Sulforaphane, a Naturally Occurring Isothiocyanate ...

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Amy V Gasper, Ahmed Al-janobi, Julie A Smith, James R Bacon, Paul Fortun, ..... Gerhauser,C., You,M., Liu,J., Moriarty,R.M., Hawthorne,M., Mehta,R.G., Moon ...
http://cancerres.aacrjournals.org/cgi/content/full/60/5/1426

Sulforaphane, a Naturally Occurring Isothiocyanate, Induces Cell Cycle Arrest and Apoptosis in HT29 Human Colon Cancer Cells Laurence Gamet-Payrastre1, Pengfei Li, Solange Lumeau, Georges Cassar, Marie-Ange Dupont, Sylvie Chevolleau, Nicole Gasc, Jacques Tulliez and François Tercé INRA, Laboratoire des Xénobiotiques, 31931 Toulouse Cedex, France [L. G-P., P. L., S. L., S. C., N. G., J. T.]; INSERM U326, 31059 Toulouse Cedex, France [F. T.]; INSERM U395, Service Commun d’Analyse et de Tri Cellulaire, 31024, Toulouse Cedex, France [G. C.]; and Laboratoire de Biologie Moléculaire des Eucaryotes, 31062, Toulouse Cedex, France [A. D., M-A. D.]

Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.

http://mct.aacrjournals.org/cgi/content/abstract/5/3/575

Inhibition of angiogenesis and endothelial cell functions are novel sulforaphane-mediated mechanisms in chemoprevention Elisabeth Bertl, Helmut Bartsch and Clarissa Gerhäuser Division of Toxicology and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany Requests for reprints: Clarissa Gerhäuser, Division of Toxicology and Cancer Risk Factors, German Cancer Research Center, C010-2 Chemoprevention, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone: 49-6221-42-33-06; Fax: 49-6221-42-33-59. E-mail: [email protected]

Sulforaphane, an aliphatic isothiocyanate, is a known cancer chemopreventive agent. Aiming to investigate antiangiogenic potential of sulforaphane, we here report a potent decrease of newly formed microcapillaries in a human in vitro antiangiogenesis model, with an IC50 of 0.08 µmol/L. The effects of sulforaphane on endothelial cell functions essential for angiogenesis were investigated in HMEC-1, an immortalized human microvascular endothelial cell line. Molecular signaling pathways leading to activation of endothelial cell proliferation and degradation of the basement membrane were analyzed by reverse transcription-PCR. Sulforaphane showed time- and concentration-dependent inhibitory effects on hypoxia-induced mRNA expression of vascular endothelial growth factor and two angiogenesis-associated transcription factors, hypoxia-inducible factor-1 and c-Myc, in a concentration range of 0.8 to 25 µmol/L. In addition, the expression of the vascular endothelial growth factor receptor KDR/flk-1 was inhibited by sulforaphane at the transcriptional level. Sulforaphane could also affect basement membrane integrity, as it suppressed transcription of the predominant endothelial collagenase matrix metalloproteinase-2 and its tissue inhibitor of metalloproteinase-2. Migration of HMEC-1 cells in a wound healing assay was effectively prevented by sulforaphane at submicromolar concentrations, and we determined an IC50 of 0.69 µmol/L. In addition, within 6 hours of incubation, sulforaphane inhibited tube formation of HMEC-1 cells on basement membrane matrix at 0.1, 1, and 10 µmol/L concentrations. These effects were not due to inhibition of HMEC-1 cell proliferation; however, after 72 hours of incubation, sulforaphane nonselectively reduced HMEC-1 cell growth with an IC50 of 11.3 µmol/L. In conclusion, we have shown that sulforaphane interferes with all essential steps of neovascularization from proangiogenic signaling and basement membrane integrity to endothelial cell proliferation, migration, and tube formation. These novel antiangiogenic activities of sulforaphane are likely to contribute to its cancer chemopreventive and therapeutic potential. [Mol Cancer Ther 2006;5(3):575–85]

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. Note: The current address for E. Bertl is RCC Ltd., Zelgliweg 1, CH-4452 Itingen, Switzerland. Received 8/15/05; revised 12/15/05; accepted 1/11/06.

http://cancerres.aacrjournals.org/cgi/content/abstract/66/3/1740

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Sulforaphane Sensitizes Tumor Necrosis Factor–Related ApoptosisInducing Ligand (TRAIL)–Resistant Hepatoma Cells to TRAILInduced Apoptosis through Reactive Oxygen Species–Mediated Upregulation of DR5 Heesue Kim1, Eun Hee Kim1, Young Woo Eom1, Wook-Hwan Kim2, Taeg Kyu Kwon3, Soo Jae Lee4 and Kyeong Sook Choi1 1

Institute for Medical Sciences and 2 Department of Surgery, Ajou University School of Medicine, Suwon, South Korea; 3 Department of Immunology, School of Medicine, Keimyung University, Taegu, South Korea; and 4 Laboratory of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea Requests for reprints: Kyeong Sook Choi, Institute for Medical Sciences, Ajou University School of Medicine, Suwon, South Korea 442-749. Phone: 82-31-219-4552; Fax: 82-31-219-4503; E-mail: [email protected] .

Sulforaphane is a chemopreventive agent present in various cruciferous vegetables, including broccoli. Here, we show that treatment with tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) in combination with subtoxic doses of sulforaphane significantly induces rapid apoptosis in TRAIL-resistant hepatoma cells. Neither TNF- - nor Fas-mediated apoptosis was sensitized in hepatoma cells by cotreatment with sulforaphane, suggesting that sulforaphane can selectively sensitize cells to TRAIL-induced apoptosis but not to apoptosis mediated by other death receptors. We found that sulforaphane treatment significantly up-regulated mRNA and protein levels of DR5, a death receptor of TRAIL. This was accompanied by an increase in the generation of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine and overexpression of catalase inhibited sulforaphane-induced up-regulation of DR5 and almost completely blocked the cotreatment-induced apoptosis. Furthermore, the sulforaphane-mediated sensitization to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs for DR5. These results collectively indicate that sulforaphane-induced generation of ROS and the subsequent up-regulation of DR5 are critical for triggering and amplifying TRAIL-induced apoptotic signaling. We also found that sulforaphane can sensitize both Bcl-xL- and Bcl-2-overexpressing hepatoma cells to TRAIL-induced apoptosis, indicating that treatment with a combination of TRAIL and sulforaphane may be a safe strategy for treating resistant hepatomas. (Cancer Res 2006; 66(3): 1740-50)

http://www.ajcn.org/cgi/content/abstract/82/6/1283

ORIGINAL RESEARCH COMMUNICATION

Glutathione S-transferase M1 polymorphism and metabolism of sulforaphane from standard and high-glucosinolate broccoli1,2,3 Amy V Gasper, Ahmed Al-janobi, Julie A Smith, James R Bacon, Paul Fortun, Clare Atherton, Moira A Taylor, Christopher J Hawkey, David A Barrett and Richard F Mithen 1

From the Phytochemicals and Health Programme, Institute of Food Research, Norwich Research Park, Colney, Norwich, United Kingdom (AVG, JRB, and RFM) and the Center for Analytical Bioscience, School Pharmacy (DAB, AA, and AVG) and Biomedical Sciences (MAT and AVG) and the Wolfson Digestive Diseases Centre (JAS, CA, PF, and CJH), University of Nottingham, Nottingham, United Kingodm

Background: Broccoli consumption is associated with a reduction in the risk of cancer, particularly in persons with a functional glutathione S-transferase M1 allele, as opposed rotrose whose GSTM1 gene has been deleted. Sulforaphane, the major isothiocyanate derived from 4-methylsulfinylbutyl glucosinolate, is thought to be the main agent conferring protection. Objective: We compared sulforaphane metabolism in GSTM1-null and GSTM1-positive subjects after they consumed standard broccoli and high-glucosinolate broccoli (super broccoli). Design: Sixteen subjects were recruited into a randomized, 3-phase crossover dietary trial of standard broccoli, super broccoli, and water. Liquid chromatography linked to tandem mass spectrometry was used to quantify sulforaphane and its thiol conjugates in plasma and urine. Results:GSTM1-null subjects had slightly higher, but statistically significant, areas under the curve for sulforaphane metabolite concentrations in plasma, a greater rate of urinary excretion of sulforaphane metabolites during the first 6 h after broccoli consumption, and a higher percentage of sulforaphane excretion 24 h after ingestion than did GSTM1-positive subjects. Consumption of high-glucosinolate broccoli led to a 3-fold greater increase in the areas under the curve and maximum concentrations of sulforaphane metabolites in plasma, a greater rate of urinary excretion of sulforaphane metabolites during the first 6 h after consumption, and a lower percentage of sulforaphane excretion after its ingestion than did the consumption of standard broccoli. Conclusions:GSTM1 genotypes have a significant effect on the metabolism of sulforaphane derived from standard or high-glucosinolate broccoli. It is possible that the difference in metabolism may explain the greater protection that GSTM1-positive persons gain from consuming broccoli. The potential consequences of consuming glucosinolate-enriched broccoli for GSTM1-null and -positive persons are discussed. Key Words: Sulforaphane • glucosinolates • broccoli • super broccoli • GSTM1 • chemoprotection

http://jn.nutrition.org/cgi/content/abstract/134/9/2229

Nutrition and Cancer

Sulforaphane Inhibits Human MCF-7 Mammary Cancer Cell Mitotic Progression and Tubulin Polymerization1,2 Steven J. T. Jackson and Keith W. Singletary3 Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801 3

To whom correspondence should be addressed. E-mail: [email protected] .

Sulforaphane (SUL), an isothiocyanate derived from hydrolysis of glucoraphanin in broccoli and other cruciferous vegetables, was shown to induce phase II detoxification enzymes, inhibit chemically induced mammary tumors in rodents, and more recently, to induce cell cycle arrest and apoptosis in colon cancer cells. In the present study, we demonstrate that SUL also acts to inhibit proliferation of MCF-7 adenocarcinoma cells from the human breast. Treatment of synchronized MCF-7 cells with 15 µmol/L SUL resulted in significant (P < 0.05) G2/M cell cycle arrest (167% of control) and elevated cyclin B1 protein (175% of control) within 24 h. Moreover, 15 µmol/L SUL significantly (P < 0.05) induced phosphorylation of histone H1 (167% of control), blocked cells in early mitosis ( 10-fold increase over control), and disrupted polymerization of mitotic microtubules in vivo. Subsequent exposure of purified bovine brain tubulin to relatively high doses of SUL significantly (P < 0.05) inhibited both tubulin polymerization rate (51% of control) and total tubulin polymerization (78% of control) in vitro. Additionally, polymerization of purified tubulin exposed to isothiocyanate-containing analogs of SUL was similarly inhibited. Taken together, these findings indicate that SUL has mammary cancer suppressive actions involving mitotic cell cycle arrest and suggest a mechanism linked to the disruption of normal tubulin polymerization and/or more subtle effects on microtubule dynamics.

KEY WORDS: • mitotic arrest • microtubule polymerization • human mammary carcinoma

ht t p: / / ict.sagepub.co m / cgi /co nt ent / a bst r act / 3 / 1 / 5

Cruciferous Vegetables: Cancer Protective Mechanisms of Glucosinolate Hydrolysis Products and Selenium Anna-Sigrid Keck, PhD United States Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota

John W. Finley, PhD United States Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota, [email protected]

Dietetic professionals urge Americans to increase fruit and vegetable intakes. The American Institute of Cancer Research estimates that if the only dietary change made was to increase the daily intake of fruits and vegetables to 5 servings per day, cancer rates could decline by as much as 20%. Among the reasons cited for this health benefit are that fruits and vegetables are excellent sources of fiber, vitamins, and minerals. They also contain nonnutritive components that may provide substantial health benefits beyond basic nutrition. Examples of the latter are the glucosinolate hydrolysis products, sulforaphane, and indole-3-carbinol. Epidemiological studies provide evidence that the consumption of cruciferous vegetables protects against cancer more effectively than the total intake of fruits and vegetables. This review describes the anticarcinogenic bioactivities of glucosinolate hydrolysis products, the mineral selenium derived from crucifers, and the mechanisms by which they protect against cancer. These mechanisms include altered estrogen metabolism, protection against reactive oxygen species, altered detoxification by induction of phase II enzymes, decreased carcinogen activation by inhibition of phase I enzymes, and slowed tumor growth and induction of apoptosis. Key Words: cruciferous veget ables • glucosinolat es • cancer prot ectio n • isot hiocyanat es sulfo r ap hane • indole-3-car binol • seleniu m

http://jn.nutrition.org/cgi/content/abstract/135/8/1865

Nutrient-Gene Interactions

Transcriptome Analysis of Human Colon Caco-2 Cells Exposed to Sulforaphane1,2,3 Maria Traka*, Amy V. Gasper*, Julie A. Smith , Chris J. Hawkey , Yongping Bao*,** and Richard F. Mithen*,4 *

Nutrition Division, Institute of Food Research, Colney Lane, Norwich, NR4 7UA, UK; Wolfson Digestive Diseases Centre, University Hospital, Nottingham, NG7 2UH, UK; and ** School of Medicine, Health and Policy, University of East Anglia, Norwich, NR4 7TJ, UK 4

To whom correspondence should be addressed. E-mail: [email protected] .

Sulforaphane (SF), a dietary phytochemical obtained from broccoli, has been implicated in several physiological processes consistent with anticarcinogenic activity, including enhanced xenobiotic metabolism, cell cycle arrest, and apoptosis. In this study, we report changes in global gene expression in Caco-2 cells exposed to physiologically appropriate concentrations of SF, through the use of replicated Affymetrix array and RT-PCR experiments. After exposure to 50 µmol/L SF, 106 genes exhibited a >2-fold increase in expression and 63 genes exhibited a >2-fold decrease in expression. There were fewer changes in gene expression at lower SF concentrations. The majority of these genes had not previously been shown to be modulated by SF, suggesting novel mechanisms of possible anticarcinogenic activity, including induction of differentiation and modulation of fatty acid metabolism. The changes in the expression of 10 of these genes, together with 4 additional genes of biological interest, were further quantified in independent studies with RT-PCR. These genes include several that have recently become associated with carcinogenesis, such as Krüppel-like factor (KLF)4, a gut-enriched transcription factor associated with induction of differentiation and reduction in cellular proliferation; DNA (cytosine-5-)-methyltransferase 1, associated with methylation; and -methylacyl-CoA racemase (AMACR), a marker associated with the development of colon and prostate cancer. The expression of 5 of these genes [caudal type homeo box transcription factor 2 (CDX-2), KLF4, KLF5, cyclin-dependent kinase inhibitor 1A (p21), and AMACR] was additionally studied after in vitro exposure to SF of surgically resected healthy and cancerous colon tissue from each of 3 patients. The study suggests the complex effects that SF has on gene expression and highlights several potential mechanisms by which the consumption of broccoli may reduce the risk of carcinogenesis.

KEY WORDS: • colon cancer • chemoprevention • sulforaphane • microarray • KLF4 • AMACR • p21

Cancer Prev Res (Phila Pa). 2009 Apr;2(4):353-60.

Dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate gastritis in Helicobacter pylori-infected mice and humans. Yanaka A, Fahey JW, Fukumoto A, Nakayama M, Inoue S, Zhang S, Tauchi M, Suzuki H, Hyodo I, Yamamoto M.

Division of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, ChibaKen, Tokyo, Japan. [email protected]

The isothiocyanate sulforaphane [SF; 1-isothiocyanato-4(R)-methylsulfinylbutane] is abundant in broccoli sprouts in the form of its glucosinolate precursor (glucoraphanin). SF is powerfully bactericidal against Helicobacter pylori infections, which are strongly associated with the worldwide pandemic of gastric cancer. Oral treatment with SF-rich broccoli sprouts of C57BL/6 female mice infected with H. pylori Sydney strain 1 and maintained on a high-salt (7.5% NaCl) diet reduced gastric bacterial colonization, attenuated mucosal expression of tumor necrosis factor-alpha and interleukin-1beta, mitigated corpus inflammation, and prevented expression of high salt-induced gastric corpus atrophy. This therapeutic effect was not observed in mice in which the nrf2 gene was deleted, strongly implicating the important role of Nrf2-dependent antioxidant and anti-inflammatory proteins in SF-dependent protection. Forty-eight H. pylori-infected patients were randomly assigned to feeding of broccoli sprouts (70 g/d; containing 420 micromol of SF precursor) for 8 weeks or to consumption of an equal weight of alfalfa sprouts (not containing SF) as placebo. Intervention with broccoli sprouts, but not with placebo, decreased the levels of urease measured by the urea breath test and H. pylori stool antigen (both biomarkers of H. pylori colonization) and serum pepsinogens I and II (biomarkers of gastric inflammation). Values recovered to their original levels 2 months after treatment was discontinued. Daily intake of sulforaphane-rich broccoli sprouts for 2 months reduces H. pylori colonization in mice and improves the sequelae of infection in infected mice and in humans. This treatment seems to enhance chemoprotection of the gastric mucosa against H. pylori-induced oxidative stress.

http://www.oxfordjournals.org/

SHORT COMMUNICATION

Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate Fung-Lung Chung1,3, C.Clifford Conaway1, C.V. Rao2 and Bandaru S. Reddy2 1 2

Division of Carcinogenesis and Molecular Epidemiology and Division of Nutritional Carcinogenesis, American Health Foundation, Valhalla, NY 10595, USA

Epidemiological studies have linked consumption of broccoli to a reduced risk of colon cancer in individuals with the glutathione S-transferase M1 (GSTM1) null genotype. GSTs are involved in excretion and elimination of isothiocyanates (ITCs), which are major constituents of broccoli and other cruciferous vegetables and have cancer chemopreventive potential, so it is speculated that ITCs may play a role in protection against human colon cancer. However, there is a lack of data from animal studies to support this. We carried out a bioassay to examine whether sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), major ITCs in broccoli and watercress, respectively, and their corresponding N-acetylcysteine (NAC) conjugates, show any chemopreventive activity towards azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats. Groups of six male F344 rats were treated with AOM subcutaneously (15 mg/kg body wt) once weekly for 2 weeks. SFN and PEITC and their NAC conjugates were administered by gavage either three times weekly for 8 weeks (5 and 20 µmol, respectively) after AOM dosing (post-initiation stage) or once daily for 3 days (20 and 50 µmol, respectively) before AOM treatment (initiation stage). The bioassay was terminated on week 10 after the second AOM dosing and ACF were quantified. SFN, SFNNAC, PEITC and PEITC-NAC all significantly reduced the formation of total ACF from 153 to 100–116 (P < 0.01) and multicrypt foci from 52 to 27–38 (more than four crypts/focus; P < 0.05) during the postinitiation treatment. However, only SFN and PEITC were effective during the initiation phase, reducing the total ACF from 153 to 109–115 (P < 0.01) and multicrypt foci from 52 to 35 (more than four crypts/focus; P < 0.05). The NAC conjugates were inactive as anti-initiators against AOM-induced ACF. These findings provide important laboratory evidence for a potential role of SFN and PEITC in the protection against colon cancer.

INTRODUCTION A recent case–control study reported that broccoli consumption is linked to a lowered risk of colon cancer and that the protective effect is especially evident in individuals with a glutathione S-transferase (GST) M1 null genotype (1). Because GSTs facilitate the conjugation of isothiocyanates (ITCs), resulting in their excretion as N-acetylcysteine (NAC) conjugates via the mercapturic acid pathway, it has been suggested that the ITC compounds in broccoli may play a role in protection against human colon cancer (1,2). Sulforaphane (SFN) is the predominant ITC found in broccoli. It has been studied for its chemopreventive potential primarily due to its activity as an inducer of phase II enzymes involved in carcinogen detoxification and elimination (3). Although it has been reported that SFN inhibits carcinogen-induced mammary gland tumorigenesis (4,5), no animal data are yet available regarding the effects of SFN on colon tumorigenesis. Several laboratory animal studies have shown that phenethyl ITC (PEITC), a principal constituent of watercress, is a potent chemopreventive agent for cancers of the breast, lung, and esophagus (6–8). However, there is only limited information about its protective effect against colon cancer. Given current data supporting ITCs as chemopreventive agents in a variety of tumor models, it is possible that SFN and PEITC would protect against colon tumor development. The present bioassay was designed to determine whether SFN and PEITC can inhibit the formation of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in

Fischer 344 rats. ACF have been recognized as an early preneoplastic lesion of colon cancer (9–11) and agents that inhibit colonic ACF formation generally show chemopreventive activity against colon cancer (12). We also included in this study the NAC conjugates of SFN and PEITC since our previous studies showed that certain ITC–NAC conjugates are promising chemopreventive agents for lung tumorigenesis, probably by a gradual release of the parent ITCs via a dissociation reaction (13,14). The structures of SFN and PEITC and their NAC conjugates are shown in Figure 1 . SFN and PEITC or their NAC conjugates were administered in two treatment regimens, either after AOM treatment (post-initiation) or before AOM treatment (initiation) in order to define better the possible mechanism of action.

Fig. 1. Structures of SFN and PEITC and their NAC conjugates.

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Male F344 rats were obtained from Charles River (Kingston, NY). They were fed AIN-76A diet (5% corn oil) and tap water ad libitum. Animals were maintained under standard conditions (12 h light/12 h dark cycle, 50% relative humidity at 21°C). At 6 weeks of age, they were randomly divided into groups of six rats as shown in Table I , except for the control groups (groups 10–14), which included three animals each. PEITC and D,L-SFN (purity >99% and >97%, respectively) were purchased from Aldrich Chemical Co. (Milwaukee, WI) and LTK Labs, Inc. (St Paul, MN). The corresponding NAC conjugates were prepared by a published method (15). The structures were verified by their proton NMR spectra. The purity of the conjugates was >97% as determined by high-performance liquid chromatography. AOM obtained from Ash-Stevens (Detroit, MI) was dissolved in saline and administered at 15 mg/kg body wt subcutaneously twice (7 days between the two injections), a previously established treatment regimen for the induction of colon tumor (16). SFN and PEITC in corn oil and NAC conjugates in saline (10% dimethylsulfoxide) were given by gavage. The high cost of SFN prohibited administration of the compound in the diet. For the post-initiation treatment, groups 2–5 were given 5 µmol SFN or PEITC or 20 mmol of the NAC conjugates, three doses each week for 8 weeks, beginning two days after the last dose of AOM. The higher doses of the NAC conjugates were given because of their gradual release of parent compounds and lower toxicity. For the assay of anti-initiation activity, groups 6–9 were treated with three doses of ITC compounds (20 µmol ITC or 50 µmol ITC–NAC) once daily; the last dose was given 2 h before AOM dosing. This dosing regimen was repeated during the second week of AOM dosing. This pre-treatment protocol was adapted based on earlier studies on mammary tumor and lung tumor models (17,18). Groups 10–13 were treated with ITCs (5 µmol/dose) or the conjugates (20 µmol/dose) only, three times weekly for 8 weeks. The bioassay was terminated at week 10 after the second AOM treatment. All animals were killed by CO2 asphyxiation. The colons were excised, fixed in buffered formalin and processed for microscopic examination; ACF were recorded using a standard procedure described previously (19). ACF were distinguished from the surrounding normal crypts by their increased size, significantly increased distance from lamina to basal surface of cells and the easily discernible pericryptal zone. For statistical analysis, means were compared among the groups using one-way analysis of variance (ANOVA) followed by Fisher's protected t-test.

View this table: Table I. Effects of SFN and PEITC on the formation of aberrant crypt foci induced [in this window] by AOM [in a new window]

No significant differences in body weights were seen in any of the treated groups compared with the control group, indicating that doses of ITCs and the conjugates used did not cause overt toxicity (Figure 2 ). This bioassay showed that post-treatment with SFN and PEITC by oral administration at 5 µmol three times weekly for 8 weeks and their NAC conjugates at 20 µmol by the same regimen inhibited formation of ACF. These treatments reduced the total number of ACF from 153 to 100–116 (P < 0.01) and reduced the number of multicrypt foci (more than four crypts/focus) from 52 to 27–37 (P < 0.05) as shown in Table I (groups 1– 5). Because the ITC conjugates are presumably less toxic than the parent ITCs, the doses of the conjugates were four times that of SFN and PEITC (5,20). However, we did not find significant differences in the inhibition of ACF between the ITCs and their NAC conjugates, suggesting that the conjugates themselves are not as active. Similar dose–effect relationships were observed previously between parent ITCs and their conjugates towards the inhibition of lung tumorigenesis (13). These results, together with those from our earlier studies (21,22), suggest that ITC conjugates render their inhibitory activity in part by deconjugation to the parent ITCs. Although the mechanism of inhibition of ACF at the post-initiation phase is not currently clear, studies have shown that PEITC and related ITCs induce p53-dependent or c-Jun kinase-mediated apoptosis in cultured tumor cells (23–25). More recently it has been reported that SFN induces cell cycle arrest in HT29 human colon cancer cells (26). The growth arrest, followed by cell death via apoptosis, appeared to be associated with expression of cyclins A and B1. In addition, NAC produced by deconjugation is a known antioxidant and has been recently shown to inhibit mouse fibroblast cell proliferation by blocking cell in G1 phase (27). All these activities could have contributed to the inhibition of ACF formation during post-initiation.

Fig. 2. Growth curves of the treated and the control groups during the bioassay.

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As shown previously by us and others, pretreatment of animals with PEITC and other related ITCs blocked chemical-induced tumorigenicity by inhibiting cytochrome P450 (CYP) enzymes responsible for the activation of carcinogens, and consequently reducing DNA damage (28–30). Using the pretreatment regimen, we investigated the effects of SFN and PEITC on AOM-induced colonic ACF formation in Fischer rats. Pretreatment with SFN or PEITC significantly decreased the total number of ACF from 153 to 109 (P < 0.001) or 115 (P < 0.01), respectively, and multicrypt foci (more than four crypts) from 52 to 35 (P < 0.05) for both compounds (Table I ). We have previously shown that PEITC and PEITC–NAC inhibit N,Ndimethylnitrosamine (NDMA) demethylase (CYP2E1) in rat liver microsomes (21). Like NDMA, AOM is metabolically activated by CYP2E1 to methyazoxymethanol, which can yield a DNA methylating species (31). Thus, inhibition of CYP2E1 by these agents in the rat liver may constitute an important mechanism of inhibition, because CYP enzyme activity in colonic tissue is low compared with that in liver (32). SFN–NAC also reduced total ACF to 120 (P < 0.01); however, it had no significant effect on multicrypt foci (those with more than four crypts/focus; 15% inhibition). PEITC–NAC, on the other hand, appeared to enhance ACF formation [total number of ACF was 198 (P < 0.001); number of multicrypt foci was 74 (P < 0.05)] (Table I ). The adverse effect caused by PEITC–NAC is somewhat unexpected in view of the inhibition by its parent ITC. At present, it is not clear why there is such a sharp contrast in ACF formation between PEITC–NAC and the other ITC compounds studied here. Previously, it has been reported that PEITC given in the diet at 0.1% for 32 weeks appeared to promote bladder cancer development in rats pretreated with carcinogens (33). This dose is considerably higher than the doses used in the present study. Phenylhexyl ITC, a synthetic homolog of PEITC, is a potent inhibitor of lung tumorigenesis in rats and mice, whereas it enhanced colon and esophagus tumorigenesis in rats, possibly due to its tissue cytotoxicity at the dose level studied (34–37). These results illustrate the importance of choosing the appropriate dose range for specific tissues in chemoprevention studies. Evidence from epidemiological studies suggests an association between consumption of cruciferous vegetables and a reduced risk of colon cancer (38). Although the beneficial effects may result from the combined effects of a number of compounds in these vegetables, the exact role of each compound that contributes to the protective effects needs to be identified more clearly. Ample data from laboratory animal studies have shown that ITCs, major constituents of cruciferous vegetables, are promising chemopreventive agents against cancers at various sites, including lung, esophagus, liver, mammary, pancreas and bladder (3,28,39). These laboratory results support a potential role of dietary ITCs in reducing the risk of certain human cancers. Considering the natural abundance of SFN and PEITC in broccoli and watercress [it is estimated that an individual's intake of SFN or PEITC after ingesting 100 g (wet weight) of broccoli or watercress can range from 50 to 200 µmol (40–43)], and their potential as chemopreventive agents, it is surprising that little is known about the effects of these agents on colon tumorigenesis. A lower homolog of PEITC, benzyl ITC (BITC) has been shown to reduce colon tumor incidence in AOM treated rats during the initiation phase, but not during the post-initiation phase (44). Another short-term study, however, has reported that both PEITC and BITC given in the diet at similar doses throughout the bioassay did not affect ACF formation (12). By contrast, the present study has demonstrated that both PEITC and SFN inhibit colonic ACF, independent of whether they are administered before or after exposure to carcinogen. These data, thus, support a potential protective role of SFN and PEITC in colon cancer and are consistent with the epidemiological observation that consumption of certain cruciferous vegetables reduces the risk of colon cancer in individuals with GSTM1 null genotype. These findings warrant further investigations of the mechanisms of action and preclinical efficacy of these agents.

Notes

3

To whom correspondence should be addressed Email: [email protected]

Acknowledgments We would like to thank Joanne Braley and Joel Reinhardt of the Research Animal Facility at the American Health Foundation for their expert work in animal handling and care. We also thank Barbara Simi for her excellent technical assistance on quantifying aberrant crypt foci and Brian Pittman for statistical analysis. This work was supported by NCI grant CA46535. This is paper number 31 in the series `Dietary Inhibitors of Chemical Carcinogenesis'.

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Received March 8, 2000; revised August 18, 2000; accepted August 29, 2000