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Supplement: International Research Conference on Food, Nutrition, and Cancer

Poster Abstracts¹

	Angiogenesis

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Se-(Methyl)selenocysteine Causes Tumor Vessel Maturation Leading to Synergistic Antitumor Effect in Human Xenografts. Arup Bhattacharya,¹ Károly Tóth,¹ Shousong Cao,¹ Rami G. Azrak,¹ Farukh A. Durrani,¹ Youcef M. Rustum,¹ and Lakshmi Pendyala.² Departments of ¹Cancer Biology and ²Medicine, Roswell Park Cancer Institute, Buffalo, NY.

Pretreatment with 5-methylselenocysteine (MSC) before chemotherapy leads to synergistic chemomodulation with a wide range of anticancer drugs including taxanes, taxols, fluoropyrimidines, irinotecan (CPT-11), platinum compounds, and cyclophosphamide. This synergy is neither drug, tumor, nor host specific and was not seen in vitro. MSC down-regulates vascular endothelial growth factor, cyclooxygenase-2, inducible nitric oxide synthase, hypoxia-inducible factor-1, and prostaglandin E₂ in tumors, leading to an antiangiogenic effect. The hallmarks of tumor vasculature lacking smooth muscle and pericytes are that they are leaky; are chaotic; and shut off and on intermittently, imparting a tortuous tumor blood flow and distribution. The resultant adverse intratumoral interstitial fluid pressure retards intratumoral drug delivery and distribution. Studies were performed using female athymic nude mice bearing different xenografts that were treated daily for up to 14 d with MSC at 0.2 mg/d p.o. to determine whether antiangiogenic effects of MSC involve tumor vessel maturation and whether vessel maturation leads to higher intratumoral drug delivery, leading in turn to better therapeutic synergy. Immunohistochemical study for vessel maturation used CD31 and -smooth muscle actin double staining on frozen tumor sections. Immunofluorescence evaluation for vessel functionality used mice bearing tumors that were injected i.v. with Hoechst 33342 in sterile phosphate-buffered saline at 15 mg/kg and 20 min later with DiOC₇ dissolved in DMSO at 1 mg/kg; tumors were harvested after 5 min. Vessel lumen areas were measured using CD31 immunostaining. After MSC therapy, the xenografts demonstrated tumor vessel maturation, as indicated by increased pericyte coverage and uptake of both fluorescent dye, and a reduced vessel lumen area compared with untreated tumors (146 vs. 82 µm in FaDu and 66 vs. 32 µm in A253; P < 0.0001). Thus, antiangiogenic MSC leads to tumor vessel maturation and results in higher intratumoral drug delivery (CPT-11, doxorubicin) without any risk of additive host tissue toxicity. Better drug delivery leads to better therapeutic efficacy.

Sulforaphane Suppresses Angiogenesis and Disrupts Endothelial Mitotic Progression and Microtubule Polymerization. Steven J.T. Jackson,¹ Keith W. Singletary,² and Richard C. Venema.^{1 1}Vascular Biology Center, Medical College of Georgia, Augusta, GA; and ²Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL.

Sulforaphane (SUL), an isothiocyanate derived from broccoli and other cruciferous vegetables, induces phase II detoxification enzymes, disrupts cancer cell microtubule polymerization, and triggers cell cycle arrest in breast and colon cancer cells. Here, we provide the first evidence that SUL also inhibits angiogenesis via suppression of endothelial cell proliferation. Bovine aortic endothelial (BAE) cells were exposed to concentrations of SUL up to 15 µmol/L before cell cycle analysis and mitotic index quantification. Within 24 h, 15 µmol/L of SUL clearly induced G₂/M accumulation and premetaphase arrest in BAE cells. Moreover, immunofluorescence tubulin staining indicated that this same SUL concentration not only disrupted mitotic progression but also perturbed normal polymerization of mitotic (and cytoplasmic) microtubules. Furthermore, daily administration of SUL (100 nmol/d, i.v. for 7 d) to female BALB/c mice bearing VEGF-impregnated Matrigel plugs strongly and significantly (P < 0.05) suppressed angiogenesis progression as measured by hemoglobin concentration. Taken together, these findings suggest that the endothelial cell population is a novel target of sulforaphane action both in vitro and in vivo. This mechanism of SUL-induced endothelial microtubule disruption and early mitotic arrest may further discern a potential role of SUL as a chemopreventive agent.

In Vivo Imaging and Characterization of Hypoxia-Induced Neovascularization and Tumor Invasion. Gina F. Lungu,¹ Meng-Lin Li,² Xueyi Xie,¹ Lihong V. Wang,² and George Stoica.^{1 1}Department of Pathobiology, College of Veterinary Medicine, and ²Optical Imaging Laboratory, Department of Biomedical Engineering, Texas A&M University, College Station, TX.

A diet rich in fruits, vegetables, and other plant foods may reduce the risk of cancer, but few links to cancer have been established. Finding a method to determine the effect of dietary compounds on cancer and to monitor the disease progression will be of interest. This study provides an integrated approach to define oxygen status (hypoxia) of intracranial tumor xenograft using a novel spectroscopic photoacoustic tomography technology (SPAT). This approach is supported by our molecular biology investigation. Brain tumors can be identified by their distorted vascular architecture and oxygen saturation images. Noninvasive in vivo tumor oxygenation imaging using SPAT is based on the spectroscopic absorption differences between oxyhemoglobin and deoxyhemoglobin. Sprague-Dawley rats inoculated intracranially with ENU1564, a carcinogen-induced rat mammary adenocarcinoma cell line, were imaged with SPAT 3 wk postinoculation. Proteins important in tumor hypoxia and invasion were detected in hypoxic brain foci identified by SPAT and were elevated compared with normal brain. Immunohistochemistry, Western blotting, and semiquantitative RT-PCR showed that hypoxia-inducible factor-, vascular endothelial growth factor-A, and vascular endothelial growth factor-R2 protein and mRNA expression levels were significantly higher (P < 0.05) in brain tumor tissues than in normal brain. Gelatin zymography and RT-PCR demonstrated the up-regulation of matrix metallopeptidase-9 in tumor foci compared with normal brain. Together these results suggest the critical role of hypoxia in driving tumor angiogenesis and invasion through up-regulation of target genes important for these functions. Moreover, this report validates our hypothesis that SPAT is suitable for detecting tumors, hypoxia, and angiogenesis and for monitoring a cell-targeted therapeutic approach to brain malignancies. We hope that SPAT will be used in the near future to monitor the effect of different dietary compounds on brain malignancy.

	Apoptosis

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Inhibition of Na⁺/K⁺-ATPase by Ouabain Potentiates Apoptosis by Inducing Perturbations in Cell Calcium Homeostasis: A Protective Role Selective for Bcl-2. Michail Panagiotidis and John A. Cidlowski. Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Apoptosis is a well-defined programmed process with distinctive characteristics including cellular shrinking, chromatin condensation, and formation of membrane-bound apoptotic bodies. Cell shrinkage occurs early in apoptosis and is accompanied by changes in the activity of ion channels and plasma membrane transporters. Na⁺/K⁺-ATPase couples the energy released from the hydrolysis of ATP to the transport of Na⁺ and K⁺ ions, thus controlling cell volume and plasma membrane potential. Additionally, the Na⁺/K⁺-ATPase is the only known receptor for cardiac glycosides including ouabain. We hypothesize that Na⁺/K⁺-ATPase is essential in modulating apoptosis, and thus, its inhibition with ouabain should potentiate the apoptotic process. We report that ouabain potentiated apoptosis in wild-type Jurkat cells exposed to Fas ligand (FasL) by increasing the percentage of shrunken cells, percentage of degraded DNA content, and caspase 3/7-like activity. When cells were treated with ouabain and then exposed to other apoptotic agents [H₂O₂, staurosporine, TNF-related apoptosis-inducing ligand (TRAIL), thapsigargin], only exposure to TRAIL showed potentiation of apoptosis, indicating that such potentiation is restricted to the death receptor pathway (FasL and TRAIL). In addition, we observed that ouabain induced perturbations in cell calcium homeostasis on both FasL and TRAIL treatments. Ouabain-induced potentiation of apoptosis was abolished in Bcl-2–overexpressing cells by inhibiting perturbations in cell calcium homeostasis, whereas Bcl-X_L overexpression had no effect. To conclude, our data indicate a novel role for calcium in modulating ouabain-induced potentiation of apoptosis and that Bcl-2 selectively modulates calcium homeostasis.

Alterations in Cellular Levels of Inositol Polyphosphates during Apoptosis. Rakhee Agarwal, Aparna Konidala, and Nawab Ali. Graduate Institute of Technology, University of Arkansas at Little Rock, Little Rock, AR.

Inositol polyphosphates (InsPs) regulate diverse cellular processes including calcium homeostasis, vesicular trafficking, chromatin remodeling, and nuclear export of mRNA. Exogenous InsPs inhibit cellular growth by inducing apoptosis in certain cancer cell lines, perhaps by interfering with the pleckstrin homology domain of the phosphatidylinositol 3-kinase, and thus have potential as anticancer agents. However, whether exogenous InsPs enter the intact cells is debatable. Changes in cellular levels of InsPs may bring about apoptotic changes; higher InsP levels increased during apoptosis. We examined apoptosis induced by InsPs in mouse osteoblast-like MC3T3 cells. InsP₆, InsP_5, and InsP₄ dose-dependently (10–50 µmol/L) increased apoptosis as measured by acridine orange/ethidium bromide and caspase-3 staining. At these concentrations, InsP₆ was more potent than InsP₅ and InsP₄ in promoting apoptosis. To study the effects of endogenous higher InsPs on apoptosis, we used NaF and antimycin to change cellular levels of InsPs. NaF increases InsP₇ and InsP₈ levels by inhibiting InsP phosphatase, a process that is reversible by antimycin because InsP kinase is inhibited as a result of depletion of cellular ATP pools. Apoptosis was induced in MC3T3 cells in a NaF dose–dependent and time-dependent manner; 50% apoptosis was observed at 1 mmol/L NaF in 8 h. Prior treatment with 10 µmol/L antimycin for 30 min significantly reduced the NaF-induced apoptosis as compared with its control. The data indicated that NaF-induced apoptosis could be caused by an increase in InsP levels that was reversed by antimycin treatment. Additionally, we measured changes in lower InsP (InsP₁–InsP₄) levels during etoposide-induced apoptosis. Inositol tris- and tetrakisphosphate levels decreased dose dependently with a maximum decrease of 24% and 47%, respectively, at 50 µmol/L etoposide. Inositol mono- and bisphosphate levels either remained unchanged or increased marginally. The significance of these changes in InsP levels remains to be investigated. [Supported by Arkansas Space Grant Consortium grant to N. Ali.]

	Biomarkers

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Association of Hydroxynonenal- 1,N²-Propano-DNA-Adduct Levels in Human Lymphocytes with DNA Repair Gene XPA and Nutritional Markers in the Bavarian Nutrition Survey. Erwin Eder,¹ Odilia Popanda,² Paul Wanek,¹ Ellen Biskup,¹ and Jakob Linseisen.^{3 1}Department of Toxicology, University of Würzburg, Würzburg, Germany; ²Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany; ³Clinical Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

1,N²-Propanodeoxyguanosine adducts of 4-hydroxynonenal are appropriate biomarkers for cancer risk from lipid peroxidation. Adduct levels were measured in human lymphocytes in the Bavarian Nutrition Survey. The study investigated dietary behavior and lifestyle in a representative population sample in Bavaria: 1050 participants aged 13–80 y. In a substudy, 568 adult participants of both genders aged 18–80 y with complete dietary information were invited for blood sampling. Here we present the results of 278 samples evaluated to date. A significant inverse correlation of adduct levels was observed with eicosapentaenoic acid [20:5(n-3)] concentration in red blood cell membranes. No clear correlations were found between adduct levels and other fatty acids. Surprisingly, a positive correlation was seen between plasma vitamin C concentrations and adduct levels. No significant correlations were observed between adduct levels and plasma concentrations of other antioxidants. Socioeconomic status significantly correlated with adduct levels: the higher the status, the higher the adduct levels. The individual 1,N²-propanodeoxyguanosine adduct level can be modulated not only by lifestyle and nutrition factors but also by the individual DNA repair capacity. We also studied the influence of repair polymorphisms. With the XPD Asp³¹²Asn variants, no changes in adduct levels were seen. Adduct levels increased in the XPD Lys⁷⁵¹Gln variants from 54.3 mean adducts/10⁹ nucleotides in Lys/Lys (n = 126) to 60.6 in the Lys/Gln (n = 126) and to 63.2 in Gln/Gln (n = 40). In the XPA (–4G/A) variants, the adduct levels increased from 56.4 in variant G/G (n = 112) to 58.3 in G/A (n = 126) and to 64.4 in A/A (n = 40). After adjustment for nutrition factors, a significantly increased odds ratio of 2.5 (CI 1.05, 5.8) was determined for having high adduct levels in XPA A/A carriers, suggesting a higher cancer risk in these individuals.

DNA from Food as a Biomarker for Human Diet. Douglas Spangler,¹ Richard Rivlin,² and David S. Thaler.^{1 1}Raymond and Beverly Sackler Laboratory of Molecular Genetics and Informatics, New York, NY; ²Clinical Nutrition Research Unit, Strang Cancer Research Laboratory, Weill Medical College of Cornell University, New York, NY.

There is a recognized need to develop new, sensitive, quantitative, and objective biomarkers of human dietary intake (1). Species- and group (e.g., vegetable)-specific DNA sequences are potential biomarkers. We have developed a methodology of food-specific DNA analysis by Q(quantitative)PCR that can be applied to a wide range of clinical samples including blood, urine, saliva, and stool. Analyzing DNA in human biological fluids has the potential to provide novel biomarkers of dietary intake. Use of these biomarkers in clinical contexts will require and inform the understanding of individual variation in rates of digestion, intestinal transit, absorption, microbial processes, and excretion that are likely to be under genetic, physiological, and microecololgical controls. A clinical study is ongoing at the Rockefeller University Hospital. Our first analyses were consistent with the hypothesis that DNA sequences unique to vegetables and to fish were present in the blood of normal humans who ingest these foods. However, DNA from these blood samples was isolated with commercial kits and columns manufactured by Qiagen. Our subsequent sequencing of the PCR amplicons indicates that at least some of the "food DNA" signals derived from purification reagents, most likely the silica-containing spin columns. Others have reported finding related contaminants in commercial DNA purification reagents (2,3). Contamination of silica columns may be difficult to avoid because of the way they are currently manufactured. Taken together, we believe that these findings call into question the use of currently available commercial silica-based purification columns for certain contamination-sensitive DNA purifications for research, medical-diagnostic, and forensic applications. [Supported by NCI and by The Sloan Foundation.]

1. Johnson RK. Dietary intake—how do we measure what people are really eating? Obes Res. 2002;10: Suppl 1:63S–8S.[Medline]

2. van der Zee A, Peeters M, de Jong C, Verbakel H. Qiagen DNA extraction kits for sample preparation for Legionella PCR are not suitable for diagnostic purposes. J Clin Microbiol. 2002;40:1126–37.[Free Full Text]

3. Peters RP, Mohammadi T, Vandenbroucke-Grauls CM, Danner SA, van Agtmael MA, Savelkoul PH. Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination? FEMS Immunol Med Microbiol. 2004;42:249–53.[Medline]

Dietary Carbohydrate as a Biomarker of Red Meat and Dairy Intake. Eric I. Park. Department of Nutrition, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC.

High consumption of red meat has been associated with increased risk of colorectal cancer. However, the precise dietary component or the mechanism that accounts for this increased risk is unknown. One abundant component of red meat that is gaining interest is a type of carbohydrate moiety known as N-glycolylneuraminic acid (NGNA). NGNA accumulates on the cell surfaces of colon cancer, breast cancer, and hepatocellular carcinoma cells, whereas the surrounding normal cells have only trace amounts. Even more intriguing is how this accumulation occurs, because normal and cancerous human cells cannot synthesize NGNA. One likely source of NGNA is the consumption of red meat and dairy products, which are abundant in NGNA, and the subsequent metabolism of the NGNA by the same intracellular enzymes and transporters that metabolize endogenous sialic acids. Despite the possibility of NGNA serving as a biomarker of red meat and diary intake and for tagging and identifying cancer cells, little is known about the metabolism of dietary NGNA. Toward this goal, pilot studies were performed to determine the feasibility of using NGNA as a marker of red meat and dairy intake. First, a human cell line experiment showed that the amount of NGNA accumulation on the surface of cells depends on the NGNA concentration in the cell medium. Second, HPLC analyses showed that concentrations of NGNA were the same in raw and cooked meats. Third, the amounts of NGNA in human serum and urine samples were detected by HPLC and NMR methods, respectively. These studies suggest that the NGNA concentration in human samples may be useful as a marker of red meat and dairy consumption and a measurement to validate the real consumption levels of self-reported red meat and dairy intakes.

	Food Composition

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Sources of Flavonoids in the U.S. Diet Using USDA's Updated Database on the Flavonoid Content of Selected Foods. D.B. Haytowitz, S. Bhagwat, J.M. Holden, S.E. Gebhardt, and J. Harnly. USDA-ARS, Nutrient Data Laboratory and Food Composition Laboratory, Beltsville, MD.

Flavonoids are biologically active polyphenolic compounds widely distributed in plants and have been linked to various chemoprotective effects (1). USDA first released a database on the flavonoid content of foods in 2003. The database was recently updated using data from USDA analysis of 20 different flavonoid monomers from a nationwide sampling of 59 fruits, nuts, and vegetables, providing high-quality U.S. data not available in the earlier database. Data from 102 scientific papers were also added. The new database contains flavonoid data for 395 food items and is available on Nutrient Data Laboratory's (NDL's) Web site (2). These data were combined with data from NDL's Key Foods list derived from consumption data from the National Health and Nutrition Examination Survey to ascertain the intake of 5 classes of flavonoids—anthocyanidins, flavanones, flavonols, flavones, and flavan-3-ols—on a population basis. Black tea provided the largest amount of flavonols to the diet (32%), followed by onions (25%). Parsley was the largest contributor of flavones. Dried parsley contains a large amount—13.53 mg/100 g—though rarely is 100 g consumed at one time. Oranges (53%) and grapefruit juice (16%) provide significant amounts of the flavanones. Brewed tea provides the largest quantities of flavan-3-ols to the diet. Blueberries contributed the largest amount of anthocyanidins (31%), followed by bananas (21%) and strawberries (14%). Even though bananas contain considerably less anthocyanidins than any of the berries, U.S. consumption of bananas is much higher than that of individual berries. Daily per capita intake of flavonoids in the United States using these data were as follows: anthocyanidins, 5 mg; flavanones, 4 mg; flavones, 1 mg; flavonols, 10 mg; and flavan-3-ols, 112 mg. This expanded database provides researchers with new values on the flavononoid content of many more foods and should enable better assessment of the effect of flavonoid consumption on various chronic diseases.

4. Nichenametla SN, Taruscio TG, Barney DL, Exon JH. A review of the effects and mechanisms of polyphenolics in cancer. Crit Rev Food Sci Nutr. 2006;46:161–83.[Medline]

5. Nutrient Data Laboratory. Home page. 2006. Last Modified: 06/24/2006. Available from: http://www.ars.usda.gov/nutrientdata.

Nuts and Seeds as Sources of - and -Tocopherols. Robin G. Thomas and Susan E. Gebhardt. USDA-ARS Nutrient Data Laboratory, Beltsville, MD.

Some nuts and seeds are among the highest natural sources of vitamin E in the U.S. food supply. In its chief function as an antioxidant, vitamin E prevents free radical reactions, which is important in protecting cells from oxidative damage. Vitamin E has been associated with reduced risk of certain cancers such as colon, bladder, and prostate cancers. Recent studies have focused on effects of -tocopherol as well as -tocopherol. Of the 4 tocopherols (, ß, , and ), -tocopherol is the only one used to estimate the current Recommended Dietary Allowance (RDA) for vitamin E. The other tocopherols are absorbed and may have other functions but are not converted to -tocopherol in the body. The RDA for vitamin E is 15 mg/d of -tocopherol for adults. According to the National Health and Nutrition Examination Survey 2001–2002, >90% of adults do not meet the Estimated Average Requirement of 12 mg/d. Nuts and seeds are often cited as good sources of vitamin E. USDA recently updated tocopherol values in several nuts and seeds in the USDA National Nutrient Database for Standard Reference (SR). One-ounce portions of almonds, hazelnuts, and sunflower seeds provide >20% of the RDA for vitamin E, and Brazil nuts and pine nuts provide 10–20% of the RDA. One-ounce portions of cashews, macadamias, pecans, pistachios, black and English walnuts, flaxseed, and sesame seeds all provide 1–4% of the RDA. The highest nut and seed sources of -tocopherol are black walnuts and sesame seeds (28 mg/100 g), pecans (24 mg/100 g), pistachios (22 mg/100 g), and English walnuts and flaxseed (20 mg/100 g). These tocopherol values are derived from data from USDA studies, the food industry, and the scientific literature. Keeping SR up to date allows researchers to more accurately estimate nutrient intake, thus enabling them to study the relationships between diet and disease more effectively.

	Dietary Pattern and Cancer Risk

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Diet and Cancer Prevention Knowledge, Beliefs, and Barriers among Students in Historically Black Colleges and Universities. Adelia C. Bovell-Benjamin, Peter N. Gichuhi, Chellani S. Hathorn, and Elaine Bromfield. Food and Nutritional Sciences, Tuskegee University, Tuskegee, AL.

In the United States, African Americans (AAs) have the highest cancer incidence and lowest survival rate. The reasons for this disparity have not been elucidated, but dietary factors, which are largely modifiable, have been implicated. Potentially adverse dietary practices implicated in cancer risk have been associated with AA diets. This study examined nutrition knowledge, dietary beliefs, and perceived barriers related to cancer that prevent AA college students from adopting eating behaviors consistent with National Cancer Institute (NCI) dietary guidelines. A cross-sectional, in-depth structured interview was administered to students attending two historically black colleges and universities. Four hundred fifty-six students (238 male; 216 female) aged 18–30 y participated. A little more than half of the students (56.4%) correctly identified the food lowest in fiber from a given list, and the majority (84.7%) knew that lower fat intake reduces cancer risk. Although 38% of the students believed that cancer may be related to what people eat, 34.6% did not believe that cancer was related to diet, and 35% did not know that consumption of salt-cured, salt-pickled, and smoked foods is associated with cancer. Most students (83%) were unaware of NCI dietary guidelines. However, 82% of the students indicated that nutrition counseling and specific instructions on how to make dietary changes to prevent cancer could help them to modify their eating behaviors. Students believed that fatty and fast foods increased cancer risk. Availability, food preferences, convenience, cost, lack of support from family members, and little knowledge of healthy foods and their preparation were identified as barriers to healthy eating.

Disparities in Environmental Risk Factors for Cancer Prevention in 2 Rural Cities. Chellani S. Hathorn, Peter N. Gichuhi, Elaine Bromfield, Samia Ibrahim, and Adelia C. Bovell-Benjamin. Department of Food and Nutritional Sciences, Tuskegee University, Tuskegee, AL.

The burden of cancer is unequally distributed among populations and geographic locations. Minority populations, especially low-income African Americans, have higher mortality rates from cancer than other ethnic groups. Environmental factors explain some of the disparity in cancers among low-income minority groups; therefore, assessment of environmental risk factors (such as diet and physical activity) in communities may be important in cancer-prevention efforts. This study compared the availability of healthy food and physical activity choices that encourage cancer risk reduction in 2 rural cities: 1 high income and 1 low income. An unobtrusive, observational cross-sectional study was conducted using the United States Department of Agriculture Thrifty Food Basket Checklist. Four convenience stores, 9 fast-food restaurants, 7 restaurants, 7 large supermarkets, and 3 mass merchandisers located within a 4-mile radius in both cities were inventoried. In the low-income city, none of the 3 large supermarkets carried low-sodium vegetables. The fast-food restaurant inventoried offered no fruit items in the low-income city, whereas 2 in the high-income city did. Low-fat milk was available in all the large supermarkets. Low-sodium, low-fat cheese was available in only 2 supermarkets in the low-income city and 5 supermarkets in the high-income city. Convenience stores and restaurants were the most common food outlets in the low-income city, and fast-food restaurants and supermarkets were most common in the high-income city. Of the 28 physical activity outlets inventoried, public recreational areas were common and mostly free in the high-income city but limited and had a fee in the low-income city. Community members in the low-income city had limited access to healthy food and physical activity choices.

Meat Consumption, N-Acetyl Transferase 1 and 2 Polymorphism, and Risk of Breast Cancer in Danish Postmenopausal Women. Rikke Egeberg,¹ Anja Olsen,¹ Herman Autrup,² Jane Christensen,¹ Connie Stripp,¹ Kim Overvad,³ and Anne Tjønneland.^{1 1}Institute of Cancer Epidemiology, The Danish Cancer Society, Copenhagen, Denmark; ²Department of Environmental and Occupational Medicine, Institute of Public Health, University of Aarhus, Denmark; ³Department of Clinical Epidemiology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark.

In 1997 the World Cancer Research Fund concluded that meat intake might have a possible association with the risk of breast cancer. However, recent epidemiologic evidence concerning this relationship is controversial. We conducted a nested case-control study with 24,697 postmenopausal women included in the Diet, Cancer, and Health cohort study (1993–2000) to investigate the association between meat intake and breast cancer risk and whether polymorphisms in N-acetyltransferase 1 (NAT1) and 2 (NAT2) modified this association. A total of 378 breast cancer cases were identified and matched to 378 control subjects. The analyses were based on a conditional logistic regression analysis corresponding to a Cox proportional hazard model because of the study design used. All models were adjusted for baseline values of established risk factors for breast cancer. The incidence rate ratio (IRR) (95% CI) for breast cancer was 1.09 (1.02, 1.17) for total meat, 1.15 (1.01, 1.31) for red meat, and 1.23 (1.04, 1.45) for processed meat per 25 g/d increment in intake. The IRR (95% CI) in fast NAT1 acetylators compared with NAT1 slow acetylators was 1.43 (1.03, 1.99) and in intermediate to fast NAT2 acetylators compared with slow NAT2 acetylators was 1.13 (0.83, 1.54). Interaction analyses revealed that the positive associations between total meat intake and red meat intake and breast cancer risk were confined to intermediate –to fast NAT2 acetylators (P_interaction = 0.03 and 0.04), whereas no significant interaction was shown for NAT1 acetylator phenotypes. In conclusion, our findings support an association between meat consumption and breast cancer risk and a modifying effect of NAT2 polymorphism on the association.

Diet Quality Predicts BRCA-Associated Breast Cancer Risk. André Nkondjock and Parviz Ghadirian. Epidemiology Research Unit, RC, CHUM, Montreal, Canada.

Although it has been suggested that dietary energy intake restriction may be related to reduced BRCA-associated breast cancer risk, it is currently not known whether overall diet quality could predict the risk among women with deleterious mutations in BRCA1 and BRCA2 genes who already have an elevated breast cancer risk. To assess possible relationships between diet quality—reflected by the Alternate Healthy Eating Index (AHEI), the Diet Quality Index-Revised (DQI-R), the alternate Mediterranean Diet Index (aMED), and the Canadian Healthy Eating Index (CHEI)—and BRCA-associated breast cancer risk, a case-control study was carried out within a cohort of 80 French-Canadian families involving 89 carriers of BRCA genes affected by breast cancer, 48 nonaffected carriers, and 46 nonaffected noncarriers. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in unconditional logistic regression models. After adjustment for age, physical activity, and total energy intake, we did not detect any association between AHEI or aMED and breast cancer. However, a strong and significant inverse relationship was apparent between DQI-R and CHEI and BRCA-associated breast cancer risk. ORs comparing the highest and lowest tertiles of diet quality scores were 0.35 (95%CI 0.12 to 1.02; P_trend = 0.034) for DQI-R and 0.18 (95%CI 0.05 to 0.68; P_trend = 0.006) for CHEI, respectively. These inverse associations were not the result of a link with any specific component of the diet quality indexes. Findings from this study suggest that dietary guidelines reflected by DQI-R and CHEI may constitute preventive strategies for reducing BRCA-associated breast cancer risk.

The PRECISE Pilot Trial: Investigating the Relation among Selenium, Homocysteine, and Folate in an Elderly UK Population. Bram Bekaert,¹ Matthew Lewis Cooper,¹ Fiona Green,¹ John Scott Jr,² and Margaret P. Rayman.^{1 1}School of Biomedical and Molecular Sciences, University of Surrey, Guildford, UK; ²School of Biochemistry and Immunology, Trinity College, Dublin, Ireland.

Colorectal cancer risk is associated with folate status (1), whereas selenium was shown to modify the risk of several cancers (2). Both play an important role in 1-carbon metabolism that involves the remethylation of homocysteine to methionine, which in turn is an important modifier of DNA methylation in neoplasia (3). We investigated the interaction among the 3 dietary factors in 501 healthy UK volunteers aged 60–74 y to determine the effect of selenium (Se) supplementation on folate and homocysteine (tHcy) concentrations. Volunteers were randomly assigned to receive placebo or 100, 200, or 300 µg/d of Se-enriched yeast for 6 mo in a double-blind, placebo-controlled trial. There was no significant effect of Se supplementation on the change in tHcy in any treatment groups in accordance with the findings of Venn et al. (4). At 6 mo, folate levels remained inversely correlated with tHcy in all treatment groups. Baseline folate concentration was significantly different between men and women (11.44 vs. 13.35 nmol/L, respectively, P = 0.013), and men had higher tHcy concentrations than women (mean ± SD, 11.59 ± 4.75 vs. 10.56 ± 4.68 µmol/L, P = 0.0002). Baseline plasma tHcy correlated inversely with both plasma folate and plasma Se in men, with folate accounting for 2.6% and Se for 1.8% of the variance. The inverse correlation of baseline Se and homocysteine in this elderly population agrees with results of other human studies (4–6), although the magnitude of the effect explained by Se when folate is taken into account was much lower and was seen only in men. Protein intake was inversely correlated with tHcy (7) and could explain the inverse relation between Se and tHcy at baseline. Dietary protein intake of the population will be investigated because high-protein foods such as meat and poultry are the major sources of Se in the UK. [Research supported by Cancer Research UK.]

6. Van Guelpen B, Hultdin J, Johansson I, Hallmans G, Stenling R, Riboli E, Winkvist A, Palmqvist R. Low folate levels may protect against colorectal cancer. Gut. 2006;55:1461–6.[Abstract/Free Full Text]

7. Clark LC, Combs GF Jr, Turnbull BW, Slate EH, Chalker DK, Chow J, Davis LS, Glover RA, Graham GF, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA. 1996;276:1957–63.[Abstract/Free Full Text]

8. Uthus EO, Ross SA, Davis CD. Differential effects of dietary selenium (Se) and folate on methyl metabolism in liver and colon of rats. Biol Trace Elem Res 2006;109:201–14.[Medline]

9. Venn BJ, Grant AM, Thomson CD, Green TJ. Selenium supplements do not increase plasma total homocysteine concentrations in men and women. J Nutr. 2003;133:418–20.[Abstract/Free Full Text]

10. Gonzalez S, Huerta JM, Alvarez-Uria J, Fernandez S, Patterson AM, Lasheras C. Serum selenium is associated with plasma homocysteine concentrations in elderly humans. J Nutr. 2004;134:1736–40.[Abstract/Free Full Text]

11. Klapcinska B, Poprzecki S, Danch A, Sobczak A, Kempa K. Selenium levels in blood of upper Silesian population: evidence of suboptimal selenium status in a significant percentage of the population. Biol Trace Elem Res. 2005;108:1–15.[Medline]

12. Stolzenberg-Solomon RZ, Miller ER 3rd, Maguire MG, Selhub J, Appel LJ. Association of dietary protein intake and coffee consumption with serunm homocysteine concentrations in an older population. Am J Clin Nutr. 1999;69:467–75.[Abstract/Free Full Text]

	Calorie Restriction

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Glucose Restriction Prevents Epigenetic Changes Associated with Carcinogenesis. Joel B. Berletch, Liang Liu, Lucy G. Andrews, and Trygve O. Tollefsbol. Department of Biology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL.

It is well documented that calorie restriction (CR) is an effective experimental means for decreasing cancer risk in various model organisms. It is unlikely, however, that most humans would be willing to maintain a 30–50% reduction in calorie intake for the bulk of their adult life span, even if it means reduced cancer risk. A better option is to identify critical CR mimetics, agents that can induce the same beneficial effects as CR but without dieting, that can be used in dietary intervention. Dietary glucose may be the key factor determining the beneficial effects of CR in reducing the risk of cancer because reduced glucose metabolism is thought to have health- and longevity-promoting effects without actually decreasing food intake in rats. In fact, our preliminary studies indicated for the first time that glucose-restricted human fibroblasts undergo senescence at a slower rate than cells receiving normal levels of glucose. Early studies indicated that CR can change the level of gene products that are involved in epigenetic modifications of important cellular processes such as acetylation of histone proteins. DNA methylation is another important epigenetic process that plays a critical role in gene regulation. Genome-wide hypomethylation occurs in aging cells and is associated with cancer. We have found that the induction of the DNA methyltransferase 1 gene, which is associated with cancer, is markedly decreased in glucose-restricted MCF-7 breast cancer cells. Taken together, these studies indicate that dietary glucose restriction may have important cancer-preventive implications.

The Effect of Intermittent Versus Chronic Energy Restriction on Breast Cancer Risk Biomarkers in Premenopausal Women: A Randomized Pilot Trial. Michelle Harvie,¹ Mary Chapman,¹ Jack Cuzick,² Alan Flyvbjerg,³ Penny Hopwood,⁴ Susan Jebb,⁵ Gaenor Parfitt,⁶ and Anthony Howell.^{4 1}School of Medicine, Withington Hospital, The University of Manchester, Manchester, UK; ²CRUK Department of Epidemiology and Statistics, Wolfson Institute, London, UK; ³Medical Research Laboratories, Aarhus University, Denmark; ⁴CRUK Department of Medical Oncology, Christie Hospital, Manchester, UK; ⁵MRC Human Nutrition Research Group, Cambridge, UK; ⁶Scholl School of Sport and Health Science, University of Exeter, UK.

Postmenopausal breast cancer risk increases 2-fold in women who gain significant amounts of weight (1), and there is evidence that energy restriction may reduce risk (2). Animal studies indicate that intermittent energy restriction (IER) reduces risk and may be superior to continuous energy restriction (CER) (3). We showed that chronic energy restriction reduces biomarkers of breast cancer in women at risk but is hard to maintain. We hypothesize that IER may be superior to CER in reducing biomarkers of breast cancer risk and may also be more acceptable to women. We are undertaking a 6-mo randomized trial to compare the 2 approaches in 104 premenopausal women aged 30–45 y, at high risk for breast cancer because of family history and adult weight gain of >7 kg. Women will be randomly assigned to either CER (75% estimated energy requirements: 1500 kcal 7 d/wk) or IER (75% estimated energy requirements: 650 kcal for 2 d and 1800 kcal 5 d/wk) over 6 mo. Study endpoints will be measures of insulin sensitivity (homeostasis model assessment, sex hormone binding globulin, testosterone), potential breast cancer growth factors (insulin-like growth factor axis, leptin, adiponectin), inflammatory markers (C-reactive protein, sialic acid), oxidative stress marker (urinary F2 isoprostane), weight and body composition (waist-hip circumference, fat-free and total fat mass). The relative acceptability of IER and CER will be assessed using quality of life questionnaire (RAND SF-36) and scales of behavior change and adherence. The relative efficacy and acceptance of IER and CER will inform future weight loss programs to prevent breast cancer. Currently 14 women have been recruited to the study (5 CER and 9 IER). This pilot study will be completed by December 2007.

13. Huang Z, Hankinson SE, Colditz GA, Stampfer MJ, Hunter DJ, Manson JE, Hennekens CH, Rosner B, Speizer FE, Willett WC. Dual effects of weight and weight gain on breast cancer risk. JAMA. 1997;278:1407–11.[Abstract/Free Full Text]

14. Harvie M, Howell A, Vierkant RA, Kumar N, Cerhan JR, Kelemen LE, Folsom AR, Sellers TA. Association of gain and loss of weight before and after menopause with risk of postmenopausal breast cancer in the Iowa women's health study. Cancer Epidemiol Biomarkers Prev. 2005;14:656–61.[Abstract/Free Full Text]

15. Cleary MP, Jacobson MK, Phillips FC, Getzin SC, Grande JP, Maihle NJ. Weight-cycling decreases incidence and increases latency of mammary tumors to a greater extent than does chronic caloric restriction in mouse mammary tumor virus- transforming growth factor- female mice. Cancer Epidemiol Biomarkers Prev. 2002;11:836–43.[Abstract/Free Full Text]

	Obesity

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Dietary Fat Predicts Biochemical Failure Only among Prostate Cancer Patients with Lower Gleason Scores. Yuko Yamamura,¹ Patricia Troncoso,² and Sara S. Strom.¹ Departments of ¹Epidemiology and ²Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX.

We demonstrated that obesity at diagnosis (BMI > 30) is a predictor of biochemical failure, measured by rising prostate-specific antigen, in prostate cancer (PCa) patients treated with radical prostatectomy. Given that Gleason score is the best predictor of biochemical failure, we compared patients with less aggressive PCa [LAPCa; Gleason score < 7(3+4)] vs. more aggressive PCa [MAPCa; Gleason score > 7(4+3)] with respect to factors associated with obesity, such as dietary fat intake and weight gain, along with clinicopathologic and demographic variables. We hypothesize that risk of biochemical failure in LAPCa versus MAPCa is modulated by different factors. In a well-characterized cohort of 390 Caucasian men with PCa treated initially with only radical prostatectomy, we used multivariable Cox proportional hazards models to estimate risk of progression. As expected, in both groups pathologic stage and surgical margin involvement remained independent predictors of biochemical failure. Of 213 patients with LAPCa, energy-adjusted high-fat intake [hazard ratio (HR) = 3.59, P = 0.04] was a significant independent predictor of biochemical failure; however, no association was found with weight change. In MAPCa patients, annual weight change between age 25 and diagnosis (continuous) but not dietary fat intake was associated with increased risk of biochemical failure (HR = 1.82, P = 0.03). These data suggest that different combinations of clinicopathological and environmental factors, such as diet and weight gain, play different roles in LAPCa versus MAPCa in predicting biochemical failure after radical prostatectomy. To further our understanding of these findings, we plan to identify and explore possible mechanisms underlying our results. [Supported by NCI CA84964, CA90270, NIEHS ES07784.]

	Survivorship

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The Effect of a Lifestyle Intervention on Body Weight, Psychological Health Status, and Risk Factors Associated with Disease Recurrence in Women Recovering from Breast Cancer Treatment: Study Protocol and Interim Findings. Emma Scott,¹ Amanda Daley,¹ Nicola Woodroofe,² Robert Coleman,³ Hilary Powers,⁴ Nanette Mutrie,¹ Vanessa Siddall,¹ Helen Crank,¹ and John M. Saxton.^{1 1}Centre for Sport and Exercise Science, ²Division of Genomic Medicine, ³Cancer Research Centre, and ⁴Human Nutrition Unit, Sheffield Hallam University, Sheffield, UK.

A large proportion of women gain weight after breast cancer diagnosis, and there is evidence that heavier women have an increased risk of disease recurrence and death compared with normal-weight women. After diagnosis, women can also experience high levels of emotional distress, which might affect stress hormone levels and impair immune function, thereby affecting cancer outcome. The aims of this research are to investigate the effects of a lifestyle intervention on body weight and psychological well-being in postmenopausal women recovering from breast cancer treatment and to determine the relation between the changes in these variables and circulating biomarkers associated with disease recurrence and survival. Patients are randomly assigned to 2 groups: a 6-mo lifestyle intervention or a normal-care control group. The lifestyle intervention combines dietary energy restriction and nutrition education with supervised aerobic exercise training. It was hypothesized that the lifestyle intervention would evoke a reduction in body weight and an improvement in psychological well-being. Furthermore, a decrease in body weight is expected to be associated with reductions in the circulating levels of estrogens and inflammatory mediators. Associations among changes in psychological well-being, stress hormone levels, and immune function are also being studied. Body weight and body composition, anthropometric measures, and psychological well-being are assessed at the start, midpoint, and end of the study, whereas circulating biomarkers are measured pre- and postintervention. Preliminary 12-wk findings for 8 women show greater reductions in body weight for the intervention versus control group (–1.58 ± 3.00 kg vs. –0.83 ± 2.11 kg, mean ± SD, respectively). Compliance to the intervention has been excellent, with women attending at least 87.5% of the dietary counseling and exercise sessions.

Strategies for Health: Follow-Up Care Practices and Beliefs among African American Breast Cancer Survivors. Renee Royak-Schaler,¹ Susan Racine Passmore,¹ Katherine Tkaczuk,¹ Joseph Finkelstein,¹ Jimmie Drummond,¹ Peggy D. Nicholson,² Alva P. Hutchison,³ and Shahinaz M. Gadalla.^{1 1}University of Maryland School of Medicine, Baltimore, MD; ²Sisters Network Baltimore Chapter, Baltimore, MD; ³American Cancer Society, South Atlantic Division Inc., Atlanta, GA.

There is a gap in scientific understanding regarding the psychosocial and health concerns and follow-up care practices of African American breast cancer survivors after primary treatment is complete. Evidence-based guidelines for survivorship care are not readily available to clinicians (1). To address this gap we investigated 1) patient perceptions of patient-physician communication and follow-up care practices and 2) strategies for developing follow-up care plans that address the special needs of African American breast cancer survivors. Four focus groups were conducted with 39 African American women, mean age 55 y, to investigate the following themes: patient-physician communication and decisionmaking about follow-up care; other sources of information used in developing follow-up care plans (e.g., Internet or print material); and preferences and avenues for information delivery to survivors. Group members also completed survey questionnaires designed to support the focus group discussions. Participants identified no clear plan valued over others as a means to reduce their risk of breast cancer recurrence. They reported receiving minimal information from their physicians about state-of-the-art strategies for reducing their risk of recurrence, for example, weight management by low-fat diet and physical activity. Although 87% said they were satisfied with the information received from medical professionals, they acknowledged that it did not specifically target the health concerns of African American women (e.g., healthy eating in the context of traditional high-fat diets). African American survivors had a critical need for guidance from healthcare professionals in developing feasible plans of self-care that target the context of their lifestyles for improving breast cancer outcomes. [Supported by grants from the Lance Armstrong Foundation and the Susan G. Komen Breast Cancer Foundation, Maryland Affiliate.]

16. Institute of Medicine. From cancer patient to cancer survivor, lost in transition. Washington, DC: National Academies Press; 2006.

	Colorectal Cancer

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Novel Combinations of Chemopreventive Agents Demonstrate a Synergistic Effect against Cell Proliferation in Colon Cancer Cell Lines. Naveen Kanthamneni and Sunil Prabhu. Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.

We studied synergistic chemopreventive effects of a dual combination of agents (aspirin with folic acid; aspirin with calcium) against cell proliferation in colon cancer cell lines, SW-480 and HT-29. These cells were grown according to previously established protocols. On reaching 75–80% confluence, 5 x 10³ cells were transferred into each well of a 96-well microplate to which different concentrations of drug alone (aspirin, folic acid, or calcium) or its dual combinations (aspirin with folic acid or aspirin with calcium) were added. The plates were then incubated for 24–48 h. Using an XTT assay method, we measured cell viability with a microplate reader at wavelength 450 nm. With the SW-480 cell line, the combination of aspirin (5 mmol/L) with folic acid (0.5 mmol/L) demonstrated a 22% decrease in cell viability when compared with the individual drugs at the same concentrations after 24 h. With the HT-29 cell line, a similar phenomenon was observed with a 20% decrease in cell viability at low concentrations of aspirin (5 mmol/L) and folic acid (0.5 mmol/L). With aspirin and calcium combinations tested on SW-480, no synergistic effects on cell viability during the first 24 h of incubation were observed. However, after 48 h a dose-dependent relationship was apparent as progressively increasing amounts of aspirin (5, 10, and 15 mol/L) demonstrated a 28–62% decrease in cell viability, respectively, when mixed with calcium (15 mmol/L). Aspirin, folic acid, and calcium are currently being investigated individually as chemopreventive agents in the fight against colon cancer. Combining these agents and demonstrating significant synergism provide promising data for developing future chemopreventive strategies in colon cancer. These results will also be useful for investigating combined chemoprevention of colon cancer using a nanotechnology-based therapeutic system.

Combination of Fish Oil and Pectin Suppressed ß-Catenin Nuclear Translocation, an Important Molecular Event in Colon Carcinogenesis. J. Vanamala,¹ A. Glagolenko,² R.J. Carroll,³ M.E. Murphy,⁴ S.S. Taddeo,¹ R.S. Chapkin,¹ N.D. Turner,¹ and J.R. Lupton.^{1 1}Faculty of Nutrition, ²Department of Nuclear Engineering, and ³Department of Statistics, Texas A&M University, College Station, TX.

We previously showed that a fish oil and pectin diet enhances spontaneous colonocyte apoptosis when compared with a corn oil and cellulose diet. We also demonstrated that the fish oil and pectin diet suppresses prostaglandin E synthase-2 (prostaglandin pathway), ß-catenin (Wnt pathway), and peroxisome proliferator-activated receptor ß/ (nuclear receptor) levels in rats exposed to radiation and chemical carcinogen. We hypothesized that a diet containing fish oil and pectin may also suppress ß-catenin nuclear translocation. To test this hypothesis, 40 rats were provided diets containing either fish oil and pectin or corn oil and cellulose. Rats were exposed to either 0 or 1 Gy, 1 GeV/nucleon Fe-ion irradiation 3 wk after starting the diets. All rats were injected with azoxymethane (15 mg/kg body wt), a colon-specific carcinogen, 10 and 17 d after irradiation. Thirty-one weeks after the last azoxymethane injection, rats were killed, and the proportion of cells with nuclear ß-catenin staining was determined within 26 crypt columns/rat by immunohistochemistry. The fish oil–pectin diet suppressed (P 0.01) nuclear translocation of ß-catenin compared with the corn oil–cellulose diet. However, irradiation did not influence the nuclear translocation of ß-catenin. These data suggest that enhanced apoptosis observed in rats consuming a fish oil–pectin diet could be attributed to both the suppressed levels of ß-catenin in the colonocytes and the attenuated nuclear translocation. Thus, this study provides evidence that the mechanism of action by which the fish oil and pectin diet induces apoptosis involves the prostaglandin and Wnt signaling pathways. [Funded by AICR 05B094, NIH CA61750, CA82907, NSBRI NASA NCC 9–58, CA59034, and NIEHS P30-ES09106.]

Dietary Flax Products Suppress the Formation of Azoxymethane-Induced Colon Cancer in Fisher 344 Male Rats. Darlene S. Williams,¹ Martha Verghese,¹ Judith Boateng,¹ Lloyd T. Walker,¹ Louis Shackelford,¹ Janak Khatiwada,¹ David Asiamah,¹ and Chandramohan B. Chawan.^{2 1}Nutrition and Carcinogenesis Laboratory, Department of Food and Animal Sciences, Alabama A&M University, Normal, AL; ²USDA/AMS/PY, Washington, DC.

Flax is a rich source of bioactive components such as PUFA (fat); dietary fiber; -linolenic acid (ALA), the essential (n-3) fatty acid; and lignans, which are phytoestrogens and antioxidants. This study was designed to determine the anticarcinogenic effect of flaxseed meal (FSM; 10% and 20%) and flaxseed oil (FSO; 7% and 14%) on azoxymethane (AOM)-induced colon cancer in Fisher 344 male rats at the initiation (I), promotion (P), and initiation plus promotion (I + P) stages of carcinogenesis. After an acclimatization period of 1 wk, 14 groups of Fisher 344 male weanling rats (15 per group) were created; 2 control groups were fed AIN 93G diet and AIN 93G + 14% soybean oil. The remaining 12 groups were assigned to 10% and 20% FSM (I, P, and I+P) and 7% and 14% FSO (I, P, and I+P). All rats received 16 mg/kg body weight of AOM at ages 7 and 8 wk. At age 45 wk rats were killed by CO₂ asphyxiation. Tumor incidence and size, respectively, were significantly (P < 0.05) lower in groups fed FSM (10% and 20%) and FSO (7% and 14%) compared with controls. The ratio of tumors to tumor-bearing rats for groups fed the control diet, 10% FSM, and 20% FSM (I, P, and I+P) were 3.80; 1.60, 1.10, 1.0; and 1.66, 1.1, 1.0, respectively. In rats fed the 7% FSO, and 14% FSO (I, P, and I+P), the ratios were 1.40, 1.25, 1.42 and 1.90, 1.40, 1.42, respectively. The activity of glutathione-S-transferase (a phase II detoxification enzyme) was significantly (P < 0.05) higher in rats fed 10% and 20% FSM and 7% and 14% FSO compared with controls. The results of this study indicated that dietary flaxseed products may suppress colon tumors, particularly at the promotion stage, and may therefore be a potential chemopreventive agent.

Antitumor and Cytotoxic Properties of Red Kidney Beans (Phaseolus vulgaris): An In Vitro and In Vivo Model. Judith Boateng,¹ Martha Verghese,¹ Lloyd T. Walker,¹ Louis Shackelford,¹ Janak Khatiwada,¹ Chandramohan B. Chawan,² Darlene S. Williams,¹ and David Asiamah.^{1 1}Nutrition and Carcinogenesis Laboratory, Department of Food and Animal Sciences, Alabama A&M University, Normal, AL; ²USDA/AMS/PY, Washington, DC.

Dry beans are a good source of fiber and phytochemicals such as flavonoids and phenolic compounds and may be responsible for preventing the onset of chronic diseases. We examined the antitumor effects of red kidney beans (RKB) on azoxymethane (AOM)-induced colon tumors [aberrant crypt foci (ACF) and endpoint tumor model (EPTM)] and the cytotoxic effects of aqueous RKB extracts on colon cancer cell line CaCo2. For the ACF study, 12 Fisher 344 male rats were fed AIN-93G control diet (C; n = 6) and 20% RKB (n = 6) for 13 wk. For EPTM, 2 groups of rats (n = 14) were fed C and 20% RKB. All rats received 2 s/c injections of AOM at ages 7 and 8 wk (16 mg/kg body weight in saline). At 17 wk (ACF) and 45 wk of age (EPTM), rats were killed by CO₂ asphyxiation. For the lactate dehydrogenase assay, cells were maintained in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum. Cells were seeded and incubated at 37°C, 7% CO₂, until a monolayer developed. Cells were incubated (24 and 48 h) with selected concentrations (0.5–25 mol/L) of RKB extract. The total number of ACF was 183 and 51 for C and 20% RKB, respectively. Activity of glutathione-S-transferase, a phase II detoxifying enzyme, was 20.38 and 48.84 in rats fed C and 20% RKB, respectively. Tumor size per rat (mm) and tumors per tumor-bearing rat for C and 20% RKB were 13.13 and 1.16, and 3.39 and 0.78, respectively. Lactate dehydrogenase activity in CaCo2 cells after 24 and 48 h incubation with RKB extracts ranged from 13.8% to 62.8% and 23.5% to 84.1%, respectively. RKB significantly (P < 0.05) reduced the incidence of chemically induced colon tumors, and RKB extracts exhibited cytotoxic effects on colon cancer cell lines and thus may play a significant role in chemoprevention.

	Breast Cancer

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In Utero and Prepubertal Exposures to Indole-3-Carbinol Have Opposing Effects on Mammary Tumorigenesis in Rats. Leena Hilakivi-Clarke,¹ Bin Yu,¹ Moira B. Hilsher,¹ and William Helferich.^{1,2 1}LCCC, Department of Oncology, Georgetown University, Washington, DC; and ²Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL.

The age when an individual is exposed to various hormones or dietary compounds has a profound effect on how these compounds alter susceptibility to developing breast cancer. We used an animal model to investigate whether a maternal dietary exposure during pregnancy or prepubertally during postnatal weeks 2 and 3 to indole-3-carbinol (I3C), present in cruciferous vegetables, affects DMBA-induced mammary tumorigenesis in rats. The dose of I3C used, 2000 ppm, was previously used in several cancer prevention studies. Results indicated that offspring of dams fed I3C during pregnancy developed more mammary tumors than control rats fed AIN93 diet (increase in mammary tumor incidence P < 0.06, tumor multiplicity P < 0.01), whereas prepubertal exposure was highly protective (reduction in incidence P < 0.001). Changes were also seen in circulating estradiol (but not progesterone) concentrations and mammary gland morphology, but these changes were not consistent with the observed differences in mammary tumorigenesis. Furthermore, as previously reported in animals exposed to I3C in adulthood, levels of estrogen receptor and aryl hydrocarbon receptor were reduced, and apoptosis was increased in the mammary glands of the rats exposed to I3C early in life; however, these changes were not consistent with the changes in mammary tumorigenesis. Maternal exposure to I3C significantly down-regulated tumor suppressor gene APC (P < 0.03) and, in agreement with this result, increased nuclear localization of ß-catenin (P < 0.04) and TCF4 expression (P < 0.05) in the offspring's mammary gland, suggesting that activation of the Wnt signaling pathway in the I3C offspring may explain why in utero exposure increased later susceptibility to developing mammary tumors. We are now investigating whether prepubertal I3C exposure down-regulated the Wnt pathway.

Phytoestrogens and Breast Cancer Progression: A Prospective Study. Louiza Velentzis, Ann Titcomb, Anna McConnochie, Annie Perkins, Paula Stavrinos, Patricia Leathem, and Tony Leathem. Department of Surgery, University College, London, UK.

Phytoestrogens are chemicals commonly found in foods of plant origin, and they can mimic the female sex hormone estrogen. The effects can be dramatic, such as the infertility in sheep from eating clover plants. Breast cancers are generally driven by estrogen, and effective treatments are based on removing or blocking this hormone. Can phytoestrogens in the diet or in dietary supplements act like estrogen and stimulate breast cancer growth, or can they block estrogen receptors like treatment with tamoxifen and slow down cancer growth? We are studying phytoestrogen levels in women with breast cancer to help answer this. In our study design, 2000 women in the United Kingdom with invasive breast cancer are recruited at diagnosis at 40 regional cancer centers, and their diet, phytoestrogen excretion, and clinical progress are monitored annually for 5 y. The aim is to seek any association between phytoestrogen intake and time to death or time to first recurrence. Urine is collected to measure 9 phytoestrogens, and blood is collected to measure biomarkers and polymorphisms. Each year patients complete dietary questionnaires—the food frequency and 7-d food diary questionnaires developed for the European Prospective Investigation into Cancer and Nutrition—plus lifestyle, quality-of-life, and complementary therapies questionnaires. Phytoestrogen intake is measured from 24-h and random urine samples with mass spectrometry and an immune assay. The phytoestrogens currently measured are enterodiol, enterolactone, secoisolariciresinol, equol, daidzein, genistein, naringenin, glycitein, and O-desmethylangolensin. We have recruited 950 patients so far.

Alcohol Drinking, Acetaldehyde Accumulation, and Oxidative Stress Promotion in Mammary Tissue: Their Potential Role in Ethanol-Induced Breast Cancer. Gerardo D. Castro, Aurora M.A. Delgado de Layño, Silvia L. Fanelli, Maria E. Maciel, Maria I. Díaz Gómez, and Jose A. Castro. Centro de Investigaciones Toxicológicas, CITEFA-CONICET, Buenos Aires, Argentina.

Previous studies from our laboratory reported the presence of several pathways of metabolic activation of ethanol to acetaldehyde and hydroxyl free radicals as well as the promotion of oxidative stress. In the present studies, we tested the possibility that after alcohol drinking, acetaldehyde accumulates in mammary tissue to reach concentrations higher than in blood. Three different doses of alcohol (low, medium, and high) were tested, and acetaldehyde concentrations in breast, liver, and blood were measured at times ranging from 1 to 24 h. We also measured alcohol dehydrogenase, aldehyde dehydrogenase, and CYP2E1-mediated p-nitrophenol hydroxylase activities. Oxidative stress induced hydroperoxide formation as determined with the xylenol orange procedure; -tocopherol and glutathione content determinations are in course for each dose at different times of exposure. Hydroperoxide levels were increased at 6 h for the highest dose tested. Acetaldehyde concentrations at the 3 alcohol doses tested were always higher than those in blood. Peak concentrations of acetaldehyde in liver, although higher than those in breast, appeared to decrease to blood levels following a similar time sequence. Limited activities of alcohol dehydrogenase and aldehyde dehydrogenase in mammary tissue were observed. The microsomal CYP2E1-mediated p-nitrophenol hydroxylase in mammary tissue was several times smaller than that in liver. In summary, results suggest that the mutagen acetaldehyde, either formed in situ or in small amounts continuously arriving via blood, tends to accumulate in mammary tissue as a consequence of a limited capacity of mammary tissue for detoxification. [Supported by grants from ANPCyT-SECyT (PICT 05–6045) and from UNSAM (PIB S05/03), Argentina.]

	Prostate Cancer

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Toxic Effects of Ethanol Administration on Rat Prostate and the Controversies about the Role of Alcohol Drinking in Prostate Cancer Induction. Maria I. Díaz Gómez, Carmen Rodríguez de Castro, Silvia L. Fanelli, Aurora M.A. Delgado de Layño, Martin H. Costantini, Jose A. Castro, and Gerardo D. Castro. Centro de Investigaciones Toxicológicas, CITEFA-CONICET, Buenos Aires, Argentina.

The potential correlation between alcohol consumption and prostate cancer led to controversial findings. To shed some light on the problem, it is critical to learn whether interaction with ethanol leads to alterations in prostate tissue similar to those attributed to alcohol-promoted liver cell injury or cancer. To that end, previous studies from our laboratory showed that cytosolic and microsomal fractions from the rat ventral prostate are able to biotransform ethanol to acetaldehyde and 1-hydroxyethyl free radicals. Present biochemical and ultrastructural studies were performed in Sprague Dawley male rats fed with a nutritionally adequate liquid diet (1) containing ethanol for 28 d and compared with adequately pair-fed controls. Prostate microsomal fractions were found to exhibit a CYP2E1-mediated p-nitrophenol hydroxylase metabolism, and that activity was induced by repetitive ethanol drinking. Cytosolic activation of acetaldehyde led to acetyl radical, as detected by spin trapping with PBN and GC-MS analysis. Low activities of alcohol dehydrogenase and aldehyde dehydrogenase were observed in prostate tissue, and acetaldehyde accumulation occurred after ethanol administration. An increased oxidizability of prostatic lipids was detected by the t-butylhydroperoxide–promoted chemiluminescence emission test and by the increased levels of lipid hydroperoxides (shown by the xylenol orange method). Ultrastructural alterations in the epithelial cells were observed. They consisted of marked condensation of chromatin around the perinuclear membrane, moderate dilatation of their endoplasmic reticulum, and some epithelial cells undergoing apoptosis. In summary, alcohol drinking leads to the formation of mutagenic acetaldehyde and to tumor-promoting oxidative stress. However, it exerts direct and indirect proapoptotic effects in the prostate epithelial cells. The balance between these 2 actions might explain, at least in part, many controversial results observed in epidemiologic studies. [Supported by grants from CONICET (PIP 02323) and the University of San Martín (PIDA UF013), Argentina.]

17. Lieber CS, De Carli LM. Liquid diet technique of ethanol administration: 1989 update. Alcohol Alcohol. 1989;24:197–211.[Abstract/Free Full Text]

Insulin-like Growth Factor-I Stimulates Lipid Metabolism in Prostate Cancer Cells. Shihua Wang,¹ Lei Shen,² Valerie L. DeGroff,¹ and Steven K. Clinton.^{1 1}Division of Hematology and Oncology, Department of Internal Medicine, and ²Division of Epidemiology and Biometrics, The Ohio State University College of Medicine and Public Health, Columbus, OH.

The hormone and growth factor insulin-like growth factor-I (IGF-I) has emerged as a potential promoter of prostate carcinogenesis. IGF-I exhibits pleiotropic activities including the stimulation of prostate tumor cell proliferation, survival, and resistance to apoptosis. To better understand how IGF-I modulates these processes and to identify novel biomarkers for future rodent and human studies, we examined global gene expression profiles (Affymetrix GeneChip - Rat Genome U34A) in AT6.3 rat prostate cancer cells after treatment for 24 h in serum-free medium (SFM), SFM with 10% fetal bovine serum, or SFM with 50 µg/L IGF-I (n = 6 each). The microarray data were analyzed by dChip and SAM software. Lipids are important structural components for cells and serve as signaling mediators associated with many critical cellular functions. This study reports our findings regarding IGF-I–regulated pathways involving lipid metabolism. Our results showed that the majority of genes involved in de novo cholesterol synthesis from mevalonate were significantly up-regulated by IGF-I and frequently down-regulated by serum. These genes included HMG-CoA synthase 1 (q = 1.2), HMG CoA reductase (q = 1.1), mevalonate kinase (q = 0), farnesyl-PP synthase (q = 0), farnesyl diphosphate farnesyl transferase 1 (q = 0.4), squalene epoxidase (q = 0), and 7-dehydrocholesterol reductase (q = 0). In addition, several enzymes related to fatty acid metabolism, such as acyl-CoA synthetase 3 (q = 0.9), and stearoyl CoA desaturase 1 (q = 0), were also up-regulated by IGF-I. These microarray observations were validated by subsequent RT-PCR and Western blots. Mevinolin (lovastatin), an inhibitor of HMG-CoA reductase, inhibited IGF-I–stimulated AT6.3 cell proliferation with an IC₅₀ <10 µmol/L. We previously observed that IGF-I increased anaerobic glycolysis, which is known as the Warburg effect, and that glucose uptake and metabolism were essential for IGF-I stimulation of prostate cancer cell proliferation and survival. Our findings suggest that IGF-I stimulation of lipid and glucose metabolism may be a critical mechanism whereby this hormone may contribute to prostate carcinogenesis. [Supported by AICR #05A131.]

Effects of Dietary Tomato, Broccoli, or Lycopene or Androgen Ablation on Proliferation and Apoptosis Biomarkers in Dunning R3327-H Prostate Adenocarcinomas. Kirstie Canene-Adams,¹ Shihua Wang,² Steven K. Clinton,² and John W. Erdman, Jr.^{1 1}Division of Nutritional Sciences, University of Illinois, Urbana, IL; and ²Department of Internal Medicine, Division of Hematology and Medical Oncology, The Ohio State University, Columbus, OH.

The androgen-dependent Dunning R3327-H transplantable rat prostate adenocarcinoma model was used to examine dietary interventions on tumor growth. Diets were initiated 1 mo before tumor inoculation (20 mg fresh tumor in Matrigel) and continued for 18 wk in 6 cohorts of animals. Diets containing 10% tomato (P < 0.05), 10% broccoli (P < 0.01), or 10% tomato plus 10% broccoli (P < 0.001) or castration (P < 0.001) significantly reduced tumor weights compared with control tumors. Reduction in tumor weights were 34% in rats fed 10% tomato, 40% in rats fed 10% broccoli, 52% in rats fed broccoli with tomato, and 62% in castrated rats. Finasteride and the low- and high-lycopene diets (0.025 or 0.25 g/kg diet, respectively) did not significantly reduce tumor weights. Tumor tissue proliferation and apoptosis rates were estimated using proliferating cell nuclear antigen (PCNA) staining and ApopTag peroxidase in situ kits, respectively. Tumor sizes were substantially larger in 3 of the 6 cohorts. In these 3 cohorts lycopene, tomato, or broccoli (P = 0.01) caused a decrease in PCNA staining compared with controls. PCNA staining was most suppressed with the tomato plus broccoli diet (P = 0.001), suggesting that one of the mechanisms behind the >50% decreased tumor growth from consuming both tomato with broccoli was a decreased rate of proliferation. Castration, tomato, broccoli, and the combination of tomato and broccoli all showed significant increases in apoptosis rates. We can conclude that diets containing tomato and broccoli significantly decrease prostate tumor growth by slowing the rate of proliferation. [Supported by AICR 01B061/ IFAFS 00–52101–9695.]

Se-Methylselenocysteine, but Not Lycopene or -Tocopherol, Decreases Growth of Transplantable Dunning R3327-H Prostate Tumors. Brian L. Lindshield, Nikki A. Ford, Kirstie Canene-Adams, Matthew A. Wallig, and John W. Erdman, Jr. Division of Nutritional Sciences, University of Illinois, Urbana, IL.

Lycopene, selenium, and vitamin E are 3 commonly consumed and supplemented micronutrients that have been associated with a decreased risk of prostate cancer. The potential protective effect of selenium (selenomethionine) and vitamin E (all-rac--tocopherol acetate) against prostate cancer is currently being evaluated in the Selenium and Vitamin E Cancer Prevention Trial (SELECT). However, there are few data on the effectiveness of these compounds or lycopene in vivo. Therefore, we evaluated the effects of lycopene, selenium, and vitamin E alone and in combination on the growth of Dunning R3327-H tumors. The dietary sources of the supplemented compounds were selenium as Se-methylselenocysteine (2 mg/kg diet), vitamin E as -tocopherol (200 mg/kg diet), and lycopene (0.025 mg/kg diet) in the form of water-soluble beadlets. Isoenergetic and isonitrogenous AIN-93G–based diets were prefed for 4–7 wk to Copenhagen rats before tumor implantation by subcutaneous injection. Tumors were allowed to grow for 18 wk before study termination. Multiple linear regression analysis showed that Se-methylselenocysteine consumption resulted in highly significant decreases in tumor weight and tumor areas. Lycopene intake resulted in minor, nonsignificant reductions in tumor weights and tumor areas, whereas -tocopherol consumption led to nonsignificant increases in tumor weights and tumor areas. There were no significant interactions among nutrient combinations. In conclusion, among the 3 micronutrients studied, only Se-methylselenocysteine consumption reduced growth of transplantable Dunning R3327-H prostate tumors. [Supported by AICR grant 05A021.]

	Herbs

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Black Cohosh Lacks an Estrogenic Effect in Breast. Rachel L. Ruhlen,¹ John K. Tracy,² Jenny Haubner,¹ Weizhu Zhu,¹ Wenyi Qin,¹ William R. Lamberson,³ George E. Rottinghaus,² and Edward R. Sauter.^{1 1}Department of Surgery, ²Veterinary Medical Diagnostic Laboratory, and ³Department of Animal Sciences, University of Missouri-Columbia, Columbia, MO.

The Women's Health Initiative indicates that hormone replacement therapy may increase breast cancer and cardiovascular disease. Herbal alternatives, such as black cohosh extract (BCE), reduce menopausal symptoms in clinical trials. The estrogenic properties of BCE have been a matter of controversy. The influence of BCE on estrogenic endpoints in breast has not been investigated. Triterpenes present in black cohosh are used for standardization. The study goals were to 1) determine the triterpene content of 2 commercially available black cohosh preparations, 2) confirm the efficacy of standardized BCE in relieving menopausal symptoms, and 3) determine effect of BCE on estrogenic markers in serum and nipple aspirate fluid. Two BCE preparations were analyzed by HPLC for triterpene content. Postmenopausal women took BCE for 12 wk followed by a 12-wk washout. To confirm efficacy of BCE, menopausal symptoms were assessed using the Kupperman Menopausal Index (KMI) before and during BCE treatment. To maximize recruitment, a placebo group was not included, so menopausal symptoms were evaluated again after a 12-wk washout. KMI and estrogen markers in serum and nipple aspirate fluid were measured at all times. One BCE contained trace amounts, and the other contained 2.5% of triterpenes. Menopausal symptoms are typically reduced 20–30% by placebo. Women taking 40 mg BCE containing 2.5% triterpenes twice daily for 12 wk experienced a 50% reduction in KMI, with reversion toward baseline after washout, confirming the efficacy of BCE. BCE had no effect on luteinizing hormone, follicle-stimulating hormone, estradiol, or trefoil factor in serum, and no effect on cellular morphology in nipple aspirate fluid. Triterpene content in commercially available BCE preparations varies. BCE standardized to 2.5% triterpenes effectively relieved menopausal symptoms without systemic or breast-specific estrogenic effects.

	Soy

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Radiosensitization of Prostate Cancer by Soy Isoflavones Targets Survival Pathways In Vitro and In Vivo. Gilda G. Hillman,¹ Julian J. Raffoul,¹ Sanjeer Banerjee,² and Fazlul H. Sarkar.² Departments of ¹Radiation Oncology and ²Pathology, Barbara Ann Karmanos Cancer Institute at Wayne State University School of Medicine, Detroit, MI.

Epidemiologic studies showed that men who consume diets rich in soy isoflavones have lower incidence of prostate cancer. We previously demonstrated that genistein, the major bioactive component of soy isoflavones, acts as a radiosensitizer and potentiates prostate tumor cell killing by radiation in vitro and in animal orthotopic metastatic tumor models in vivo. However, when administered alone in vivo, pure genistein promoted increased lymph node metastasis. Recently, we found that a mixture of soy isoflavones, consisting of genistein, daidzein, and glycitein, was safer because it did not promote increased metastasis to lymph nodes in vivo. Furthermore, the combination of soy isoflavones with prostate tumor irradiation potentiated inhibition of primary tumor growth and metastasis, as observed with pure genistein and radiation. Soy isoflavones also increased radiation-induced tumor cell killing in vitro like pure genistein. Investigation of molecular mechanisms by which soy isoflavones potentiate radiotherapy revealed that soy isoflavones inhibited activation of nuclear factor (NF)-B, a transcription factor critically involved in cancer cell survival. Like pure genistein, soy isoflavones also abrogated radiation-induced activation of NF-B, probably driving the cells to apoptotic pathways as confirmed by induction of cleaved poly(ADP-ribose) polymerase in these cells. APE1/Ref-1, a DNA repair-redox protein implicated in tumor cell survival and radioresistance, functioned as a redox activator of transcription factors, including NF-B. We demonstrated that soy isoflavones decreased APE1/Ref-1 expression in vitro, whereas radiation up-regulated it. Pretreatment with soy isoflavones followed by radiation inhibited APE1/Ref-1 expression in correlation with the decreased activation of NF-B. The down-regulation of APE1/Ref-1 and NF-B by isoflavones in vitro could be recapitulated in vivo, supporting our hypothesis that these markers represent biological targets of isoflavones. Their down-regulation by isoflavones could potentially radiosensitize prostate tumor cells. Our studies demonstrate the safety and potential for the use of soy isoflavones to improve clinical strategies for prostate cancer radiotherapy.

Genetic Influences on Interindividual Variability in Soybean Isoflavone Absorption and Metabolism. Luisa Wakeling and Dianne Ford. Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle, UK.

Consumption of soybean isoflavones may have a number of health benefits, including reduced risk of cardiovascular disease and cancer. In unfermented plant sources, isoflavones occur as ß-glucosides. Absorption requires cleavage by intestinal ß-glucosidase enzymes lactase phlorizin hydrolase (LPH) and cytosolic ß-glucosidase (CBG). Intestinal and hepatic UDP-glucuronosyl transferases (UGTs) including UGT1A1 are involved in phase II conjugation. We hypothesize that nonsynonymous coding single nucleotide polymorphisms (SNPs) in LPH and CBG may contribute to variability in isoflavone absorption and that an insertion polymorphism [(TA)₇] in the promoter region of UGT1A1 may alter isoflavone bioavailability. The Soya Isoflavone Metabolism Study (SIMS) is designed to establish whether genotype with respect to these polymorphisms influences the absorption and metabolism of a single oral dose of isoflavones. Healthy premenopausal women (n = 100) were genotyped for polymorphisms in LPH (G666A, A1095G, A4926G), CBG (T1417A), and UGT1A1 (UGT1A1*28 A(TA)₇TAA). ß-Glucosidase activity from shed oral epithelial cells was also measured for each participant. Analysis by univariate ANOVA indicated that the interactions LPH666G x CBG1417T (P = 0.024) and LPH666G x LPH4926A (P = 0.022) were significant for oral epithelial ß-glucosidase activity, indicating a possible effect of genotype on the rate of isoflavone aglycone release in the oral cavity. Thirty volunteers selected according to genotype participated in the second stage of SIMS. A fasting blood sample was taken 3 h after soy administration, and urine was collected over 24 h. Sample analysis by RP-HPLC for isoflavones and glucuronides is in progress. Any effects of genotype on soybean isoflavone metabolism indicated in this study may guide personalized dietary advice concerning the likely health benefits of isoflavone consumption and will indicate whether studies designed to investigate effects of interindividual genetic variability on health outcomes of isoflavone consumption should include analysis of the SNPs studied herein.

	Phytochemicals

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Caffeine Activates the Multidrug Resistance Gene Leading to Increased Drug Efflux and Chemoresistance. Pellegrino G. Magro, Michael V. Mandola, Jia Shi, Zhen Hu, Brijesh Patel, Kirk Pabon, and Kathleen W. Scotto. The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ.

Caffeine is the most widely consumed psychoactive substance in the world. It elicits multiple cellular functions including the inhibition of the adenosine receptor, induction of calcium release through the ryanodine receptor, and inhibition of the ATM/ATR protein kinases. Surprisingly, despite the seemingly pancellular cascades that caffeine induces, the effect of caffeine on gene expression has attracted little attention. We now show that caffeine, in doses equivalent to those contained in everyday consumables, is a potent transcriptional activator of the MDR1 gene. There is a dose-dependent activation of an MDR1 promoter-driven luciferase reporter construct in the presence of increasing concentrations of caffeine, concomitant with an increase in the expression of endogenous MDR1 mRNA and the MDR1 gene product, P-glycoprotein (P-gp). The overexpressed P-gp is properly translocated to the plasma membrane and is functional as evidenced by an increase in doxorubicin efflux in cells pretreated with caffeine. This increased doxorubicin efflux is correlated with an increase in cellular resistance to doxorubicin in SW620 cells. Furthermore, we show that the induction of MDR1 gene expression by caffeine is p53-independent and occurs in various cell types derived from different tissues. These findings suggest that caffeine consumption could play a role in the development of an MDR phenotype in vivo and could thus affect the outcome of chemotherapeutic regimens in the treatment of cancer.

Sulforaphane and Erucin Increase the Expression of Multidrug Resistance Protein 1 in A549 Human Lung Carcinoma Cells. Kristin E. Harris and Elizabeth H. Jeffery. Division of Nutritional Sciences, University of Illinois, Urbana, IL.

Multidrug resistance proteins (MRPs) have been termed the phase III detoxification system because they protect against xenobiotic insult by actively secreting foreign compounds from tissues such as liver and colon. However, MRP overexpression in tumors can lead to drug resistance, a major obstacle in the treatment of many cancers, including lung cancer. Isothiocyanates, such as sulforaphane (SF) and erucin (ER), which are derived from cruciferous vegetables, enhance the expression of phase II detoxification enzymes. Initial studies suggest that SF and ER may alter MRP transporter expression as well. The ability of SF and ER to modulate MRP expression is evaluated in this study. The expression of P-glycoprotein (P-gp), MRP1, and MRP2 in liver, colon, and lung cancer cell lines treated with ER or SF was analyzed by Western blotting. Sulforaphane and ER increased the level of MRP1 in HepG2 liver and A549 lung cells as well MRP2 in HepG2 and Caco-2 colon cells in a dose-dependent manner. Neither SF nor ER affected P-gp expression in any of the cell lines tested. Alterations in mRNA expression of MRP1 and MRP2 were evaluated by real-time PCR in SF-treated A549 cells. Treatment with SF led to a significant dose-dependent increase in MRP1 and MRP2 mRNA in A549 cells. These data suggest that SF and ER can increase MRP1 and MRP2 expression through a transcriptional mechanism. [Supported by USDA/IFAFS 2000–04766.].

Epigenetic Mechanisms Lead to Telomerase Inhibition in Breast Cancer Cells Treated with the Green Tea Component EGCG. Joel B. Berletch, Canhui Liu, Lucy G. Andrews, and Trygve O. Tollefsbol. Department of Biology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL.

Dietary polyphenols are common in the human diet and have chemopreventive and anticancer activities. Tea is one of the most consumed beverages worldwide, and a major constituent of green tea is the polyphenol (–)-epigallocatechin gallate (EGCG). Many studies have shown that EGCG has potent anticancer properties, but the mechanisms for these effects are unknown. Recent studies indicated that this dietary polyphenol alters the activity of DNA methyltransferase I, leading to changes in the expression of methylation-controlled cancer genes. The gene for telomerase is important in oncogenesis that is controlled through DNA methylation, and we found that EGCG down-regulates transcription of hTERT (the catalytic subunit of telomerase) in cancer cells, leading to apoptosis. The hTERT gene is paradoxically hypermethylated in cancer cells that express this gene; we found that treating MCF-7 breast cancer cells with EGCG leads to hypomethylation of the hTERT promoter, presumably through DNMT1 inhibition and apoptosis of these cells. The hypomethylation of the hTERT promoter is especially pronounced in the E2F-1 methylation-sensitive binding sites. In rare cancer cells, such as multipotent HL60 cells, the hTERT promoter is hypomethylated but telomerase positive; we have found that EGCG has no effect on hTERT regulation in these cells. These studies collectively indicate that EGCG works through epigenetic mechanisms in most telomerase-positive cancer cells to lead to the down-regulation of telomerase and apoptosis of cancer cells. Ultimately, completion of this study may lead to elucidation of the mechanisms through which dietary polyphenols mediate their chemopreventive and anticarcinogenic effects, thereby facilitating more effective uses of green tea and polyphenols in cancer prevention and therapy.

Pharmacokinetics of Curcumin as Glucuronide and Sulfate Conjugates. Shaiju K. Vareed,¹ Zora Djuric,² Bill C. Frame,³ Daniel Normolle,³ and Dean E. Brenner.^{1 1}Department of Internal Medicine, Hematology-Oncology Division, ²Department of Family Medicine, and ³Biostatistics Unit, and ⁴Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI.

Chemoprevention of cancer using natural products has the potential to be efficacious and yet nontoxic. Curcumin, the yellow pigment extracted from turmeric, has chemopreventive properties in vitro and in vivo. Curcumin has been used as a food additive and an herbal medicine for centuries because of its antioxidant, antimicrobial, and anticancer properties. We designed a clinical trial to 1) determine whether curcumin in a capsule formulation is absorbed and 2) assess the pharmacokinetics of curcumin at doses of 10 and 12 g. To assess bioavailability and pharmacokinetics, a trial was performed with 6 subjects per dose level using 10 and 12 g curcumin. After the single dose of curcumin was taken with a standardized fatty meal, blood specimens were collected at 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 24, 36, 48, and 72 h. A reverse-phase gradient HPLC method was developed for identifying and quantifying curcumin in plasma. The assay was linear from 0.2 to 27 µmol/L. At doses of 10 and 12 g, no free curcumin was detected in human plasma. Essentially all the curcumin in plasma was conjugated, and enzymatic hydrolysis with ß-glucuronidase and sulfatase was necessary to quantify curcumin. Our data indicate that the maximal concentration of the conjugated forms was achieved 2–10 h after oral administration with a concentration range of 4.1–10.1 µmol/L. Oral curcumin is absorbed in humans and circulates in the blood as conjugates; deconjugation at the target tissue may be responsible for its biological effects. [Supported by NCI-N01-CN-35160 and AICR#06A035.]

3,3'-Diindolylmethane, a Naturally Occurring Compound Found in Cruciferous Vegetables, Reduces HER-2/neu Signaling and Inhibits Growth of HER-2/neu–Positive Breast Cancer Cells. Susan Lanza-Jacoby, Kandace McGuire, and Noel Ngoubilly. Department of Surgery, Thomas Jefferson University, Philadelphia, PA.

Breast tumors with overexpression of the oncogene erbB-2/neu/HER-2, a member of the epidermal growth factor receptor (EGFR) family, occur in 25–30% of human breast cancers and are associated with chemoresistance resulting in poor clinical outcome. Although an antibody against the HER-2/neu receptor is available to treat breast tumors overexpressing HER-2/neu, many cardiotoxic effects have been noted, which complicates their use. Several studies documented that intake of indole-3-carbinol and 3,3'-diindolylmethane (DIM) present in the cruciferous family have antitumor effects against several types of cancer including breast cancer. This study investigated whether DIM has a growth-inhibitory effect on breast cancer cells with elevated expression of HER-2/neu by examining cell survival, apoptosis, and potential downstream EGFR-activated pathways. DIM inhibited growth of human MDA-MB-435eB and BT474 cells and MMTV-HER2/neu mammary tumor NMF11.2 cells in a concentration- and time-dependent manner. Results from the M30 CytoDeath and TUNEL assays showed that DIM induced apoptosis in NMF11.2 and 435eB cells in a concentration-dependent manner. The growth-inhibitory effects of DIM were associated with decreased EGF-induced phosphorylation of the HER-2/neu receptor, extracellular signal-regulated kinase1/2, and AKT. Feeding MMTV-HER-2/neu mice 550 mg/kg body weight of absorption-enhanced DIM for 12 mo reduced tumor incidence by 50% (P < 0.01, n = 6) and increased tumor latency with no toxic side effects. These findings suggest that DIM has potential benefits for use as adjuvant therapy for prevention and treatment of HER-2/neu–overexpressing breast cancer.

Proteomic and Genomic Analysis of Human Tumor Cells Treated with Diallyl Disulfide. Nagathihalli S. Nagaraj¹ and Om V. Singh.^{2 1}Department of Medicine, James Graham Brown Cancer Center, University of Louisville, Louisville, KY; and ²Department of Pediatrics, The Johns Hopkins School of Medicine, Baltimore, MD.

Diallyl disulfide (DADS), an important component of garlic, has a potential chemopreventive activity against human cancers. Evidence is also mounting to indicate that DADS can contribute to apoptosis in cancer cells, but the proteomic and genomic data leading to the proapoptotic effect of DADS are very poor. Using proteomic and genomic means, we systematically studied the responses of protein and gene expression simultaneously in breast cancer line MCF-7, prostate cancer cell line PC-3, and lung cancer cell line calu-1 induced by DADS. Protein profiles were analyzed by 2-dimensional gel electrophoresis and mass spectrometry. Differential gene expression patterns were determined in DADS-exposed versus nonexposed cells using high-density microarray RNA expression profiling. A total of 36 unique proteins and a set of 43 genes that were specifically up- or down-regulated in MCF-7, PC-3, and calu-1 cells were detected. Of these, 7 are typically expressed for apoptosis: S100A11, KRT8, FADD, CASP2, CASP3, CSTB, and APAF1. These were further examined by Western blotting and RT-PCR, resulting in data coincident with the proteomic and genomic evidence. Interestingly, 60–70% of proteins and genes in all the cell lines studied were highly associated with the apoptotic pathway. Furthermore, DADS induced a marked amount of Bax release, mitochondrial membrane potential, cytochrome c release, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. DADS-treated cells triggered mitochondria-mediated signaling pathways that lead to apoptosis. The proteomic and genomic results presented support the hypothesis that garlic is a strong inducer of apoptosis in tumor cells.

Dietary Flavonoids Induce MLL Translocations in Human CD34⁺ Hematopoietic Stem Cells. Sahar Barjesteh van Waalwijk van Doorn-Khosrovani,¹ Jannie Janssen,² Lou Maas,¹ Roger W.L. Godschalk,¹ Jan G. Nijhuis,³ and Frederik-Jan van Schooten.^{1 1}Department of Health Risk Analysis and Toxicology, The Nutrition and Toxicology Research Institute Maastricht, Maastricht University, the Netherlands; ²Department of Clinical Genetics and ³Department of Obstetrics and Gynecology, Academic Hospital, Maastricht, the Netherlands.

Genetic abnormalities leading to infant leukemias already occur during fetal development and often involve rearrangements of the mixed-lineage-leukemia (MLL) gene. These rearrangements resemble the aberrations observed in therapy-related leukemias following treatment with topoisomerase II (topoII)-inhibiting agents such as etoposide. Because flavonoids present in food (e.g., soy, fruits, vegetables, red wine, beer, and tea), herbal medicines, and dietary supplements are potent topoII inhibitors, we examined the effect of three widely consumed flavonoids (quercetin, genestein, and kaempferol) in primary hematopoietic CD34⁺ stem cells isolated from umbilical cord blood. Using neutral Comet assay, we demonstrated a dose-dependent double-strand-break formation after exposure to flavonoids, particularly genistein and quercetin. An incorrect repair of these double-strand breaks resulted in chromosomal abnormalities, as determined by fluorescence in situ hybridization (FISH) and an inverse PCR approach. Surprisingly, genistein and quercetin seemed to be more clastogenic than etoposide and kaempferol. A concentration of genestein as low as 1 µmol/L resulted in MLL translocations. The estimated frequency of MLL translocations after exposure to 50 µmol/L genestein or quercetin was 500 per 10⁶ cells. Most of these translocations were formed by microhomology-mediated nonhomologous end joining. Moreover, in all but one translocation, SINE/Alu or LINE/L1 repetitive elements were present in at least 1 side of the breakpoint junction. Beside MLL translocations, FISH analysis demonstrated monosomy or trisomy of MLL in 8–10% of the quercetin-exposed stem cells. Our study demonstrates that biologically relevant doses of flavonoids can induce MLL abnormalities in hematopoietic stem cells. This is particularly alarming because differences in metabolism and excretion rate between mother and fetus can lead to a higher flavonoid concentration in the fetus. Therefore, it is important to raise public awareness and set guidelines for marketing flavonoid supplements to reduce the risk of infant leukemias.

Nutritive Evaluation and Anticlastogenic Potential of Gracilaria sp. Kumvan Wiravan,¹ Kupradinun Piengchai,² Kangsadalampai Kaew,¹ Tepsuwan Anong,² Kusamran R. Wannee,² and Butryee Chaniphun.^{1 1}Institute of Nutrition, Mahidol University, Nakhon Pathom, Thailand; and ²National Cancer Institute, Bangkok, Thailand.

A dietary red seaweed, Gracilaria sp., is consumed by Thai people as a food ingredient. The aims of this study were to determine the nutritive value and the clastogenic and anticlastogenic potentials of Gracilaria sp. induced by cyclophosphamide (CP), an indirect-acting carcinogen, and mitomycin C (MMC), a direct-acting carcinogen, by using the erythrocyte micronucleus assay in the mouse. The results, expressed per 100 g unprocessed and processed dry seaweed, showed that moisture content was 4.4–9.6 g; ashes, 4.0–37.9 g; protein, 9.0–9.4 g; fat, 0.6 g; total dietary fiber, 50.9–53.8 g; carbohydrate, 50.9–76.4 g; and iodine, 10.723–21.269 mg. The iodine content in Gracilaria sp. was very high when consumed in one serving, so high that consumption could lead to harm. Feeding diets containing 1% and 2% Gracilaria sp. for 2 wk had no significant effect in the erythrocyte micronucleus assay. For the anticlastogenicity test, male mice were fed diets containing 1% and 2% powder of Gracilaria sp. for 2 wk. MMC and CP were given; blood was collected at 0, 24, and 48 h after administration; and the number of micronucleated peripheral reticulocytes (MNRETs) was counted. Both 1% and 2% Gracilaria sp. decreased the number of MNRETs in a dose-dependent manner. Significant inhibitory effects on carcinogens were shown by the diet containing 2% Gracilaria sp. at 48 h after carcinogen administration compared with the control group. In conclusion, this study demonstrated that Gracilaria sp. can be consumed as a source of protein, fiber, and iodine in a serving and it, interestingly, has anticlastogenic activity against MMC and CP in the erythrocyte micronucleus assay.

	Lipids

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Effect of Conjugated Linoleic Acid on Mammary Tumor Growth and Metastasis in a Transgenic Mouse Model of Breast Cancer. Margaret Flowers,¹ Cynthia Thomson,¹ Joyce Schroeder,² and Patricia Thompson.³ Departments of ¹Nutritional Sciences, ²Molecular and Cellular Biology, and ³Pathology, University of Arizona, Tucson, A.

Conjugated linoleic acid (CLA) refers to a class of natural fatty acids derived from the biohydrogenation of linoleic acid by digestive bacteria in ruminant animals. CLA inhibits chemically induced mammary tumors and induces apoptosis in several cancer cell lines; limited evidence suggests isomer-specific properties. However, the mechanism of CLA action is still largely unknown. We are interested in the role of CLA as an anti-breast-tumor agent, which we hypothesize is primarily mediated through a dependent interaction with the peroxisome proliferator-activated receptors (PPARs), a class of nuclear receptors and transcription factors that perform a variety of cellular functions including fatty acid metabolism, energy homeostasis, and cell growth. Administration of the mixed isomer formulation of CLA commonly distributed over the counter as a weight-loss product showed weak antitumor activity in a genetic model of mammary tumorigenesis (n = 6). A preliminary analysis of selected tissue samples suggested an inhibitory effect on the protein kinase B/Akt cell survival pathway. Surprisingly, we found that dietary CLA was associated with higher cellular density and a distinctive tumor phenotype (i.e., densely packed tumor cells with an intact basement membrane and prolific) among tumors that arose in CLA mice compared with control animals. Our preliminary findings suggest a complex effect of CLA on mammary epithelial cell growth in animals genetically prone to breast tumors that warrants further characterization, particularly because of increased cellular density in the presence of an unexpected inhibitory action on the protein kinase B/Akt cell survival pathway. Future work will include molecular and pathogenic characterization of the tumor type arising in animals fed CLA compared with a control diet, including characterization of their invasive potential, which we suspect to be low despite high cellularity

Eicosapentaenoic Acid and Docosahexaenoic Acid as Chemopreventive Agents against 17ß-Estradiol Epoxide Formation and the Potential to Prevent Breast Cancer Carcinogenesis at Initiation. Fu-Li Yu,¹ Roger Greenlaw,² Wanda Bender,¹ and Katarzyna Berbeka.^{1 1}Department of Biomedical Sciences and ²Department of Medicine, University of Illinois College of Medicine at Rockford, Rockford, IL.

We found that 17ß-estradiol (E₂) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane to inhibit nuclear RNA synthesis and to bind DNA, forming DNA adducts both in vitro and in vivo. Because DNA adducts can cause mutation, and mutation is the molecular basis for the initiation of carcinogenesis, we proposed that E₂ epoxidation is the underlying mechanism for initiation of breast cancer. A method to screen chemopreventive agents against breast cancer at initiation was developed. This report examines the transcriptional and DNA binding properties of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their preventive effect on E₂ epoxidation. We found that both EPA and DHA strongly prevented the formation of E₂ epoxide, as reflected by the loss of the ability of E₂ to inhibit nuclear RNA synthesis. Additionally, we found that EPA and DHA prevented the binding of ³H-labeled E₂ to DNA. This preventive effect of EPA and DHA on the binding of E₂ to DNA was further confirmed when liver microsomes were used for the activation. We believe that this is the first report on the transcriptional and DNA binding properties of EPA and DHA and their inhibitory effect on the formation of E₂ epoxide. As dietary supplements, EPA and DHA may have the potential to prevent E₂-induced breast cancer carcinogenesis at initiation. [Partially supported by an Excel Academic Award from SwedishAmerican Hospital.]

Effect of Walnut Consumption on Cancer Growth. W. Elaine Hardman, Hyeong-Chan Jo, and Gabriela Ion. Department of Biochemistry and Microbiology, Marshall University School of Medicine, Huntington, WV.

Walnuts contain a high level of (n-3) fatty acids (18C, -linolenic acid), phytosterols, vitamin E, and melatonin. Consumption of long-chain (n-3) fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and phytosterols and antioxidants (vitamin E and melatonin) has been shown to slow cancer growth. We studied whether consumption of walnuts could affect growth of MDA-MB 231 human breast cancers implanted into nude mice. One million MDA-MB 231 cells were injected sc between the scapulae of 40 nude mice consuming an AIN-76A diet slightly altered to contain 10% corn oil. After the tumors reached a measurable size (3–5 mm diameter), the tumor-bearing mice were divided into 2 groups with similar mean tumor size and equal numbers of larger and smaller tumors. The diet of 1 group of mice was changed to contain ground walnuts at the human equivalent of 28 g/d. Diets were balanced to contain equal amounts of protein, carbohydrate, fat, and fiber. Tumors were measured and mice were weighed 3 times per week. About 10 d after the diet change, tumor sizes began to diverge. Linear regression analyses from day 10 until day 35 showed that the growth rate of tumors of the walnut group was significantly less than that of the control group. At the end of the study, the mean size of the tumors of the walnut group was half the mean size of the tumors of the control group. EPA and DHA content of the liver of the walnut group was significantly higher than that of the control group, indicating that the -linolenic acid was efficiently converted to EPA and DHA. Additional analyses are ongoing. Consumption of walnuts may be an effective way to increase the (n-3) fatty acid content of the diet for benefit against cancer growth.

Transgenic fat-1 Mice Convert Dietary (n-6) PUFAs to (n-3) PUFAs in Colonic Cells. Claudia Gravaghi,¹ Martha Peppas,¹ Jing X. Kang,² Krista M.D. La Perle,³ Fred Quimby,⁴ Lotus Altholtz,⁴ Lisa C. Hudgins,⁵ and Sergio A. Lamprecht.^{1 1}Strang Cancer Research Laboratory at The Rockefeller University, New York, NY; ²Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA; ³Animal Resource Center, Memorial Sloan-Kettering Cancer Center, New York, NY; ⁴Laboratory Animal Research Center, The Rockefeller University, New York, NY; and ⁵The Rogosin Institute, Iris & B. Gerard Cantor Clinical Research Laboratory, The Rockefeller University, New York, NY.

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in either promoting [(n-6) class] or inhibiting [(n-3) class] inflammation and neoplasia. In contrast with plant and nematode cells, mammalian cells are devoid of a fatty acid (n–3) desaturase, an enzyme able to convert (n-6) PUFAs to (n-3) PUFAs, and consequently essential fatty acids must be supplied in the diet. In this study we have used transgenic mice engineered to carry the Caenorhabditis elegans fat-1 gene encoding a (n-3) desaturase (1) to assess PUFA production in colonic cells. Presence of (n-3) desaturase transcript was confirmed by RT-PCR analysis. To measure the conversion of dietary (n-6) PUFAs to (n-3) PUFAs, fat-1 mice and control littermates were fed for 7 wk AIN-76A standard diet supplemented with 10% (w/w) safflower oil rich (76%) in (n-6) polyunsaturated linoleic acid. The fatty acid profile in colons of control and transgenic mice was determined by lipid methylation and capillary gas chromatography. The results show that the (n-6)/(n-3) PUFA ratio in colonic cells of fat-1 mice is markedly lower (9.83 ± 2.62) than that of wild-type littermates (54.5 ± 9.24, P < 0.001); this process was associated with a significant decrease (P < 0.01) in (n-6) PUFA–derived prostaglandin E₂ production assayed by prostaglandin-E₂ monoclonal enzyme immunoassay. Cumulatively our results indicate that colonic cells of fat-1 mice have acquired the novel biochemical competence to convert (n-6) PUFAs to (n-3) PUFAs. These transgenic mice represent a unique model for studying dietary PUFA modulation of the inflammatory process and carcinogenesis. [Supported by AICR Grant 04B036.]

18. Kang JX, Wang J, Wu L, Kang ZB. Transgenic mice: fat-1 mice convert n-6 to n-3 fatty acids. Nature. 2004;427:504.[Medline]

	Vitamins

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
Dietary Vitamin C and Risk of Stomach Cancer: A Systematic Literature Review. Victoria J. Burley, Darren C. Greenwood, Doris Chan, Janet E. Cade, Jennifer Moreton, Yu-Kang Tu, and David Forman. Centre for Epidemiology and Biostatistics, University of Leeds, Leeds, UK.

In preparation for the second report from WCRF/AICR, Food, Nutrition, Physical Activity and the Prevention of Cancer: a Global Perspective, a systematic review of the literature relating to stomach cancer has been undertaken. As part of this review, 1 objective was to assess the relation between vitamin C and risk of gastric cancer. All original, etiological, peer-reviewed studies published before January 2006 were considered with no exclusions on the basis of language or quality. Separate random-effects meta-analyses were performed for cohort and case-control studies based on derived linear dose-response curves. Data on dietary vitamin C intakes were extracted from 4 cohort and 28 case-control studies, with 2 and 17 of each study type providing sufficient information for meta-analysis. The pooled estimates of relative risk per 30 mg/d were 0.92 (95% CI 0.85, 1.00; P = 0.04) and 0.85 (95% CI 0.80, 0.89; P < 0.001) for cohort and case-control studies, respectively. Meta-regression suggested there was no excess heterogeneity within the cohorts, but there was a substantial amount of unexplained heterogeneity within the case-control studies (I² = 90%, Q = 184, df = 18, P < 0.001). Additionally, for these latter studies, Egger's test suggested significant small-study bias (P = 0.002). Meta-analyses of data on blood vitamin C from 2 cohort and 3 case-control studies indicate consistency with the results on dietary vitamin C. The pooled estimates of risk from these studies were 0.98 (95% CI 0.95, 1.01, P = 0.3) and 0.85 (95% CI 0.80, 0.90; P < 0.001) per 1 µmol/L, respectively. These data indicate a statistically significant reduction in risk of stomach cancer associated with increasing levels of dietary and blood levels of vitamin C. However, the small size of the effect does not rule out the possibility that the association is caused by uncontrolled confounding or bias.

Supplementary and Dietary Vitamin D Intake and Renal Cell Cancer Risk. Robin Taylor Wilson,¹ Jiangyue Wang,² Vernon Chinchilli,² John Richie,¹ Lee E. Moore,³ and Demetrius Albanes.^{3 1}Pennsylvania State College of Medicine, Penn State Cancer Institute, Hershey, PA; ²Pennsylvania State College of Medicine, Division of Biostatistics, Hershey, PA; ³National Cancer Institute, Division of Cancer Epidemiology and Genetics, Bethesda, MD.

Renal cell cancer (RCC) is the third most rapidly increasing cancer in the United States. Vitamin D has a suspected preventive role. We sought to determine the risk of RCC associated with supplemental and dietary vitamin D intake. Cases were identified through the Alpha Tocopherol Beta Carotene (ATBC) trial cohort (1985–2002). A detailed dietary history, fasting serum sample, and height and weight measurements were obtained at recruitment. Individuals diagnosed with prior cancer or using vitamin E, vitamin A, or ß-carotene supplements were excluded. Person years of observation were counted from the date of randomization until diagnosis of RCC, death, or end of study. Dietary intake variables were energy adjusted. The hazard ratio (HR) of RCC was determined by stepwise Cox proportional hazards regression including all suspected variables. Multiplicative interactions were determined between vitamin D intake and each variable remaining in the final model. There were 228 RCC cases among 26,758 men. The highest quartile of total vitamin D intake was associated with an increased risk of RCC (HR = 1.6, 95% CI 1.13, 2.41, > 7.4 µg/d, P-trend = 0.008) compared with the lowest quartile, adjusting for BMI, hypertension, cholesterol, quercetin, and pack years of smoking. Supplemental vitamin D at the highest intake was also associated with increased risk (HR = 3.1, 95% CI 1.14, 8.35, P-trend = 0.282). Interaction terms for cholesterol and quercetin with total vitamin D intake were statistically significant (P < 0.05). We observed an increased risk of RCC in male smokers with the highest levels of vitamin D intake. These findings do not support a preventive role of dietary vitamin D in relation to kidney cancer incidence.

Pharmacologic Ascorbate Concentrations Selectively Kill Cancer Cells: Ascorbic Acid as a Prodrug for Ascorbate Radical or H₂O₂ Delivery to Tissues. Qi Chen,¹ Je-Hyuk Lee,¹ Liqun Zhang,¹ Andrew Sun,¹ Murali C. Krishna,² Michael G. Espey,² Chaya Pooput,¹ Kenneth Kirk,¹ Peter Choyke,² Garry R. Buettner,³ Emily Shacter,⁴ and Mark Levine.^{1 1}National Institute of Diabetes and Digestive and Kidney Diseases and ²National Cancer Institute, National Institutes of Health, Bethesda, MD; ³College of Medicine, University of Iowa, Iowa City, IA; and ⁴Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD.

Pharmacokinetics data in humans indicate that i.v. but not oral ascorbate could have an unanticipated role in cancer treatment. We hypothesized that millimolar ascorbate extracellular concentrations achieved only by i.v. administration could serve as a prodrug for ascorbate radical or H₂O₂ delivery to tissues with minimal formation of the prodrug in blood. Under in vitro conditions mimicking clinical i.v. use, some cancer cells were killed by millimolar ascorbate, whereas normal cells were unaffected. Apoptosis and pyknosis-necrosis were caused by extracellular ascorbate independent of intracellular concentrations and were critically dependent on H₂O₂ formation. H₂O₂ generation was dependent on ascorbate concentration and linearly related to ascorbate radical formation, with 0.5–10% serum required. In contrast, millimolar ascorbate additions to hemolyzed and nonhemolyzed blood generated no detectable H₂O₂ and only trace ascorbate radical. To test these concepts in vivo, i.v. ascorbate was administered to rats (0.25–0.5 mg/g), a dose on a weight basis that could readily be given to humans. Extracellular fluid (ECF) was collected by microdialysis from muscles. After i.v. administration, ascorbate reached 2–8 mmol/L for about 2 h in both blood and ECF. Ascorbate radical increased from below detectable to 20 nmol/L in blood but >200 nmol/L in ECF and was dependent on ascorbate concentration. In ECF, H₂O₂ increased from <2 µmol/L before i.v. administration to about 20–30 µmol/L after i.v. administration. Similar data were obtained from urine. With oral rather than i.v. administration of the same dose, ascorbate concentrations in blood and ECF were <190 µmol/L, and there was no ascorbate radical or H₂O₂ formation in either fluid. Taken together, these findings provide support for the hypothesis and plausibility for i.v. ascorbate use in cancer treatment and possibly in treatment of some infections in which H₂O₂ or reactive oxygen species may have therapeutic roles.

Vitamin A Signaling in Prostate Epithelium. Max E. Gottesman,¹ Lesley Wassef,² Silke Vogel,¹ and Loredana Quadro.^{2 1}Department of Medicine, Columbia University, New York, NY; ²Department of Food Science, Rutgers University, New Brunswick, NJ.

Alterations in vitamin A availability and metabolism are known to have profound effects on prostate growth and prostate cancer development. We used mice lacking retinol-binding protein (RBP) to investigate this issue. RBP is a specific serum transport protein that delivers retinol (vitamin A alcohol) from liver stores to target tissues. RBP^–/– mice have low circulating retinol levels and rely predominantly on dietary vitamin A to maintain normal physiological functions. Although expressed mainly in the liver, RBP is also synthesized in extrahepatic tissues, where its function is largely unknown. In this context, we found that RBP is expressed in mouse prostate. Surprisingly, RBP^–/– mice display elevated prostate retinol and retinyl ester levels at age 15 wk. This suggests that RBP synthesized in prostate epithelium may prevent accumulation of vitamin A by secreting excess retinol back into the bloodstream. In addition, the prostate epithelium of 15-wk-old RBP^–/– mice showed increased cellular proliferation compared with wild-type age-matched controls, as assessed by proliferating cell nuclear antigen (PCNA) levels. However, total p27 levels were also increased. Phospho-Akt2 concentrations were elevated, whereas phospo-Akt1 levels remained unchanged. Androgen receptor and cyclin D1 levels were also unaffected. We also introduced the probasin-regulated transgene expressing the SV40 early genes (T/t antigens; TRAMP) into the RBP^–/– background and generated a mouse strain with both increased prostate cancer incidence and altered retinoid metabolism (RBP^–/– TRAMP). As expected, the transgene increased PCNA levels in prostate epithelium. However, PCNA levels were reduced in RBP^–/– TRAMP mice compared with RBP^+/+ TRAMP controls. These data suggest that vitamin A may suppress proliferation in premalignant cells. In summary, alteration of vitamin A metabolism seems to have distinct effects on the prostate of wild-type mice and of mice prone to developing prostate cancer. Further analyses are ongoing to determine how vitamin A exerts these effects.

Use of Vesiculated -Tocopheryl Succinate and Dendritic Cell Vaccination to Treat Metastatic Murine Breast Cancer. Emmanuel T. Akporiaye,¹ Lalitha Ramanathapuram,¹ Tobias Hahn,¹ and Sharon Dial.^{2 1}Departments of Microbiology and Immunology and ²Veterinary Science and Microbiology, University of Arizona, Tucson, AZ.

-Tocopheryl succinate (-TOS) is a semisynthetic analog of vitamin E that preferentially induces apoptotic death of cancer cells. The ability of -TOS to suppress tumor growth in preclinical animal models has led to increased interest in its potential use in combination with immunotherapy for treating cancer. We combined vesiculated -TOS (V-TOS) with nonmatured dendritic cells (DCs) to treat metastatic murine breast cancer. Treatment with V-TOS plus nonmatured DCs inhibited the growth of established tumors and prolonged survival. The combination treatment dramatically reduced formation of lung metastases after resection of primary tumor. We also compared V-TOS with -tocopheryloxyacetic acid (-TEA) which unlike -TOS is resistant to intestinal esterases and can therefore be administered orally without concern for conversion to vitamin E. Comparison of intraperitoneal versus oral gavage administration of both agents revealed that only -TEA suppressed tumor growth when administered by oral gavage. To make -TEA treatment more clinically relevant, we administered -TEA in mouse chow to prevent or treat established disease and metastases. Dietary -TEA suppressed primary tumor growth and dramatically reduced lung metastases. In trying to understand the mechanism of action of the combination treatment, we showed that V-TOS– and V-TEA–treated tumor cells caused DC maturation in vitro evidenced by increased expression of costimulatory molecules, IL-12p70 secretion, and antigen presentation. These effects are mediated in part by heat shock proteins released from tumor cells after -TOS or -TEA treatment. These findings demonstrate enhancement of in vivo antitumor effects of -TEA and -TOS when combined with DC vaccination. The results also suggest that combining DC vaccination with dietary -TEA may be a viable approach for treating metastatic breast cancer.

Evaluation of the Anticancer Actions of Natural Vitamin E Forms, RRR--Tocopherol and -Tocopherol. Weiping Yu. Molecular Genetics and Microbiology, College of Natural Sciences, University of Texas at Austin, Austin, TX.

Focus of previous studies of vitamin E compounds as antitumor agents has been on natural RRR--tocopherol (T), the major vitamin E form in tissues, and synthetic vitamin E, all-rac--tocopherol, found in supplements. Data supporting these vitamin E forms as potent anticancer agents are inconclusive. Recent data suggest that -tocopherol (T), the most abundant form of vitamin E in the American diet, exhibits anticancer properties. Studies reported here examined the proapoptotic properties of T and T in human estrogen-responsive MCF-7 and non-responsive MDA-MB-435 breast cancer cells, focusing on prodeath and prolife signaling pathways. T but not T induced both cell types to undergo apoptosis in a dose- and time-dependent manner while sparing human mammary epithelial cells from apoptosis. T sensitized both cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)–induced apoptosis. T increased death receptor DR5 mRNA and protein expression. siRNA knockdown of DR5 blocked both T-induced apoptosis and sensitization of breast cancer cells to TRAIL-induced apoptosis. T induced activation of JNK/c-Jun, activation of pro-apoptotic Bax, processing of Bid to its truncated form (tBid), and cleavage of caspase-8 and -9. Furthermore, T down-regulated antiapoptotic phospho-Akt (pAkt) and survivin protein expression. Knockdown of pAkt as well as survivin enhanced T-induced apoptosis. In summary, these studies showed that T but not T induced apoptosis and sensitized human breast cancer cells to TRAIL/DR5–induced apoptosis via JNK/c-Jun/Bax and caspase-8/tBid/Bax, leading to mitochondria-dependent caspase 9 activation as well as suppression of antiapoptotic pAkt and survivin. [Supported by AICR grant 26–7693 and The Foundation for Research.]

	Minerals

TOP Angiogenesis Apoptosis Biomarkers Food Composition Dietary Pattern and Cancer... Calorie Restriction Obesity Survivorship Colorectal Cancer Breast Cancer Prostate Cancer Herbs Soy Phytochemicals Lipids Vitamins Minerals

 
The Inflammatory Response and Breast Cancer Metastasis to Bone: A Role for Selenium? Andrea M. Mastro,¹ K. Sandeep Prabhu,² Hema Vunta,² Leah J. Novinger,¹ and C. Channa Reddy.^{2 1}Departments of Biochemistry and Molecular Biology and ²Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA.

The skeletal system is a frequent target of metastatic breast cancer. Once in the bone, the cancer cells activate osteoclasts to degrade bone. Nonetheless, the osteoblasts appear unable to restore the bone even in the presence of drugs that inhibit osteoclasts. We propose that in the presence of metastatic breast cancer, the osteoblasts no longer function normally as bone-building cells. In support of this idea, we found that human metastatic breast cancer cells, MDA-MB-231 and MDA-MB-435, increase osteoblast apoptosis, suppress osteoblast differentiation, and affect osteoblast morphology. In addition, we found that in the presence of breast-cancer-cell-conditioned medium, osteoblasts undergo an inflammatory stress response exemplified by the production of IL-6, KC (IL-8), macrophage chemoattractant protein-1, and cyclooxygenase-2, which attract and activate osteoclasts. Our studies are based on the premise that the cancer cells bring about this inflammatory response through a change in the oxidative state of the osteoblasts that is controlled by cellular selenium status through the redox-regulated transcription factor, nuclear factor-B. Nuclear factor-B was activated in osteoblasts in breast-cancer-cell-conditioned medium. Exogenous addition of selenium or catalase inhibited this activation, suggesting a role for reactive oxygen species. Because dietary selenium has been linked to various stages of cancer progression including metastasis, it is important that the mechanisms be elucidated. This study may provide evidence to show how selenium status may affect metastatic bone loss.

Maternal Iron Overload Stimulates Oxidative Stress and Cell Damage of Hepatic Tissues in Rats. Mi-Na Park and Yeon-Sook Lee. Department of Food and Nutrition and Research Institute of Human Ecology, Seoul National University, Seoul, Korea.

Although iron is an essential micromineral, excess iron may promote oxidative stress and be a risk factor for chronic disease such as cancer. We investigated the long-term effects of maternal iron overload on oxidative stress in hepatic tissues in female rats and their offspring. Ten-week-old female Sprague-Dawley rats were mated with male rats. Pregnant dams were fed diets with normal iron (35 mg/kg diet), high iron (350 mg/kg diet), and excess iron (1050 mg/kg diet) during pregnancy and lactation. Dams and their pups were killed on postnatal day 16. There was no significant difference in weight gain and diet intake according to iron intake levels. However, the concentration of serum iron, hematocrit, and transferrin saturation were significantly increased in pups from dams fed the excess iron diet but not in their dams. With increasing maternal iron intake, the iron and ferritin content of the liver in both dams and their pups were significantly increased. Maternal excess iron intake significantly increased the content of malondialdehyde and protein carbonyl of the liver in both dams and their pups. Excess iron intake also decreased the activity of antioxidant enzymes glutathione peroxidase, glutathione reductase, and catalase in the liver of pups. Cell swelling, vacuolation, necrotic foci, and iron deposition showed cell damage in the liver of high-iron and excess-iron diet groups. In addition, the protein level of Bcl-2 as an antiapoptotic factor was remarkably decreased with increased maternal iron intake. Although iron overload during pregnancy and lactation had little effect on hematology measurements of dams, it led to the deposit of iron, the decline of activity of antioxidant enzymes, and cell death in the liver tissues in both dams and pups. These results suggest that hepatic iron accumulation as a result of excess iron intake may enhance susceptibility to cancer development related to oxidative stress.

	FOOTNOTES

 
¹ Published in a supplement to The Journal of Nutrition. Presented as part of the International Research Conference on Food, Nutrition, and Cancer held in Washington, DC, July 13–14, 2006. This conference was organized by the American Institute for Cancer Research and the World Cancer Research Fund International and sponsored by (in alphabetical order) the California Walnut Commission; Campbell Soup Company; Cranberry Institute; Hormel Institute; IP-6 International, Inc.; Kyushu University, Japan Graduate School of Agriculture; National Fisheries Institute; and United Soybean Board. Guest editors for this symposium were Vay Liang W. Go, Susan Higginbotham, and Ivana Vucenik. Guest Editor Disclosure: V.L.W. Go, no relationships to disclose; S. Higginbotham and I. Vucenik are employed by the conference sponsor, the American Institute for Cancer Research.

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