JOURNAL OF MEDICINAL FOOD J Med Food 17 (11) 2014, 1177–1182 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.0145

Enhancement of by Methyl Jasmonate Treatment and Effects on Prostate Carcinogenesis

Ann G. Liu,1 John A. Juvik,2 Elizabeth H. Jeffery,1 Lisa D. Berman-Booty,3 Steven K. Clinton,4 and John W. Erdman, Jr.1

1Division of Nutritional Sciences, Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. 2Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. 3Department of Veterinary Biosciences and 4Division of Medical Oncology, The Ohio State University, Columbus, Ohio, USA.

ABSTRACT Broccoli is rich in bioactive components, such as and indole-3-carbinol, which may impact cancer risk. The profile of broccoli can be manipulated through treatment with the plant stress hormone methyl jasmonate (MeJA). Our objective was to produce broccoli with enhanced levels of indole glucosinolates and determine its impact on prostate carcinogenesis. Brassica oleracea var. Green Magic was treated with a 250 lM MeJA solution 4 days prior to harvest. MeJA-treated broccoli had significantly increased levels of , neoglucobrassicin, and (P < .05). Male transgenic adenocarcinoma of mouse prostate (TRAMP) mice (n = 99) were randomized into three diet groups at 5–7 weeks of age: AIN-93G control, 10% standard broccoli powder, or 10% MeJA broccoli powder. Diets were fed throughout the study until termination at 20 weeks of age. Hepatic CYP1A was induced with MeJA broccoli powder feeding, indicating biological activity of the indole glucosinolates. Following *15 weeks on diets, neither of the broccoli treatments significantly altered genitourinary tract weight, pathologic score, or metastasis incidence, indicating that broccoli powder at 10% of the diet was ineffective at reducing prostate carcinogenesis in the TRAMP model. Whereas broccoli powder feeding had no effect in this model of prostate cancer, our work demonstrates the feasibility of employing plant stress hormones exogenously to stimulate changes in profiles, an approach that may be useful for optimizing bioactive component patterns in foods for chronic-disease-prevention studies.

KEY WORDS: Brassica oleracea indole-3-carbinol indole glucosinolates neoglucobrassicin sulforaphane

INTRODUCTION brassican, which is hydrolyzed to N-methoxy-I3C, has not previously been evaluated in an animal model of cancer and pidemiological studies provide moderate support for is reported to inhibit sulforaphane action in cell culture, but the hypothesis that consumption of cruciferous vegeta- E only when both compounds were at concentrations in excess bles reduces the risk of developing prostate cancer.1 Broccoli of 5 lM.5 is the most commonly consumed cruciferous vegetable in the Glucosinolates are divided into three classes based on the United States with the average American eating 8.6 pounds amino acid precursors from which they originate. Aliphatic (3.9 kg) of fresh broccoli and broccoli products each year.2 glucosinolates are derived from methionine, indole gluco- Broccoli contains many glucosinolates including gluco- sinolates are from tryptophan, and aromatic glucosinolates , which is hydrolyzed to sulforaphane and gluco- are from phenylalanine. These are consti- brassicin, which is hydrolyzed to indole-3-carbinol (I3C), tutively present in cruciferous plant tissues, but they can also which both demonstrate anticancer activity in rodent models be rapidly induced by stresses, such as wounding, patho- and in vitro.3,4 However, studying these compounds in iso- gens, or herbivore attack. These stress conditions lead to lation may obscure any inhibitory, additive, or synergistic the endogenous biosynthesis of jasmonates, which induce actions or bioactive components that might take place when the synthesis of indole glucosinolates.6 External applica- a whole vegetable is consumed. For example, neogluco- tion of methyl jasmonate (MeJA), a volatile methylester of jasmonic acid, has been shown to induce production of 7–9 Manuscript received 10 September 2013. Revision accepted 28 May 2014. indole glucosinolates in Brassica species. MeJA treat- ment offers a way of altering the phytochemical profile of Address correspondence to: John W. Erdman, Jr., PhD, Division of Nutritional Sciences, Department of Food Science and Human Nutrition, University of Illinois at Urbana- broccoli to increase concentrations of potentially anticarci- Champaign, 905 S. Goodwin Ave., Urbana, IL 61801, USA, E-mail: [email protected] nogenic bioactives.

1177 1178 LIU ET AL.

We were particularly interested in enhancing the indole TRAMP mice on a pure C57BL/6 background [C57BL/6- glucosinolate content of whole broccoli because I3C and 3,30- Tg(TRAMP)8247Ng/J; The Jackson Laboratory, Bar Har- diindolymethane (DIM) have been shown to reduce prostate bor, ME, USA] were obtained at 5–7 weeks of age and carcinogenesis. Indole glucosinolates and their metabolites individually housed in shoe-box cages under controlled may impact the carcinogenesis process through multiple conditions (12-h light–dark cycle, 22C, 60% humidity). modes of action, including upregulation of cytochrome P450 Mice were weighed weekly throughout the study. Mice were 1A and associated carcinogen-metabolizing enzymes, inhi- randomly assigned to three experimental groups (n = 33). bition of proliferation, induction of apoptosis, and alterations AIN-93G-based experimental diets included AIN-93G in hormone metabolism.4 We chose to alter the phytochemical control, 10% control broccoli powder, or 10% MeJA-treated profile of whole broccoli because our lab had previously broccoli powder. Diets were balanced for protein, fat, en- shown that feeding a diet containing 10% freeze-dried whole ergy, and fiber. Diet formulations are shown in Table 1. tomato or broccoli powder significantly reduced tumor growth Food was replaced and intake was measured every other in a rat transplantable tumor model of prostate cancer.10 day. Fresh diets were prepared monthly, tightly sealed in Whereas previous studies using the transgenic adenocar- plastic bags, and stored in opaque waterproof plastic bins at cinoma of mouse prostate (TRAMP) model have demon- 4C. Mice were sacrificed at 20 weeks of age. At the con- strated that pharmacologic levels of isolated broccoli clusion of the study, mice were anesthetized with CO2 and bioactive compounds reduce prostate carcinogenesis, it is blood was collected by cardiac puncture. Following exsan- unknown whether dietary consumption of whole broccoli guination, lungs, liver, and genitourinary tract were re- will alter this process.11–13 Here we examined the effects of moved and weighed. The prostate was microdissected when standard broccoli and MeJA-treated broccoli on prostate possible and individual lobes were weighed and preserved in carcinogenesis in TRAMP mice. We hypothesized that both 10% formalin for histology. Lungs and sections of each liver broccoli treatments would reduce prostate carcinogenesis lobe were also preserved in 10% formalin for histology. and that MeJA-treated broccoli would further reduce carci- nogenesis compared with standard broccoli. To our knowl- Detoxification enzyme activity edge, this is only the second study to examine the effects of Hepatic microsomal and cytosolic fractions were pre- whole broccoli in an animal model of prostate cancer; the pared as previously described.14 Activity of the phase I first was our study utilizing the Dunning rat model.10 enzyme CYP1A was measured in the microsomal fraction as ethoxyresorufin O-deethylase (EROD) activity15 with slight MATERIALS AND METHODS 16

modification. Activity of the cytosolic phase II enzyme Broccoli powder production NAD(P)H-quinone oxidoreductase 1 (NQO1) was measured according to the method of Prochaska and Santamaria17 with Broccoli (Brassica oleracea var. Green Magic) was modification.16 grown on the University of Illinois campus in Urbana, Illi- nois, from May through August 2009. Broccoli plants were started and maintained in a commercial hot house for 3 weeks. Before transplanting to experimental fields, broccoli Table 1. AIN-93G-Based Diet Formulations seedlings were hardened off outside for 1 week. To produce indole glucosinolate–enriched broccoli, treatment consisted g/100 g total diet of the plant hormone MeJA (250 lM) in a 0.1% Triton X- Broccoli Broccoli 100 aqueous solution sprayed on individual plants 4 days Control (Control) (MeJA) prior to harvest.9 Plant surfaces were fully saturated with Cornstarch 39.7 36.6 36.6 MeJA, allowed to dry, and then received an additional ap- Casein 20.0 16.8 16.8 plication of MeJA. Broccoli heads were collected at harvest Maltodextrin 13.2 13.2 13.2 maturity with some stem, frozen in liquid nitrogen, stored at Sucrose 10.0 10.0 10.0 - 20C, and then freeze-dried without being allowed to Cellulose 5.0 1.6 1.6 thaw. After freeze-drying, broccoli samples were ground Mineral mixa 3.5 3.5 3.5 b into a fine powder using a commercial coffee grinder and Vitamin mix 1.0 1.0 1.0 stored at 4C. L- 0.3 0.3 0.3 Choline bitartrate 0.3 0.3 0.3 Soybean oil 7.0 6.7 6.7 Glucosinolate and sulforaphane analysis Control broccoli powderc 10.0 a Intact glucosinolates were analyzed by the method de- MeJA broccoli powder 10.0 9 scribed by Kim and Juvik. Sulforaphane formation from Diets were balanced for energy, protein, fat, and fiber. 14 broccoli powders was determined as previously described. aAIN93M-MX formulation. bAIN93-VX formulation. Experimental design cTen grams of broccoli powder provides 3.4 g total dietary fiber, 3.1 g carbohydrate, 3.2 g protein, and 3.0 g fat (estimated from the USDA National The University of Illinois Animal Care and Use Com- Nutrient Database for Standard Reference). mittee approved the animal protocol. Ninety-nine male MeJA, methyl jasmonate. BROCCOLI GLUCOSINOLATES AND PROSTATE CANCER 1179

Tissue histology and immunohistochemistry Table 2. Glucosinolate Profile of Broccoli Powders A section of each prostate lobe was preserved in 10% lmol/g dry weight formalin for 24 h and then transferred to 70% ethanol. For- malin-fixed tissues were embedded in paraffin, sectioned, Standard broccoli MeJA broccoli and transferred to slides. Four-micrometer sections for his- Glucobrassicin 2.61 – 0.07 3.06 – 0.07* tological evaluation were stained with hematoxylin/eosin. Neoglucobrassicin 5.23 – 0.67 39.77 – 0.67* Pathologic evaluation was performed by a veterinary Gluconasturtiin 0.87 – 0.19 3.79 – 0.19* pathologist as previously described.18 Each of the four 2.49 – 0.59 3.64 – 0.59 Gluconapin 0.50 – 0.03 0.41 – 0.03 prostate lobes (dorsal, ventral, lateral, and anterior) of Glucoiberin 0.32 – 0.08 0.49 – 0.08 C57BL/6 TRAMP mice were assessed individually and 0.46 – 0.07 0.49 – 0.07 assigned two grades. The first grade represented the most 0.24 – 0.02 0.22 – 0.02 severe lesion within that lobe, and the second grade identi- fied the most common lesion in the lobe. The distributions of Values are means – SE, n = 3. each of these lesions within the individual lobe were de- *Within rows it indicates significant differences between treatments termined and described as focal, multifocal, or diffuse. (P < .05). Adjusting the lesion grades (0–7) to include an indication of distribution (focal, multifocal, or diffuse) provided adjusted Diet and weight gain scores, ranging from 0 (normal) to 21 [diffuse, poorly dif- Diets were well tolerated and there were no significant ferentiated (PD) carcinoma]. The adjusted lesion scores are differences among groups in food intake or body weight. as follows: 0, normal prostate; 1–3, low-grade prostatic in- Two animals had to be put down prior to completion of the traepithelial neoplasia (PIN); 4–6, moderate-grade PIN; 7–9, study for health reasons unrelated to cancer development. high-grade PIN; 10–12, phyllodes-like tumor; 13–15, well- On average, mice consumed 4.9 – 0.1 g per day. The esti- differentiated (WD) carcinoma; 16–18, moderately differen- mated daily intakes of glucoraphanin, glucobrassicin, and tiated carcinoma; and 19–21, poorly differentiated carcinoma. neoglucobrassicin per mouse were 1.2, 1.3, and 2.6 lmol for Four-micrometer sections were stained for proliferating standard broccoli, and 1.8, 1.5, and 19.9 lmol for MeJA cell nuclear antigen (PCNA) (Abcam, Cambridge, MA, USA) broccoli, respectively. and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). Images were captured and quantified as previ- Detoxification enzyme activity ously described10 with the researchers blinded to treatments.

Two representative images without necrosis or artifacts were Broccoli and its glucosinolate hydrolysis products have been shown to induce phase I and phase II detoxification captured for the dorsal and lateral lobes of the prostate. For 19 PCNA, a proliferative index percentage, (PCNA positive/ enzymes. Glucobrassicin and neoglucobrassicin, as well as total nuclei counted) · 100, was calculated. For cleaved cas- their respective hydrolysis products I3C and N-methoxy- pase-3 the number of apoptotic nuclei was counted at 400 · I3C, are known to induce CYP1A. As expected, we ob- magnification. served a dose-dependent increase in CYP1A activity with increasing levels of indole glucosinolates in broccoli Statistical analysis (Fig. 1A). MeJA broccoli feeding resulted in hepatic EROD activity 2.5 times greater than the control group (P < .05). Data were compared among treatments by two-tailed Standard broccoli also significantly increased hepatic analysis of variance, using the Mixed Models procedure of EROD activity but to a lesser magnitude, 1.5 times greater SAS Statistical Software (version 9.2; SAS Institute, Cary, than control (P < .05). The induction of EROD activity with NC, USA). Values were considered different from controls MeJA broccoli feeding indicates biological activity of in- at P < .05 using Tukey’s procedure. dole glucosinolates in the liver. Hepatic NQO1 activity did not change with either of the broccoli treatments (Fig. 1B). RESULTS Broccoli powder characterization Tissue weights Broccoli powders were analyzed for glucosinolate con- Liver and genitourinary tract weights were not different tent (Table 2). MeJA treatment significantly increased levels among groups. Mean genitourinary tract weights were of indole glucosinolates, glucobrassicin, and neogluco- 1.00 – 0.06, 1.25 – 0.12, and 1.25 – 0.12 g for control, stan- brassicin, and the aromatic glucosinolate gluconasturtiin dard broccoli, and MeJA broccoli groups, respectively. (P < .05). Strikingly, neoglucobrassicin levels were over Proliferation and apoptosis seven times higher in the MeJA broccoli (39.77 – 0.67 lmol/g) than in the control broccoli (5.23 – 0.67 lmol/g). This induc- There were no significant differences among groups in tion was approximately two times greater than levels previ- prostatic proliferation or apoptosis as measured via PCNA ously observed with the same variety of broccoli.9 Levels of and cleaved caspase-3 (data not shown). Our values are aliphatic glucosinolates, including glucoraphanin, were simi- within the normal range for proliferative index and apoptotic lar in both broccoli powders. index values observed in TRAMP mice. 1180 LIU ET AL.

Table 4. Adjusted Lesion Scores of the Most Common Lesion Present in Prostate Lobes

Treatment group Ventral Lateral Dorsal Anterior Control 4.73 – 1.06 11.38 – 0.95 8.91 – 0.63 0.75 – 0.60 Standard broccoli 6.73 – 1.06 13.81 – 0.93 10.73 – 0.63 1.85 – 0.60 MeJA broccoli 5.23 – 1.10 11.23 – 0.96 10.00 – 0.64 1.06 – 0.60

Values are means – SE, n = 31–33. Scale ranges from 0 (normal prostate tissue) to 21 (diffuse PD carcinoma).18

tastases. Three mice (3% of all mice) developed liver me- tastases, one from each diet group. Fourteen mice (14% of all mice) developed microscopic lung metastases: 3 control, 7 standard broccoli, and 4 MeJA broccoli.

DISCUSSION We succeeded in altering the phytochemical profile of broccoli through MeJA treatment, which produced broccoli with substantially higher levels of indole glucosinolates, modestly increased levels of aromatic glucosinolates, and with relatively unchanged levels of aliphatic glucosinolates. This phytochemical profile differs both from regular broc- coli and from broccoli sprouts, which have very high levels of glucoraphanin and minor amounts of other glucosino- lates.21 We observed a significant increase in hepatic CYP1A activity, indicating biological activity of indole glucosino- lates. Surprisingly, neither of the broccoli treatments in-

FIG. 1. Impact of broccoli feeding on hepatic detoxification en- creased NQO1 activity. Sulforaphane is known to induce zymes. (A) Liver ethoxyresorufin O-deethylase activity, (B) liver NAD(P)H-quinone oxidoreductase 1 activity. Results shown are NQO1 activity in short-term studies, including work by our means – SEs, n = 8. Samples were run in triplicate. For each enzyme, group with diets containing 10% or 20% broccoli powder different letters above bars indicate significant differences between fed for 5–7 days 1,4,19 The lack of induction observed in the treatments (P < .05). present study may indicate an adaptation to chronic feeding. Previous work from our lab found no alterations in hepatic NQO1 activity after male Copenhangen rats were fed a 10% Histology broccoli powder diet for 22 weeks (unpublished data), Prostate cancer progression varied by lobe as expected.20 suggesting that NQO1 may not remain upregulated during The lateral prostate had mostly diffuse WD carcinomas long-term feeding. while the dorsal prostate had predominantly diffuse high- An alternate possibility is that high levels of neoglu- grade PIN. Within each prostate lobe, no significant differ- cobrassicin may actually inhibit Nrf2 action and suppress ences in adjusted lesion score were observed for either the induction of phase II enzymes. Work by Haack et al. dem- most severe lesion present or the most common lesion onstrated that 7.5 lM of neoglucobrassicin hydrolyzed in present, indicating that broccoli feeding did not impact situ inhibited sulforaphane’s induction of Nrf2 target genes prostate cancer progression (Tables 3 and 4). As expected NQO1 and peroxidase 2 in HepG2 cells.5 It is for TRAMP mice of this age, few animals developed me- possible that while MeJA treatment enhanced levels of total glucosinolates, the specific increase in neoglucobrassin lev- els may have abrogated sulforaphanes’s effects. However, to Table 3. Adjusted Lesion Scores of the Most Severe date this inhibitory effect has not been evaluated in vivo. Lesion Present in Prostate Lobes Our study was limited in that we did not measure serum or Treatment group Ventral Lateral Dorsal Anterior tissue levels of indole glucosinolate metabolites such as I3C. However, previous pharmacokinetic studies suggest that Control 6.76 – 1.04 14.56 – 0.45 9.18 – 0.66 8.22 – 0.31 these metabolites would likely not be detectable in our study Standard broccoli 8.70 – 1.04 15.73 – 0.44 11.45 – 0.66 8.54 – 0.30 samples since I3C is rapidly degraded in the body and falls MeJA broccoli 7.29 – 1.07 15.00 – 0.45 10.69 – 0.67 7.75 – 0.31 below detectable levels within 1 h after oral administra- 22 Values are means – SE, n = 31–33. Scale ranges from 0 (normal prostate tion. The dose provided here by a 10% MeJA broccoli diet tissue) to 21 (diffuse PD carcinoma).18 is *1000-fold lower than the bolus dose given in pharma- PD, poorly differentiated. cokinetic studies and would likely not be detectable, BROCCOLI GLUCOSINOLATES AND PROSTATE CANCER 1181 particularly since mice were fed ad libitum rather than given a bolus gavage of pure compound. Our results indicate that feeding diets containing 10% broccoli powder did not alter tumor growth or progression of prostate carcinogenesis, which was surprising given that several articles have shown that individual phytochemicals from broccoli reduced prostate carcinogenesis.11–13,23 Con- sumption of MeJA broccoli resulted in potent induction of CYP1A in the liver; however, consumption of both standard broccoli and MeJA-treated broccoli failed to reduce prostate PIN of lymph node metastases pulmonary metastases carcinogenesis. This is in contrast to previous studies that creased PIN at 28 weeks demonstrate that I3C and DIM (both breakdown products of the indole glucosinolate glucobrassicin and known CYP1A inducers) can reduce prostate carcinogenesis11,13 (Table 5). ic intraepithelial neoplasia; SF, sulforaphane; For example, a diet containing 1% I3C significantly reduced genitourinary tract weight11 and TRAMP mice gavaged with strain Outcome

3 lmol of DIM had an increased incidence of PIN and a Background decreased incidence of WD carcinoma, indicating a slowing of prostate cancer progression.13 Additionally several other studies that treated TRAMP mice with individual phytochemicals found in broccoli or broccoli sprouts have also observed reductions in prostate carcinogenesis12,23,24 (Table 5 and personal communication Age at sacrifice with Dr. Emily Ho, Oregon State University, Corvallis, OR, 20 weeks C57BL/6 No effect USA, April 16, 2011). Reduction in the incidence of PIN and WD carcinomas was observed when TRAMP mice were gavaged with 6 lmol sulforaphane every other day.12 Fur- b

ther, there was a decreased incidence of PD cancers when mol IG mol IG or mol SF 12 and 28 weeks C57BL/6 Decreased WD carcinoma and in- m m TRAMP mice received *15 lmol of phenethyl m

23 per day in their diet. These effective outcomes, compared mol SF 24 weeks C57BL/6 X FVB Decreased PIN, WD carcinoma, and m mol I3C 24 weeks C57BL/6 Decreased GU tract weight, incidence m ). with the present study, suggest that the effects of a complex mol PEITC 24 weeks C57BL/6 X FVB Decreased PD carcinoma 1 mol DIM 22 weeks C57BL/6 Decreased WD carcinoma, increased m - m mixture of glucosinolates within whole broccoli may be bioactive exposure Estimated cumulative MeJA: 2002 day

quite different from the effects of pharmacological doses of $ a pure compound such as I3C. Our study was limited in that we did not have a positive control such as pure sulforaphane or I3C that might have mol/(mouse demonstrated efficacy of the model. However, the TRAMP l model is known to be a robust model of prostate cancer that 13–15 weeks Standard: 399 16 weeks 115 19 weeks 1995 16 weeks UKN17–19 weeks 24 weeks 306–324 C57BL/6 Decreased GU tract weight has been successfully used for several dietary intervention 8 and 24 weeks 633 and 1898 studies.24 The progression of cancer observed in our control number of weeks that mice received treatment.

mice mirrored the established progression course that has ·

been well documented in this strain. We have no reason to ) Exposure a mol/g in mol m

believe that the model was ineffective and conclude that m there was no effect of broccoli treatment on this model of mol I3C/day) 16 weeks 38,864 prostate carcinogenesis. m mol IG/day; MeJA: mol gavage every

In summary, neither of the broccoli treatments signifi- m cantly reduced prostate carcinogenesis in TRAMP mice. m mol PEITC/day)

Apparently, the quantity or profile of indole glucosinolates m mol SF/day) mol IG/day) m contributed by the 10% broccoli powders in this study was m (estimated daily intake Summary of Studies with Broccoli or Broccoli Compounds in the Transgenic Adenocarcinoma of Mouse Prostate Model not sufficient to reduce prostate carcinogenesis in the Compound–delivery route -diindolylmethane: 2.4 0 5. gavage every other day (Standard: 3.8 21.0 (SF not reported) diet (15 other day (11.3 number of days treated per week

TRAMP model. Quantities of broccoli bioactive compounds · were much higher in other studies showing reduction of Broccoli sprout: 8% powder in diet I3C: 1% in diet (347 : 3 3,3 Sulforaphane: 6 carcinogenesis in this model (Table 5). Clearly more work is Table 23 necessary to determine the optimal dose and profile of . 24 -diindolymethane; GU, genitourinary tract; I3C, indole-3-carbinol; IGs, indole glucosinolates; PEITC, phenethyl isothiocyanate; PIN, prostat 12 . 0 13 et al broccoli bioactives that are necessary for prostate cancer 11 et al prevention. These studies should also evaluate dose re- et al. et al. et al. Based on daily dose sponse, phytochemical bioavailability, and tissue distribu- If applicable, based on estimated average food intake of 4.9 g to yield a b DIM, 3,3 Keum Singh Powolny Cho Broccoli powder Liu (current study) Broccoli: 10% powder in diet Study Pure compound Wu Ho (unpublished) Broccoli sprout: 15% powder in diet tion of metabolites. Our work demonstrates the feasibility of UKN, unknown because of insufficient information to make calculation; WD, well-differentiated carcinoma. 1182 LIU ET AL. employing plant stress hormones exogenously to stimulate 11. Wu TY, Saw CL, Khor TO, Pung D, Boyanapalli SS, Kong AN: changes in phytochemical profiles, an approach that may be In vivo pharmacodynamics of indole-3-carbinol in the inhibition useful for optimizing bioactive component patterns in foods of prostate cancer in transgenic adenocarcinoma of mouse for chronic-disease-prevention studies. prostate (TRAMP) mice: involvement of Nrf2 and cell cycle/ apoptosis signaling pathways. Mol Carcinog 2011. ACKNOWLEDGMENTS 12. Singh SV, Warin R, Xiao D, et al.: Sulforaphane inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice in This work was supported in part by the Illinois Council on association with increased cytotoxicity of natural killer cells. Food and Agricultural Research (CF071-006-4-SEN). The Cancer Res 2009;69:2117–2125. authors thank Kang Mo Ku at the University of Illinois for 13. Cho HJ, Park SY, Kim EJ, Kim JK, Park JH: 3,30-Diindo- overseeing the production of broccoli and performing glu- lylmethane inhibits prostate cancer development in the transgenic cosinolate analysis, Dr. Matt Wallig for providing immu- adenocarcinoma mouse prostate model. Mol Carcinog 2011;50: nohistochemistry expertise, Sonja Volker for performing 100–112. liver detoxification enzyme assays, and Ed Dosz for per- 14. Liu AG, Volker SE, Jeffery EH, Erdman JW, Jr.: Feeding to- forming sulforaphane extraction and analysis. mato and broccoli powders enriched with bioactives improves bioactivity markers in rats. J Agric Food Chem 2009;57:7304– 7310. AUTHOR DISCLOSURE STATEMENT 15. Paolini M, Perocco P, Canistro D, et al.: Induction of cytochrome No competing financial interests exist. P450, generation of oxidative stress and in vitro cell-transforming and DNA-damaging activities by glucoraphanin, the bioprecursor REFERENCES of the chemopreventive agent sulforaphane found in broccoli. Carcinogenesis 2004;25:61–67. 1. Singh SV, Singh K: Cancer chemoprevention with dietary iso- 16. Lai RH, Keck AS, Wallig MA, West LG, Jeffery EH: Evaluation mature for clinical translational research. Carcino- of the safety and bioactivity of purified and semi-purified glu- genesis 2012;33:1833–1842. coraphanin. Food Chem Toxicol 2008;46:195–202. 2. Economic Research Service USDA: Vegetables and melons 17. Prochaska HJ, Santamaria AB: Direct measurement of yearbook data. 2009. http://usda.mannlib.cornell.edu/MannUsda/ NAD(P)H:quinone reductase from cells cultured in microtiter viewDocumentInfo.do?documentID=1212 (Accessed October 20, wells: a screening assay for anticarcinogenic enzyme inducers. 2009). Anal Biochem 1988;169:328–336. 3. Juge N, Mithen RF, Traka M: Molecular basis for chemopre- 18. Berman-Booty LD, Sargeant AM, Rosol TJ, et al.: A review of

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