 2-MeOE1 4-MeOE1 In addition to the widely accepted hormonal mechanism of estrogen carcinogenesis, estrogens can be metabolized by 50 CYP450s to form redox active and/or electrophilic o-quinones, which could act as chemical carcinogens by modi- fying cellular macromolecules (5, 36; Fig. 1). An imbalance

Relative peak area ratio in estrogen metabolism results from increased CYP450- 0 0 2 4 6 8 10 catalyzed o-quinone formation and/or reduced detoxiﬁca- hops (μg/mL) tion of these o-quinones, which could affect DNA integrity and lead to transformation of normal cells into a malignant phenotype (17). In the present study, OTC botanical dietary Figure 3. Dose-dependent effect of hops on the formation of 2-MeOE1 supplements used as alternatives to estrogen replacement and 4-MeOE1. MCF-10A cells were treated with different concentrations therapy, including black cohosh and hops, were analyzed of hops in the presence of E2 (1 mmol/L) for 6 days, and cell media were collected and analyzed for the estrogen metabolites by LC/MS-MS. Each for their ability to modulate oxidative estrogen meta- value represents the average SD of 3 experiments carried out bolism in MCF-10A cells. We hypothesized that hops in independently in duplicate. particular could reduce oxidative estrogen metabolism,

www.aacrjournals.org Cancer Prev Res; 5(1) January 2012 77

Hemachandra et al.

P < 0.05 A OH

HO OH 2.5 100 XH OO 2.0 80 1.5 60 1.0

0.5 40 OH HO O Relative protein expression protein Relative 0.0 20 8-PN Days 1 3 6 1 3 6 OH O Relative peak area ratio CYP450 1B1 0 0.0 0.5 1.0 1.5 2.0 2.5 β-Actin XH or 8-PN (μmol/L) E2 +++++ + ___ Hops +++ Figure 5. Effect of XH and 8-PN on estrogen metabolism in MCF-10A P < 0.05 B cells. 2-MeOE1 (open circles) and 4-MeOE1 (closed circles) formation was plotted against different concentrations of XH and 8-PN. MCF-10A cells 2.5 were treated with E2 (1 mmol/L) in the presence and absence of different concentrations of either XH or 8-PN for 6 days. Cell media were 2.0 analyzed for estrogen metabolites by LC/MS-MS. There was a signiﬁcant

inhibition (P < 0.0005) of both 2-MeOE1 and 4-MeOE1 formation in the 1.5 presence of nanomolar concentrations of 8-PN. Each value represents an average SD of 3 experiments carried out independently in duplicate. 1.0

0.5 transformation of MCF-10A cells, and ultimately prevent

Relative protein expression protein Relative estrogen dependent cancers. 0.0 Days 1 3 6 1 3 6 Analysis of oxidative estrogen metabolites in biological samples has been reported using a variety of different CYP450 1A1 methods including LC/MS-MS, gas chromatography–mass β-Actin spectroscopy, and immunoassay (31, 37, 38). A LC/MS-MS method was used in the present study to accurately measure E2 + + + + + + Hops ___+++ and quantify 2-MeOE1 and 4-MeOE1 in MCF-10A cells (Fig. 2). Because the stability of the catechol estrogens in the cell media was low, the quantity of the methoxy ethers C 1.5 was measured instead, assuming that their production was reﬂective of catechol estrogen formation (35). Catechol estrogen formation has been measured in MCF-10F cells 1.0 although pre-exposure to 2,3,7,8- tetrachlorodibenzo-p- dioxin to induce CYP450s 1B1/1A1 was necessary as well 0.5 as including the COMT inhibitor Ro 41-0960 in the incuba- tions (39). In the present study, extraction of estrogen metabolites with dichloromethane increased extraction

Relative protein expression protein Relative 0.0 efﬁciency relative to solid phase extraction and deriv- 1 3 6 1 3 6 Days atization with dansyl chloride enhanced the ionization ability in the triple quadruple mass spectrometer leading COMT

β-Actin the treatment, and protein was extracted and analyzed by E 2 + + + + + + immunoblotting. Anti-CYP450 1B1, anti-CYP450 1A1, and anti-COMT Hops ___+++ antibodies were used in 1:200, 1:1,000, and 1:1,000 dilutions, respectively. These blots are representatives of 3 experiments done independently. The intensity of the bands was normalized to b-actin as

Figure 4. Hops inhibits E2-induced (A) CYP450 1B1 and (B) 1A1 outlined in Material and Methods and represented as relative protein expression. C, COMT expression is not signiﬁcantly affected by either E2 expression. Each lane contains 30 mg of total protein as determined by or hops. Cells were collected at different time points (1, 3, and 6 d) after bicinchoninic acid assay.

78 Cancer Prev Res; 5(1) January 2012 Cancer Prevention Research

Hops Modulates Estrogen Carcinogenesis

E was used as substrate, did not show inhibition below P < 0.001 2 300 micromolar concentrations of resveratrol (16). Similarly, hops had no effect on CYP450 1B1 estrogen 4-hydroxylase or ethoxyresoruﬁn O-dealkylase activity unless toxic con- > m 200 centrations were used ( 20 g/mL, Supplementary Fig. S1). Another possible inhibitory mechanism could involve ** inhibition of E2-induced CYP450 upregulation. It has been *** previously shown that E2 induces the expression of CYP450 100 1B1 in MCF-7 cells through a mechanism involving both estrogen responsive element and ERa (45). Furthermore,

Number colonies/well of CYP450 1A1 expression can also be induced by E2 in HEPA 0 1C1C7 cells via transcriptional regulation (46). In the DMSO E2 E2 + Hops E2 + 8-PN present study, a time dependent induction in CYP450 1B1 and CYP450 1A1 expression in MCF-10A cells was observed upon exposure to E (Fig. 4). Because MCF-10A Figure 6. Effect of hops and 8-PN on E -induced malignant transformation 2 2 cells do not respond to estrogen via classical ERa genomic in MCF-10A cells. MCF-10A cells were treated with E2 (1 mmol/L) in the presence and absence of hops (5 mg/mL) or 8-PN (50 nmol/L). Treatments pathways, the mechanism of CYP450 induction by E2 were continued for 3 weeks, and cells were passaged once a week. At could involve extranuclear ER, ER-b, or aryl hydrocarbon the end of 3 weeks, cells were plated and maintained on soft agar for receptor (AhR), as these receptors are expressed in MCF-10 3 weeks, and formation of colonies were observed and counted with an inverted microscope. Hops (P < 0.005) and 8-PN (P < 0.0001) significantly cells (47). In the present study, hops significantly inhibited inhibited E2-induced malignant transformation in MCF-10A cells. estrogen-induced CYP450 1B1 and CYP450 1A1 expression DMSO-treated (0.01%) samples were used as a negative control. Each in MCF-10A cells (Fig. 4). point represents an average of triplicate SD. Because hops showed a significant inhibition in oxidative estrogen metabolism in MCF-10A cells, isolated phenolic components from hops were further studied. XH is the most to higher sensitivity. The LC/MS-MS method used in the abundant and bioactive phytoconstituent of hops (48), present study was sufficiently sensitive to detect 2-MeOE1 which has previously been reported to have potential che- and 4-MeOE1 without any prior induction or inhibition of mopreventive properties via induction of NADPH-depen- enzymes in MCF-10A cells. dent quinone oxidoreductase (NQO1) in liver cells (26). Estrogen metabolites can be used as biomarkers of cancer However, no significant inhibition of estrogen meta- initiation and genotoxicity in MCF-10A and MCF-10F bolism by XH was observed in MCF-10A cells (Fig. 5). In cells to provide a platform to better understand the mecha- contrast, 8-PN which is a potent estrogenic compound nism of estrogen chemical carcinogenesis. These nontu- isolated from hops (21), showed a significant inhibitory morigenic human mammary epithelial cell lines have been effect (P < 0.0005) even at nanomolar concentrations used as model cellular systems for the analysis of oxidative (Fig. 5). Although 8-PN showed a significant inhibition on estrogen metabolism, malignant transformation, DNA estrogen metabolism in MCF-10A cells, it had little inhib- modification, and reactive oxygen species formation (14, itory effect on CYP450 1B1 catalyzed formation of 4-OHE2 15, 17, 40, 41). It has been shown that E2-induced oxidative in estradiol metabolism experiments using recombinant metabolism and malignant transformation can be inhibited CYP450 1B1 enzyme (Supplementary Fig. S1A). It has been by agents with antioxidant activity such as resveratrol (42). reported that 8-PN can act via ER-b to modulate gene The chemopreventive activities of hops and black cohosh expression in rat brain cells (49). The mechanism of inhi- have been investigated and ascribed to induction of detox- bition of oxidative estrogen metabolism in MCF-10A cells ification enzymes (26) and antioxidant activity (43), respec- by 8-PN could be through inhibition of E2 upregulation of tively. Black cohosh did not significantly inhibit estrogen CYP450 1B1, which could be mediated through ER-b, metabolism in MCF-10A cells at 20 mg/mL concentration extranuclear ER, or AhR. (Fig. 2A). In contrast, hops showed a significant dose- The ability to form anchorage-independent colonies in dependent inhibition towards the formation of 2-MeOE1 semisolid media is considered a characteristics of malignant and 4-MeOE1 (Fig. 2B; Fig. 3). Similar modulation of transformation (14). It has previously been shown that estrogen metabolism has been reported for resveratrol in MCF-10A and 10F cells can undergo malignant transfor- MCF-10F cells and with dietary berries in animal models mation upon exposure to E2 (15, 17), and E2 induced (44). malignant transformation could be inhibited by botanical Several potential mechanisms could explain the inhibi- components such as resveratrol (17, 42). In the present tory effect of hops on estrogen metabolism. Components of study, it was confirmed that MCF-10A cells were trans- hops could act as direct inhibitors of CYP450 1B1 and formed into a malignant phenotype upon exposure to E2 CYP450 1A1. For example, resveratrol and its derivatives (Fig. 6). Anchorage-independent colony formation in the were reported to be inhibitors of CYP450 1B1 and CYP450 E2-treated cells was significantly higher (P < 0.001) than that 1A1, with nanomolar potency (33). However, similar stud- of the negative control (0.01% DMSO; Fig. 6). There was a ies in CYP450 1B1 and CYP450 1A1 supersomes, in which significant inhibition (P < 0.005) of E2-induced malignant

www.aacrjournals.org Cancer Prev Res; 5(1) January 2012 79

Hemachandra et al.

transformation by hops (Fig. 6). 8-PN, which is a potent investigation of hops in humans as a dietary supplement inhibitor of oxidative estrogen metabolism in MCF-10A with potential cancer chemopreventive activity in breast cells (Fig. 5), also caused a significant reduction (P < epithelial cells. 0.0001) in E2-induced malignant transformation when used in nanomolar (50 nmol/L) amounts (Fig. 6). These Disclosure of Potential Conflicts of Interest data suggest that 8-PN could be responsible for the effects of No potential conflicts of interest were disclosed. hops extract on E2-induced malignant transformation of MCF-10A cells. The content of 8-PN in hops extracts was 0.084%, which in the doses used in the transformation Acknowledgments studies corresponds to approximately 15 to 20 nmol/L 8- The authors thank Maitrayee Bose, Ping Yao, and Jeff Anderson for their PN; quantitatively in accord with the potency observed for technical support and A. Menassi and G. Appendino for synthesizing 8-PN. inhibition of catechol estrogen formation. These doses are The authors also thank Harald Schwarz and Martin Biendl from Hopsteiner (S. S. Steiner, New York, NY and Steiner Hopfen GmbH, Mainburg, Ger- similar to exposure levels expected for women taking hops many) for providing the hop extracts. This paper is dedicated to the memory botanical dietary supplements (50) and the serum levels of of Norman R. Farnsworth (1930–2011), who guided the UIC/NIH Botanical prenylflavonoids expected in in vivo studies (21). Further- Center for Dietary Supplements Research from its inception until this year and was the inspiration for this research. more, at these low concentrations, classical antioxidant effects are less likely to be a significant contributor. Grant Support In conclusion, hops extract inhibited estrogen oxidative metabolism and estrogen-induced malignant transforma- The support for this work was provided by NIH CA130037 from NCI and tion in the MCF-10A model of mammary carcinogenesis. P50 AT00155 jointly provided to the UIC/NIH Center for Botanical Dietary Supplements Research by the Office of Dietary Supplements and the Nation- Theresultsareentirelyconsistent with inhibition medi- al Center for Complementary and Alternative Medicine. ated by the botanical component and estrogen agonist 8- The costs of publication of this article were defrayed in part by the payment PN via potent blockade of estrogen-induced CYP450 of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. upregulation. Further work is needed to distinguish the site of 8-PN interaction, which may be a nonclassical ER, Received July 7, 2011; revised September 2, 2011; accepted September 17, ER-b or AhR; however, these results support the further 2011; published OnlineFirst October 13, 2011.

References 1. Russo J, Hasan Lareef M, Balogh G, Guo S, Russo IH. Estrogen and its breast epithelial cells and breast tumor cells. Carcinogenesis metabolites are carcinogenic agents in human breast epithelial cells. 1998;19:291–8. J Steroid Biochem Mol Biol 2003;87:1–25. 13. Soule HD, Maloney TM, Wolman SR, Peterson WD, Brenz R, McGrath 2. Yaghjyan L, Colditz G. Estrogens in the breast tissue: a systematic CM, et al. Isolation and characterization of a spontaneously immor- review. Cancer Cause Control 2011;22:529–40. talized human breast epithelial cell line, MCF-10. Cancer Res 3. Cavalieri EL, Rogan EG. Depurinating estrogen-DNA adducts in the 1990;50:6075–86. etiology and prevention of breast and other human cancers. Future 14. Park SA, Na HK, Kim EH, Cha YN, Surh YJ. 4-Hydroxyestradiol induces Oncol 2010;6:75–91. anchorage-independent growth of human mammary epithelial cells via 4. Bolton JL. Mechanisms of estrogen carcinogenesis: modulation by activation of IkappaB kinase: potential role of reactive oxygen species. botanical natural products. In: Penning TM, editor. Chemical carcino- Cancer Res 2009;69:2416–24. genesis. Philadelphia, PA: Humana Press; 2011. p. 75–93. 15. Cuendet M, Liu X, Pisha E, Li Y, Yao J, Yu L, et al. Equine estrogen 5. Bolton JL, Thatcher GRJ. Potential mechanisms of estrogen quinone metabolite 4-hydroxyequilenin induces anchorage-independent carcinogenesis. Chem Res Toxicol 2008;21:93–101. growth of human mammary epithelial MCF-10A cells: differential gene 6. Schiff R, Chamness G, Brown P. Advances in breast cancer treatment expression. Mutat Res 2004;550:109–21. and prevention: preclinical studies on aromatase inhibitors and new 16. Chen ZH, Hurh YJ, Na HK, Kim JH, Chun YJ, Kim DH, et al. selective estrogen receptor modulators (SERMs). Breast Cancer Res Resveratrol inhibits TCDD-induced expression of CYP1A1 and 2003;5:228–31. CYP1B1 and catechol estrogen-mediated oxidative DNA damage 7. Strasser-Weippl K, Goss PE. Advances in adjuvant hormonal therapy in cultured human mammary epithelial cells. Carcinogenesis 2004; for postmenopausal women. J Clin Oncol 2005;23:1751–9. 25:2005–13. 8. Goss P. Anti-aromatase agents in the treatment and prevention of 17. Lu F, Zahid M, Wang C, Saeed M, Cavalieri EL, Rogan EG. Resveratrol breast cancer. Cancer Control 2002;9:2–8. prevents estrogen-DNA adduct formation and neoplastic transforma- 9. Burnett C, Valentini S, Cabreiro F, Goss M, Somogyvari M, Piper MD, tion in MCF-10F cells. Cancer Prev Res 2008;1:135–45. et al. Exemestane for breast-cancer prevention in postmenopausal 18. Rees M. Alternative treatments for the menopause. Best Pract Res Clin women. N Eng J Med 2011;364:2381–91. Obstet Gynaecol 2009;23:151–61. 10. Dawling S, Roodi N, Mernaugh RL, Wang X, Parl FF. Catechol-O- 19. Dog TL, Marles R, Mahady G, Gardiner P, Ko R, Barnes J, et al. methyltransferase (COMT)-mediated metabolism of catechol estro- Assessing safety of herbal products for menopausal complaints: An gens: comparison of wild-type and variant COMT isoforms. Cancer international perspective. Maturitas 2010;66:355–62. Res 2001;61:6716–22. 20. Chadwick LR, Nikolic D, Burdette JE, Overk CR, Bolton JL, van 11. Beuten J, Gelfond JA, Byrne JJ, Balic I, Crandall AC, Johnson-Pais Breemen RB, et al. Estrogens and congeners from spent hops (Humu- TL, et al. CYP1B1 variants are associated with prostate cancer in lus lupulus L.). J Nat Prod 2004;67:2024–32. non-Hispanic and Hispanic Caucasians. Carcinogenesis 2008;29: 21. Overk CR, Guo J, Chadwick LR, Lantvit DD, Minassi A, Appendino G, 1751–7. et al. In vivo estrogenic comparisons of Trifolium pratense (red clover) 12. Spink DC, Spink BC, Cao JQ, DePasquale JA, Pentecost BT, Fasco Humulus lupulus (hops), and the pure compounds isoxanthohumol MJ, et al. Differential expression of CYP1A1 and CYP1B1 in human and 8-prenylnaringenin. Chem Biol Interact 2008;176:30–9.

80 Cancer Prev Res; 5(1) January 2012 Cancer Prevention Research

Hops Modulates Estrogen Carcinogenesis

22. Overk CR, Yao P, Chadwick LR, Nikolic D, Sun Y, Cuendet MA, et al. receptor-dependent, as well as independent, genotoxic effects. Ann Comparison of the in vitro estrogenic activities of compounds from N Y Acad Sci 2009;1155:132–40. hops (Humulus lupulus) and red clover (Trifolium pratense). J Agric 37. Blair IA. Analysis of estrogens in serum and plasma from postmen- Food Chem 2005;53:6246–53. opausal women: Past present, and future. Steroids 2010;75: 23. Burdette JE, Liu J, Chen SN, Fabricant DS, Piersen CE, Barker EL, et al. 297–306. Black cohosh acts as a mixed competitive ligand and partial agonist of 38. Piwowarska J, Radowicki S, Pachecka J. Simultaneous determi- the serotonin receptor. J Agric Food Chem 2003;51:5661–70. nation of eight estrogens and their metabolites in serum using liquid 24. Piersen CE. Phytoestrogens in botanical dietary supplements: impli- chromatography with electrochemical detection. Talanta 2010;81: cations for cancer. Integr Cancer Ther 2003;2:120–38. 275–80. 25. Gerhauser C, Alt A, Heiss E, Gamal-Eldeen A, Klimo K, Knauft J, et al. 39. Lu F, Zahid M, Saeed M, Cavalieri EL, Rogan EG. Estrogen metabolism Cancer chemopreventive activity of xanthohumol, a natural product and formation of estrogen-DNA adducts in estradiol-treated MCF-10F derived from hop. Mol Cancer Ther 2002;1:959–69. cells: The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction and 26. Dietz BM, Kang YH, Liu G, Eggler AL, Yao P, Chadwick LR, et al. catechol-O-methyltransferase inhibition. J Steroid Biochem Mol Biol Xanthohumol isolated from Humulus lupulus inhibits menadione- 2007;105:150–8. induced DNA damage through induction of quinone reductase. Chem 40. Russo J, Fernandez SV, Russo PA, et al. 17-Beta-estradiol induces Res Toxicol 2005;18:1296–305. transformation and tumorigenesis in human breast epithelial cells. 27. Lamy V, Roussi S, Chaabi M, Gosse F, Schall N, Lobstein A, et al. FASEB J 2006;20:1622–34. Chemopreventive effects of lupulone, a hop {beta}-acid, on human 41. Zahid M, Gaikwad NW, Ali MF, Lu F, Saeed M, Yang L, et al. Prevention colon cancer-derived metastatic SW620 cells and in a rat model of of estrogen-DNA adduct formation in MCF-10F cells by resveratrol. colon carcinogenesis. Carcinogenesis 2007;28:1575–81. Free Radical Bio Med 2008;45:136–45. 28. Einbond LS, Su T, Wu HA, Friedman R, Wang X, Jiang B, et al. Gene 42. Zahid M, Saeed M, Beseler C, Rogan EG, Cavalieri EL. Resveratrol and expression analysis of the mechanisms whereby black cohosh inhibits N-acetylcysteine block the cancer-initiating step in MCF-10F cells. human breast cancer cell growth. Anticancer Res 2007;27:697–712. Free Radical Bio Med 2011;50:78–85. 29. Hostanska K, Nisslein T, Freudenstein J, Reichling J, Saller R. Cimi- 43. Burdette JE, Chen SN, Lu ZZ, Xu H, White BE, Fabricant DS, et al. Black cifuga racemosa extract inhibits proliferation of estrogen receptor- cohosh (Cimicifuga racemosa L.) protects against menadione-induced positive and negative human breast carcinoma cell lines by induction DNA damage through scavenging of reactive oxygen species: bioas- of apoptosis. Breast Cancer Res Treat 2004;84:151–60. say-directed isolation and characterization of active principles. J Agric 30. Geller SE, Shulman LP, van Breemen RB, Banuvar S, Zhou Y, Epstein Food Chem 2002;50:7022–8. G, et al. Safety and efficacy of black cohosh and red clover for the 44. Aiyer HS, Gupta RC. Berries and ellagic acid prevent estrogen-induced management of vasomotor symptoms: a randomized controlled trial. mammary tumorigenesis by modulating enzymes of estrogen metab- Menopause 2009;16:1156–66. olism. Cancer Prev Res 2010;3:727–37. 31. Xu X, Keefer LK, Ziegler RG, Veenstra TD. A liquid chromatography- 45. Tsuchiya Y, Nakajima M, Kyo S, Kanaya T, Inoue M, Yokoi T. Human mass spectrometry method for the quantitative analysis of urinary CYP1B1 is regulated by estradiol via estrogen receptor. Cancer Res endogenous estrogen metabolites. Nat Protocols 2007;2:1350–5. 2004;64:3119–25. 32. Kastrati I, Edirisinghe PD, Wijewickrama GT, Thatcher GRJ. Estrogen- 46. Lee SS, Jeong HG, Yang KH. Effects of estradiol and progesterone on induced apoptosis of breast epithelial cells Is blocked by NO/cGMP cytochrome P4501A1 expression in Hepa 1c1c7 cells. Biochem Mol and mediated by extranuclear estrogen receptors. Endocrinology Biol Int 1998;45:775–81. 2010;151:5602–16. 47. Chen JQ, Russo PA, Cooke C, Russo IH, Russo J. ER[beta] shifts from 33. Mikstacka R, Przybylska D, Rimando AM, Baer-Dubowska W. Inhibi- mitochondria to nucleus during estrogen-induced neoplastic trans- tion of human recombinant cytochromes P450 CYP1A1 and CYP1B1 formation of human breast epithelial cells and is involved in estrogen- by trans-resveratrol methyl ethers. Mol Nutr Food Res 2007;51: induced synthesis of mitochondrial respiratory chain proteins. Biochim 517–24. Biophys Acta 2007;1773:1732–46. 34. Chang M, Peng KW, Kastrati I, Overk CR, Qin ZH, Yao P, et al. 48. Monteiro R, Faria A, Azevedo I, Calhau C. Modulation of breast cancer Activation of estrogen receptor-mediated gene transcription by the cell survival by aromatase inhibiting hop (Humulus lupulus L.) flavo- equine estrogen metabolite, 4-methoxyequilenin, in human breast noids. J Steroid Biochem Mol Biol 2007;105:124–30. cancer cells. Endocrinology 2007;148:4793–802. 49. Amer DAM, Kretzschmar G, Muller€ N, Stanke N, Lindemann D, Vollmer 35. Rangiah K, Shah SJ, Vachani A, Ciccimaro E, Blair IA. Liquid chroma- G. Activation of transgenic estrogen receptor-beta by selected phy- tography/mass spectrometry of pre-ionized Girard P derivatives for toestrogens in a stably transduced rat serotonergic cell line. J Steroid quantifying estrone and its metabolites in serum from postmenopausal Biochem Mol Biol 2010;120:208–17. women. Rapid Commun Mass Sp 2011;25:1297–307. 50. Bolca S, Li J, Nikolic D, Roche N, Blondeel P, Possemiers S, et al. 36. Santen R, Cavalieri E, Rogan E, Russo J, Guttenplan J, Ingle J, et al. Disposition of hop prenylflavonoids in human breast tissue. Mol Nutr Estrogen mediation of breast tumor formation involves estrogen Food Res 2010;54:S284–S94.

www.aacrjournals.org Cancer Prev Res; 5(1) January 2012 81

Hops (Humulus lupulus) Inhibits Oxidative Estrogen Metabolism and Estrogen-Induced Malignant Transformation in Human Mammary Epithelial cells (MCF-10A)

L.P. Hemachandra, P. Madhubhani, R. Chandrasena, et al.

Cancer Prev Res 2012;5:73-81. Published OnlineFirst October 13, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/1940-6207.CAPR-11-0348

Supplementary Access the most recent supplemental material at: Material http://cancerpreventionresearch.aacrjournals.org/content/suppl/2011/10/12/1940-6207.CAPR-11-0348.DC 1

Cited articles This article cites 49 articles, 9 of which you can access for free at: http://cancerpreventionresearch.aacrjournals.org/content/5/1/73.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://cancerpreventionresearch.aacrjournals.org/content/5/1/73.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerpreventionresearch.aacrjournals.org/content/5/1/73. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.