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Potent Induction of Phase 2 Enzymes in Human Prostate Cells by Sulforaphane¹

Department of Urology, Stanford University Medical Center, Stanford, California 94305-5118

	Abstract

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Two population-based, case-control studies have documented reduced risk of prostate cancer in men who consume cruciferous vegetables. Cruciferae contain high levels of the isothiocyanate sulforaphane. Sulforaphane is known to bolster the defenses of cells against carcinogens through up-regulation of enzymes of carcinogen defense (phase 2 enzymes). Prostate cancer is characterized by an early and near universal loss of expression of the phase 2 enzyme glutathione S-transferase (GST)-. We tested whether sulforaphane may act in prostatic cells by increasing phase 2 enzyme expression. The human prostate cancer cell lines LNCaP, MDA PCa 2a, MDA PCa 2b, PC-3, and TSU-Pr1 were treated with 0.1–15 µM sulforaphane in vitro. LNCaP was also treated with an aqueous extract of broccoli sprouts. Quinone reductase enzymatic activity, a surrogate of global phase 2 enzyme activity, was assayed by the menadione-coupled reduction of tetrazolium dye. Expression of NQO-1, GST-, -glutamylcysteine synthetase-heavy and -light chains, and microsomal GST was assessed by Northern blot analysis. Sulforaphane and broccoli sprout extract potently induce quinone reductase activity in cultured prostate cells, and this induction appears to be mediated by increased transcription of the NQO-1 gene. Sulforaphane also induces expression of -glutamylcysteine synthetase light subunit but not the heavy subunit, and this induction is associated with moderate increases in intracellular glutathione levels. Microsomal and -class glutathione transferases were also induced transcriptionally. Sulforaphane induces phase 2 enzyme expression and activity significantly in human prostatic cells. This induction is accompanied by, but not because of, increased intracellular glutathione synthesis. Our findings may help explain the observed inverse correlation between consumption of cruciferae and prostate cancer risk.

	Introduction

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In the United States, prostate cancer is the most prevalent noncutaneous malignancy and the second leading cause of male cancer death (1) . Prostate cancer has a long latency and estimates are that 10 to 12 years are required before prostate cancer becomes clinically manifest (2) . Sakr et al. (3) have identified prostatic intraepithelial neoplasia, a prostate cancer precursor lesion, in 10% of men by 30 years of age and small foci of frank carcinoma in more than 10% of men before age 40. Prostate cancer is usually diagnosed clinically in the sixth and seventh decades of life, allowing a large window of opportunity for interventions to prevent or slow the progression of the disease.

The most common molecular genetic change in prostate cancer involves silencing of expression of GSTP1³ , a critical enzyme of carcinogen defense, through methylation of deoxycytidine residues in "CG islands" in the 5' regulatory region of the GSTP1 gene (4 , 5) . This change appears to occur early in prostate carcinogenesis, because it is found in virtually all of the cases of high-grade prostatic intraepithelial neoplasia and is a near universal finding in clinical prostate cancers regardless of grade or stage (6) . The glutathione transferases protect cells against carcinogenic oxidative stress by conjugation of electrophiles to reduced glutathione. Up-regulation of phase 2 enzymes, including the glutathione transferases, can protect cells against carcinogens and has been documented to prevent carcinogen-induced tumors in a variety of animal models (7 , 8) .

Early loss of GSTP1 may predispose prostatic cells to the damaging effects of endogenous or exogenous carcinogens and may contribute to carcinogenesis. Two recent epidemiological studies (9 , 10) suggest that such a preventive intervention may be possible. Both studies have found an association between decreased prostate cancer risk and high consumption of cruciferous vegetables. Cruciferae are known to contain high levels of the isothiocyanate sulforaphane, the most potent monofunctional phase 2 enzyme-inducing agent thus far identified (11) .

Phase 2-inducing agents have been reported to increase phase 2 enzyme activity through increased transcription at phase 2 enzyme gene loci (12) . A putative ARE in the regulatory regions of these genes is thought to be responsible for enhanced expression of many of these genes (13, 14, 15, 16, 17, 18, 19) ; e.g., sulforaphane will increase expression of a reporter gene downstream of promoter constructs containing the ARE consensus sequence and a minimal promoter. Levels of reporter gene induction parallel endogenous QR induction in the same cell line (20) .

Our hypothesis is that induction of phase 2 enzymes by sulforaphane may help explain the association between high consumption of cruciferae and decreased prostate cancer risk. Because prostate cancer lacks expression of GSTP1, induction of other phase 2 enzymes by sulforaphane may offer a mechanistically based prostate cancer-preventive strategy. Because little is known about phase 2 enzyme expression, regulation, or activity in prostatic epithelial cells, we evaluated the effect of sulforaphane on the androgen-sensitive prostate cancer cell line LNCaP, three androgen-insensitive cell lines (PC-3, TSU-Pr1, and DU-145), and a normal prostate epithelial cell strain.

	Materials and Methods

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Cell Culture.
LNCaP were obtained from the American Type Culture Collection and grown in RPMI 1640 with L-glutamine, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). PC3 and Tsu-Pr1 were a gift from William G. Nelson (Johns Hopkins University, Baltimore, MD) and grown in the same medium. MDA PCa 2a and MDA PCa 2b were kindly provided by Nora Navonne (M. D. Anderson Cancer Center, Houston, Texas) and were grown in HPC1 (BRFF) supplemented with 20% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (21) .

Reagents.
L-sulforaphane was purchased from LKT Laboratories (St. Paul, MN). All of the remaining chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Broccoli sprouts were grown from seed on sterile agar and aqueous extracts prepared as described (22) . Two separate batches of organic broccoli sprouts purchased from a local supermarket exhibited nearly identical inducer potency to those raised in the laboratory and were therefore used for subsequent experiments.

Northern Blot Analysis.
Cells were harvested at approximately 70% confluency, and mRNA was isolated using Oligotex Direct mRNA isolation kit (Qiagen). For each lane, 6 µg of polyadenylate+ mRNA were electrophoresed through a 1% agarose gel and transferred to Brightstar-Plus nylon membrane (Ambion) using the Stratagene Posiblot pressure blotter and pressure control station (Stratagene). The RNA was cross-linked to the membrane by exposure to 125 mJoules of UV light in GS Gene Linker (Bio-Rad). cDNA probes were labeled with either [³²P]dCTP using the Nick Translation System (Promega) or psoralen-biotin using the BrightStar Psoralen-Biotin Kit (Ambion). Hybridizations were performed at 50°C in a buffer containing 6 x saline-sodium phosphate-EDTA, 5 x Denhardt’s Reagent, 6% SDS, 25 µg/ml salmon testes DNA, and 50% formamide. Washes were performed at 55°C with 1 x SSC and 0.1% SSC. Northern blots hybridized with [³²P]dCTP-labeled probes were exposed to a Molecular Dynamics Phosphorimager screen and scanned ImageQuant software. Northern blots hybridized with psoralen-biotin-labeled probes were processed using the BrightStar Detection kit (Ambion) according to the recommended instructions. All of the images were analyzed using ImageQuant software.

Determination of Enzyme Activity in Cell Culture.
LNCaP, MDA PCa 2a, and MDA PCa 2b were grown in 96-well plates at a density of 8 x 10⁴ cells/ml. PC3 and Tsu-Pr1 were grown in 96-well plates at a density of 4 x 10⁴ cells/ml. After 20 h of incubation, cells were treated with L-sulforaphane dissolved in DMSO (LKT Laboratories) at the indicated concentrations. Control wells were treated with the corresponding concentration of DMSO. QR activity was assessed by the menadione-coupled reduction of tetrazolium dye as modified from Prochaska et al. (23 , 24) . After 48 h of treatment with L-sulforaphane, media was gently aspirated and cells were lysed by incubation at 37°C with 50 µl of 0.08% digitonin and 2 mM EDTA (pH 7.8) with gentle agitation. While the cells were incubating, a stock solution was prepared by combining 16.7 mg of BSA, 7.5 mg of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide, 0.6 mg of NADP, 1.25 ml of 0.5 M Tris-HCl (pH 7.4), 166.7 µl of 1.5% Tween 20, 166.7 µl of 150 mM glucose 6-phosphate, 16.7 µl of 7.5 mM FAD, 50 units of yeast glucose 6-phosphate dehydrogenase, and distilled water to a final volume of 25 ml for each plate to be assayed. Immediately before use, 25 µl of 50 mM menadione dissolved in acetonitrile was added to the stock solution. After 30-min incubation, 200 µl of the complete stock solution was added to each well. After 5 min, optical absorbance at 610 nm was determined in a LabSystems Multiscan Ascent microplate reader. GST activity was determined by reduction of CDNB in accord with methods described by Habig et al. (25) .

Toxicity of L-sulforaphane was assessed in parallel plates treated identically to those used in assays for QR activity. Cytotoxicities were monitored by the LIVE/DEAD fluorescent assay (Molecular Probes) according to the suggested protocol.

QR activity, in arbitrary units, was calculated automatically from the mean activity for all of the three wells at each concentration. Activity was corrected for toxicity at each concentration as described by Prochaska et al. (23 , 24) . Inducer potency is expressed as the ratio of corrected QR activity for treated cells to corrected QR activity for the vehicle controls.

Determination of GSH Levels in Cell Culture.
LNCaP were grown in 96-well plates at a density of 8 x 10⁴ cells/ml. After 20 h of incubation, cells were treated with L-sulforaphane at the indicated concentrations. After an additional 48 h, the medium was removed, and the relative GSH levels were determined as described by Gerhauser et al. (26) . GSH levels were determined in triplicate for each dose of sulforaphane and were corrected for toxicity as above. Reported values represent the average of two separate experiments.

	Results

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Sulforaphane Induces QR Activity in Cultured Prostate Cells.
QR (NADPH menadione:oxidoreductase; EC 1.6.99.2) protects cells from quinones and their precursors by obligate two-electron reduction of quinones to hydroquinones, thereby preventing generation of highly reactive semiquinones (that arise from single electron transfer). QR is stably expressed in vitro and is induced coordinately with other phase 2 enzymes (27) . QR has been used as a surrogate marker of global phase 2 enzyme activity in vitro and in vivo. To test whether sulforaphane has the ability to induce QR enzyme activity in prostate cells, we treated four prostate cancer cell lines and one primary prostate cell strain grown from histologically normal prostatic tissue harvested at surgery (courtesy of Donna Peehl). Cells were treated with sulforaphane or DMSO vehicle as control, and QR enzymatic activity was measured using the technique of Prochaska and Santamaria (23) . Over a range of concentrations, sulforaphane induced QR activity in all of the prostate cell lines tested (Table 1) . Sulforaphane was particularly potent at inducing QR enzymatic activity in the normal prostate cell strain with maximal induction (2.46-fold) at 1–3 µM and 1.35-fold induction occurring at 0.1 µM sulforaphane. Potent induction was also seen at micromolar doses in LNCaP, MDA PCa 2a, and MDA PCa 2b. All of these cell lines resemble human prostatic epithelia in that they express prostate-specific antigen and androgen receptor and possess relatively slow growth kinetics (21 , 28) . TSU-PR1, on the other hand, lacks these features of prostatic cells and shows somewhat diminished responsiveness to sulforaphane.

View larger version (76K): [in this window] [in a new window]   Fig. 1. Transcriptional response of phase 2 enzymes to sulforaphane in various prostate cancer cell lines. The cell lines LNCaP, MDA PCa 2A, MDA PCa 2B, PC3, and TSU-Pr1 were treated for 8 h with 10 µM sulforaphane or with the DMSO control. Northern blot analyses were performed using (A) NQO-1, (B) GSTA1, (C) microsomal GST, and (D) GAPDH cDNA probes.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Northern blot analysis of the transcriptional response to sulforaphane. LNCaP cells were incubated for 1 to 72 h with 10 µM sulforaphane or the DMSO control before the RNA was harvested. The membrane was probed with labeled (A) NQO-1 and (B) GAPDH cDNAs.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Differential effects of sulforaphane on -GCS subunits. Northern blot analyses of (A) sulforaphane-induced -GCS light chain expression over time and (B) unchanged -GCS heavy chain expression treated with 9 h of 10 µM sulforaphane treatment in LNCaP cells.

View larger version (9K): [in this window] [in a new window]   Fig. 4. QR response to sulforaphane is abolished in a reduced environment. LNCaP cells were pretreated for 2 h with 10 µM NAC followed by 8 µM sulforaphane or DMSO control before assaying QR activity. Bars, SD of triplicate data points.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sulforaphane is a potent phase 2 enzyme-inducing agent in human prostate cells in vitro. Sulforaphane produced robust and sustained transcriptional induction of NQO-1 gene expression that was accompanied by similar increases in QR enzymatic activity. Other members of the class of phase 2 enzymes were also induced transcriptionally. Intracellular levels of reduced glutathione increased after sulforaphane treatment, likely attributable to increased expression of the -GCS-L gene, an enzyme that catalyzes the rate-limiting step in glutathione synthesis. Together, the changes induced by sulforaphane buttress cellular defenses against carcinogens by increasing the reductive capacity of the cell.

All of the prostatic cells tested in vitro were affected similarly by sulforaphane, including a normal prostate cell strain, three hormone-responsive immortalized cell lines, and an androgen-insensitive cell line. Each of the cell lines demonstrated the same pattern of phase 2 enzyme response and glutathione induction, although there were quantitative differences. Regulation of the response to sulforaphane in these cell lines, therefore, appears to remain intact, although somewhat complicated. The light and heavy subunits of -GCS were not induced coordinately, despite the presence of a stereotypical ARE in the 5'-regulatory regions of both genes. Other investigators (29 , 30) have reported coordinate induction of these subunits in response to phase 2-inducing agents. Furthermore, sulforaphane was capable of inducing expression of phase 2 enzymes known to lack AREs, namely GST- and microsomal GST. Thus, regulation of these enzymes, at least in prostatic cells, is likely to involve more than binding of a protein complex to the ARE enhancer element. Detailed study of the regulatory regions of these genes will be necessary to understand the complex regulatory pathways that modulate the cellular response to sulforaphane.

Alteration of intracellular redox status may be one means by which sulforaphane acts to increase phase 2 enzyme expression. Pretreatment of prostatic cells with NAC, which is known to increase intracellular levels of reduced glutathione, completely ablated the effects of sulforaphane. Although it is possible that NAC acts directly on sulforaphane, we suspect that NAC reduces intracellular proteins mediating the phase 2 enzyme response. Understanding the role of intracellular redox in the regulation of phase 2 enzyme response has implications in the design of future clinical trials in cancer prevention; e.g., one proposed intervention strategy for prostate cancer combines NAC (to increase intracellular reduced glutathione, a GST substrate) with a phase 2 enzyme-inducing agent such as sulforaphane (34) . Our results suggest that such an approach may ablate the response to sulforaphane, at least in prostatic cells. Indeed, because sulforaphane increases intracellular glutathione pools by itself, such combined therapy may be unnecessary.

Our findings may help explain the recent observation (9 , 10) that consumption of cruciferae, naturally rich sources of sulforaphane, may lower the risk of later development of prostate cancer. Because the loss of one phase 2 enzyme, namely -class glutathione transferase, is an early and near universal finding in human prostate cancer, sulforaphane may help compensate for this loss by increasing global phase 2 enzyme activity. At first glance, it seems somewhat surprising that loss of expression of a single GST could increase risk of prostate cancer. Glutathione transferases comprise a family of enzymes with broad and overlapping substrate specificity; thus, loss of any single member should be compensated by the activity of the remaining GSTs (32) . However, several epidemiological studies (35, 36, 37, 38) have suggested that loss of individual GSTs (e.g., GSTM1-null phenotype) can confer increased susceptibility to cancer at several organ sites. Low activity GSTP1 alleles have been associated with increased prostate cancer risk (39 , 40) . Indeed, mice engineered to lack -class GST expression are more susceptible to carcinogen-induced tumors (41) . Thus, loss of expression of a single GST appears to increase cancer risk, either from global decreases in GST activity or from loss of protection against a carcinogen inactivated solely by the lost enzyme.

Could the capacity of sulforaphane to induce phase 2 enzymes compensate for or prevent loss of GSTP1 expression? An intriguing study by Lin et al. (42) suggests that induction of phase 2 enzymes may be particularly pertinent in the setting of GST enzymatic deficiency. Patients with a previous history of colonic polyps were stratified for their subsequent risk of developing colorectal polyps based on levels of consumption of cruciferous vegetables. Compared with subjects that never consumed broccoli, those in the highest quartile of broccoli consumption had an odds ratio of 0.47 (95% confidence interval, 0.30–0.73), and this protective effect was only observed in subjects with the GSTM1 null genotype. No protection was conferred in subjects with wild-type GSTM-1 alleles. A similar interaction between GSTM1 genotype and broccoli consumption has been observed in lung cancer (43) . Because GSTP1 is lost in all of the human prostate cancers, induction of global phase 2 enzyme activity and increasing intracellular reduced glutathione may be have great relevance in preventing this disease.

In summary, sulforaphane is a potent inducer of phase 2 enzymes in human prostatic cells. Induction of phase 2 enzymes is one possible explanation for the association between high consumption of cruciferae and decreased prostate cancer risk. On the basis of these findings, intervention trials may be warranted, and broccoli sprouts, a rich natural source of sulforaphane, may be appropriate for use in such a trial. Additional work will be necessary to elucidate the mechanisms of phase 2 enzyme induction in human prostate cells.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment 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.

¹ Supported by the Department of Defense New Investigator Award DAMD17-98-1-8555 and by the Doris Duke Clinician Scientist Award T98064.

² To whom requests for reprints should addressed, at Department of Urology, Room S287, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5118. E-mail: jdbrooks{at}stanford.edu

³ The abbreviations used are: GST, glutathione-S-transferase; ARE, antioxidant response element; GSH, reduced glutathione; QR, quinone reductase; -GCS, -glutamylcysteine synthetase; -GCS-L, -GCS-light chain; NAC, N-acetyl cysteine.

Received 3/12/01; revised 6/15/01; accepted 6/20/01.

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				C. S. M. Furniss, R. N. Bennett, J. R. Bacon, G. LeGall, and R. F. Mithen Polyamine Metabolism and Transforming Growth Factor-{beta} Signaling Are Affected in Caco-2 Cells by Differentially Cooked Broccoli Extracts J. Nutr., October 1, 2008; 138(10): 1840 - 1845. [Abstract] [Full Text] [PDF]

				G. A. Saxe, J. M. Major, L. Westerberg, S. Khandrika, and T. M. Downs Biological Mediators of Effect of Diet and Stress Reduction on Prostate Cancer Integr Cancer Ther, September 1, 2008; 7(3): 130 - 138. [Abstract] [PDF]

				V. A. Kirsh, U. Peters, S. T. Mayne, A. F. Subar, N. Chatterjee, C. C. Johnson, and R. B. Hayes Prospective Study of Fruit and Vegetable Intake and Risk of Prostate Cancer J Natl Cancer Inst, August 1, 2007; 99(15): 1200 - 1209. [Abstract] [Full Text] [PDF]

				G. Pappa, J. Strathmann, M. Lowinger, H. Bartsch, and C. Gerhauser Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane on proliferation of human colon cancer cells in vitro Carcinogenesis, July 1, 2007; 28(7): 1471 - 1477. [Abstract] [Full Text] [PDF]

				A. V. Gasper, M. Traka, J. R. Bacon, J. A. Smith, M. A. Taylor, C. J. Hawkey, D. A. Barrett, and R. F. Mithen Consuming Broccoli Does Not Induce Genes Associated with Xenobiotic Metabolism and Cell Cycle Control in Human Gastric Mucosa J. Nutr., July 1, 2007; 137(7): 1718 - 1724. [Abstract] [Full Text] [PDF]

				A. Herman-Antosiewicz, H. Xiao, K. L. Lew, and S. V. Singh Induction of p21 protein protects against sulforaphane-induced mitotic arrest in LNCaP human prostate cancer cell line Mol. Cancer Ther., May 1, 2007; 6(5): 1673 - 1681. [Abstract] [Full Text] [PDF]

				J.-L. Chang, J. Bigler, Y. Schwarz, S. S. Li, L. Li, I. B. King, J. D. Potter, and J. W. Lampe UGT1A1 Polymorphism Is Associated with Serum Bilirubin Concentrations in a Randomized, Controlled, Fruit and Vegetable Feeding Trial J. Nutr., April 1, 2007; 137(4): 890 - 897. [Abstract] [Full Text] [PDF]

				S. Choi, K. L. Lew, H. Xiao, A. Herman-Antosiewicz, D. Xiao, C. K. Brown, and S. V. Singh D,L-Sulforaphane-induced cell death in human prostate cancer cells is regulated by inhibitor of apoptosis family proteins and Apaf-1 Carcinogenesis, January 1, 2007; 28(1): 151 - 162. [Abstract] [Full Text] [PDF]

				S. A. Ritz, J. Wan, and D. Diaz-Sanchez Sulforaphane-stimulated phase II enzyme induction inhibits cytokine production by airway epithelial cells stimulated with diesel extract Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L33 - L39. [Abstract] [Full Text] [PDF]

				J. H. Fowke, J. D. Morrow, S. Motley, R. M. Bostick, and R. M. Ness Brassica vegetable consumption reduces urinary F2-isoprostane levels independent of micronutrient intake Carcinogenesis, October 1, 2006; 27(10): 2096 - 2102. [Abstract] [Full Text] [PDF]

				G. A. Saxe, J. M. Major, J. Y. Nguyen, K. M. Freeman, T. M. Downs, and C. E. Salem Potential attenuation of disease progression in recurrent prostate cancer with plant-based diet and stress reduction. Integr Cancer Ther, September 1, 2006; 5(3): 206 - 213. [Abstract] [PDF]

				J. Y. Nguyen, J. M. Major, C. J. Knott, K. M. Freeman, T. M. Downs, and G. A. Saxe Adoption of a plant-based diet by patients with recurrent prostate cancer. Integr Cancer Ther, September 1, 2006; 5(3): 214 - 223. [Abstract] [PDF]

				C. Xu, X. Yuan, Z. Pan, G. Shen, J.-H. Kim, S. Yu, T. O. Khor, W. Li, J. Ma, and A.-N. T. Kong Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2. Mol. Cancer Ther., August 1, 2006; 5(8): 1918 - 1926. [Abstract] [Full Text] [PDF]

				A. Herman-Antosiewicz, D. E. Johnson, and S. V. Singh Sulforaphane Causes Autophagy to Inhibit Release of Cytochrome c and Apoptosis in Human Prostate Cancer Cells Cancer Res., June 1, 2006; 66(11): 5828 - 5835. [Abstract] [Full Text] [PDF]

				C. Xu, G. Shen, X. Yuan, J.-h. Kim, A. Gopalkrishnan, Y.-S. Keum, S. Nair, and A.-N. T. Kong ERK and JNK signaling pathways are involved in the regulation of activator protein 1 and cell death elicited by three isothiocyanates in human prostate cancer PC-3 cells Carcinogenesis, March 1, 2006; 27(3): 437 - 445. [Abstract] [Full Text] [PDF]

				H. Kim, E. H. Kim, Y. W. Eom, W.-H. Kim, T. K. Kwon, S. J. Lee, and K. S. Choi Sulforaphane Sensitizes Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Resistant Hepatoma Cells to TRAIL-Induced Apoptosis through Reactive Oxygen Species-Mediated Up-regulation of DR5 Cancer Res., February 1, 2006; 66(3): 1740 - 1750. [Abstract] [Full Text] [PDF]

				A. V Gasper, A. Al-janobi, J. A Smith, J. R Bacon, P. Fortun, C. Atherton, M. A Taylor, C. J Hawkey, D. A Barrett, and R. F Mithen Glutathione S-transferase M1 polymorphism and metabolism of sulforaphane from standard and high-glucosinolate broccoli Am. J. Clinical Nutrition, December 1, 2005; 82(6): 1283 - 1291. [Abstract] [Full Text] [PDF]

				M. Traka, A. V. Gasper, J. A. Smith, C. J. Hawkey, Y. Bao, and R. F. Mithen Transcriptome Analysis of Human Colon Caco-2 Cells Exposed to Sulforaphane J. Nutr., August 1, 2005; 135(8): 1865 - 1872. [Abstract] [Full Text] [PDF]

				S. V. Singh, S. K. Srivastava, S. Choi, K. L. Lew, J. Antosiewicz, D. Xiao, Y. Zeng, S. C. Watkins, C. S. Johnson, D. L. Trump, et al. Sulforaphane-induced Cell Death in Human Prostate Cancer Cells Is Initiated by Reactive Oxygen Species J. Biol. Chem., May 20, 2005; 280(20): 19911 - 19924. [Abstract] [Full Text] [PDF]

				S. B. Jones, S. E. DePrimo, M. L. Whitfield, and J. D. Brooks Resveratrol-Induced Gene Expression Profiles in Human Prostate Cancer Cells Cancer Epidemiol. Biomarkers Prev., March 1, 2005; 14(3): 596 - 604. [Abstract] [Full Text] [PDF]

				G. K. McWalter, L. G. Higgins, L. I. McLellan, C. J. Henderson, L. Song, P. J. Thornalley, K. Itoh, M. Yamamoto, and J. D. Hayes Transcription Factor Nrf2 Is Essential for Induction of NAD(P)H:Quinone Oxidoreductase 1, Glutathione S-Transferases, and Glutamate Cysteine Ligase by Broccoli Seeds and Isothiocyanates J. Nutr., December 1, 2004; 134(12): 3499S - 3506S. [Abstract] [Full Text] [PDF]

				N.-A. Pham, J. W. Jacobberger, A. D. Schimmer, P. Cao, M. Gronda, and D. W. Hedley The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice Mol. Cancer Ther., October 1, 2004; 3(10): 1239 - 1248. [Abstract] [Full Text] [PDF]

S. J. T. Jackson and K. W. Singletary Sulforaphane Inhibits Human MCF-7 Mammary Cancer Cell Mitotic Progression and Tubulin Polymerization J. Nutr., September