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Poster Abstracts

Effects of Lycopene Extracts on Rat Prostate Carcinoma Cells. R. S. Gunasekera,* W. L. McKeehan and B. S. Patil. *University of Houston-Victoria, Victoria, TX; Center for Cancer Biology and Nutrition, Texas A&M University System Health Science Center, Houston, TX and Texas A&M University-Kingsville Citrus Center, Weslaco, TX.

Consumption of fruits and vegetables containing lycopene, a carotenoid without provitamin A activity, has been associated with a lower risk of prostate and breast cancer. Lycopene plays a role not only in the prevention of cancers but also in prevention of cardiovascular disease. The effects of lycopene on epithelial and stromal cells from both normal prostate tissue and slow-growing, androgen-sensitive tumors were examined (Yan et al. 1993 ). The effects of standard lycopene preparations on androgen-independent, malignant (Dunning R3327AT3 or AT3 cells) type II tumor cells were compared with their benign parent type I tumor epithelial cells (DTE). Type II malignant tumors are metastatic and represent the most life-threatening stage of the disease. Pure lycopene was obtained from Sigma Chemical Company. Grapefruit lycopene was extracted and purified from Rio Red grapefruit. Results showed that lycopene introduced in -cyclodextrin as a water-soluble carrier inhibits the proliferation of AT3 cells in a concentration- and time-dependent manner. Similar cell proliferation studies done using DTE cells did not show any inhibition. These results suggest that lycopene may selectively inhibit the extremely malignant AT3 prostate cancer cells relative to their benign parent. The differential results also indicate that the inhibitory effect on AT3 cells is not a general cytotoxic effect on all cells in culture. Additional experiments indicated that lycopene had little effect on the mitogenic activity of external fibroblast growth factor or transforming growth factor-ß on the different tumor cell types. Studies by others have implied an effect of lycopene on growth stimulated by insulin-like growth factor-1 in other tumor cell systems.

Lycopene, Tomato Powder and Dietary Restriction Influence Survival of Rats with Prostate Cancer Induced by NMU and Testosterone. Thomas W.-M. Boileau, Steven K. Clinton, Zhiming Liao, Marcia H. Monaco, Sharon M. Donovan and John W. Erdman, JR. Division of Nutritional Sciences, University of Illinois, Urbana, IL, and the Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Division of Hematology and Oncology, Ohio State University, Columbus, OH.

A study was undertaken to determine whether lycopene, tomato powder or 20% dietary restriction could influence prostate cancer induced by N-methyl-N-nitrosourea (NMU) and testosterone in rats. A 2 x 3 factorial design tested effects of dietary lycopene source (control beadlets, lycopene beadlets and tomato powder) and dietary restriction (unrestricted feeding vs. 20% total dietary restriction) as well as their interaction on survival and hormonal biomarkers related to prostate cancer progression. Dietary lycopene was 161 mg/kg in rats fed beadlets at 0.25% of the diet, 13 mg/kg in rats fed tomato powder at 10% of the diet and not present in rats fed control diets. Five-week-old male Wistar-Unilever rats (n = 194) were randomly assigned to receive one of three AIN-based experimental diets (control, lycopene beadlets or tomato powder) and fed for 1 wk before tumors were initiated by sequential antiandrogen, androgen and carcinogen treatment followed by the promotion of tumor formation with subcutaneous testosterone implants. Three days after carcinogen treatment, each group was randomly assigned to receive 20% dietary restriction or unrestricted feeding. Plasma testosterone concentrations were not significantly different among dietary groups, indicating equal exposure to androgens from the implants. Concentrations of plasma insulin-like growth factor (IGF) I and IGF-I binding protein 3 were reduced (11% reduction, P = 0.139, and 30% reduction, P < 0.0001, respectively) by dietary restriction but were not altered by dietary lycopene or tomato powder. Increased overall survival as analyzed by Cox’s proportional hazards model was noted for rats fed tomato powder (39% increase, risk ratio [RR] = 0.613, P = 0.0056) and lycopene (17% increase, RR = 0.837; P = 0.162) compared with control rats fed placebo beadlets. Dietary restriction also significantly increased overall survival (23% increase, RR = 0.770, P = 0.0429) as compared with unrestricted feeding. No interactions between lycopene source and dietary restriction were noted for survival. We conclude that dietary restriction and the consumption of tomato powder or lycopene enhance survival in the NMU-androgen–induced prostate cancer model. Furthermore, tomato powder may contain substances in addition to lycopene that favorably modulate prostate carcinogenesis. [Supported by National Cancer Institute RO1-CA72482 and the Ohio State University Comprehensive Cancer Center, National Cancer Institute grant P30 CA16058.]

Large-Scale Production, Purification and Chemical Characterization of ß-Carotene Oxidation Products That Inhibit Growth and Metabolism of Cancer Cells. L. M. Canfield, C. B. Brochini and A.A.L. Gunatilaka. Departments of Biochemistry and Arid Land Studies, University of Arizona, Tucson, AZ.

Diets high in ß-carotene are associated with a lowered risk for cancer, but ß-carotene supplementation does not protect against and may in fact potentiate human cancer. We hypothesize that oxidation products of ß-carotene may be responsible at least in part for its in vivo actions. In support of this hypothesis, we previously reported isolation and identification of a highly enriched fraction of ß-carotene oxidation products—Carotenox—containing 5,8-endoperoxy-2,3-dihydro-ß-apocarotene-13-one as a major component (Hu et al. 1998 ). Carotenox reversibly inhibits the growth of breast cancer cells, (50% inhibitory concentration [IC₅₀] 20 µmol/L), inhibits DNA synthesis and arrests cell division in G_O/G₁. Because our previous method yielded only trace quantities of active product, we have developed procedures 1) for producing gram quantities of ß-carotene oxidation products by reaction with ¹O₂; 2) for purifying these products in high yield by column chromatography, thin layer chromatography and HPLC and 3) for large-scale bioassay using cancer cell lines, HeLa and MCF-7. Using these procedures, we have partially purified milligram quantities of several fractions that strongly inhibit growth of cancer cells (IC₅₀ 1–10 µg/mL). Work is in progress to purify these compounds to homogeneity by using HPLC and to characterize them by nuclear magnetic resonance and mass spectroscopy. These compounds may explain in part the apparent contradictory actions of ß-carotene in vivo. [Supported by grants from Wyeth-Ayerst Nutritionals; the University of Arizona Small Grants Program, College of Agriculture and Life Sciences and Fundação de Amparo à Pesquisa do Estado de São Paulo, Sao Paulo, Brazil.]

3,3'-Diindolylmethane (DIM) Acts as a Potential Antiandrogen in LNCaP Human Prostate Cancer Cells. Hien T. Le, Gary L. Firestone* and Leonard F. Bjeldanes. Department of Nutritional Sciences and Toxicology and *Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA.

3,3'-Diindolylmethane (DIM) is a major digestive derivative of indole-3-carbinol (I3C), a naturally occurring component of dietary cruciferous vegetables. Our results indicate that DIM has antiproliferative effects and acts as a potential antiandrogen in LNCaP cells, an androgen-dependent human prostate cancer cell line. DIM functions as an extracellular growth-suppression signal by inducing a G1 cell cycle arrest of LNCaP cells and inhibits dihydrotestosterone stimulation of DNA synthesis. We examined the effect of DIM on the promoter activity of the mouse mammary tumor virus single long terminal repeat, which contains a single androgen response element (ARE) region and a prostate-specific antigen (PSA) promoter-enhancer containing three AREs, all critical to the activity of the PSA promoter. A dose-dependent inhibition of dihydrotestosterone induction by DIM was observed when these promoters were transiently transfected into LNCaP cells and followed by luciferase analysis. A similar effect of DIM was not observed in androgen-independent PC-3 cells until a wild type androgen receptor was coexpressed with the reporter plasmid. Our data strongly support the hypothesis that the androgen receptor is a major target of DIM regulation in prostate cancer cell growth. Competitive binding assays indicated that DIM does not compete with the natural ligand for androgen receptor binding. However, preliminary gel mobility shift data indicate that DIM promotes stable binding of the androgen receptor to the ARE. Thus, it seems likely that DIM is a potent antiandrogen and can potentially control the emergence and proliferation of prostate cancer cells by regulating the activation of androgen receptor–dependent gene transcription in prostate cancer epithelial cells. We plan to further examine the mode of action of DIM on the expression of genes and regulatory proteins involved in aspects of prostate tumor growth and progression to enable us to further understand the cancer preventive properties of DIM.

3,3'. Diindolylmethane (DIM) Regulates Bcl-2/Bax Family, a Central Apoptotic Regulator, at the Transcriptional and Posttranscriptional Level in Human Breast Cancer Cells (MCF-7 and MDA-231 Cells). Chibo Hong, Gary L. Firestone* and Leonard F. Bjeldanes. Departments of Nutritional Sciences and Toxicology and *Molecular Cell Biology, University of California at Berkeley, Berkeley, CA.

The naturally occurring phytochemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent. 3,3'-Diindolylmethane (DIM) is a major active derivative of I3C formed both in vivo and in vitro. However, its exact intracellular targets and mechanism of anticancer effect remain unknown. We used human breast cancer cells—MCF-7 cells (estrogen receptor positive cells) and MDA-231 cells (estrogen receptor negative cells)—to examine the cell growth inhibition and apoptotic effect of DIM. DIM inhibited cell growth and DNA synthesis in both a dose-dependent and time-dependent manner. When cells were exposed to DIM at 50 µmmol/L, apoptosis was shown by the characteristic morphology of cell nuclei with fluorescent staining. DNA fragmentation was detected with the TUNEL assay and phosphatidylserine externalization was observed with Annexin V assay. All these data from cell morphological and biochemical examinations suggest that DIM caused cell death through apoptosis. Western and coimmunoprecipitation assays were used to detect Bcl-2 and Bax protein levels in MCF-7 and MDA-231 cells. DIM decreased total Bcl-2 protein level and Bax-bound Bcl-2 protein in a dose- and time-dependent manners. DIM at 50 µmmol/L also increased Bax protein level proportional to treatment time. These results show that DIM induces apoptosis in MCF-7 and MDA-231 cells by increasing the ratio of Bax/Bcl-2 and free Bax protein. For investigation of the regulation at transcriptional level, Northern blot analysis revealed a dose-dependent and time-dependent decrease in the amount of Bcl-2 mRNA in MCF-7 cells treated with DIM. Because the Bcl-2 family is a central regulator in the apoptosis pathway, these results demonstrate that DIM may be a potential anticancer and apoptotic modulator in human breast cancer.

Measurement of 8OHdG as a Biomarker of Oxidative Damage in Healthy Women and Women Previously Treated for Breast Cancer. C. A. Thomson, D. S. Alberts, K. Z. LeWinn and C. Bogert. Arizona Cancer Center, Tucson, AZ.

8-Hydroxdy-2'deoxyguanosine (8OHdG) levels have been used as a biomarker of oxidative DNA damage in several populations. However, normal values for this biomarker in healthy women and breast cancer survivors are not available. The purpose of this study was to determine the mean level of urinary 8OHdG from a cohort of healthy women (n = 55) as well as breast cancer survivors (n = 58). This study also sought to evaluate the effect of a cancer preventive diet on oxidative DNA damage in breast cancer survivors. Urine samples for 8OHdG quantification by an enzyme-linked immunosorbent assay were collected at baseline for all subjects and at 6 mo for breast cancer survivors in the diet intervention trial. Mean baseline urinary 8OHdG levels were similar for both cohorts (healthy women and breast cancer survivors) and demonstrated wide variability (10.3 ± 16.1 and 13.9 ± 10.3, respectively). Baseline dietary intake was similar for all groups. At the end of the diet study, breast cancer survivors in the diet intervention group were consuming significantly more vegetables, vegetable juice, - and ß-carotene, lutein, folate and vitamins C and E than were the control group subjects. A nonsignificant reduction in 8OHdG excretion of 32% was demonstrated in survivors assigned to the dietary intervention (n = 28). Positive associations were shown between intake of meat (healthy women) and saturated fat (dietary intervention group) and urinary 8OHdG. Intake of fruit, vegetable juice, folate, ß-carotene and vitamins A and C were negatively associated with 8OHdG levels in the diet intervention group only. Future investigation using 8OHdG as a biomarker of oxidative damage should include larger cohorts and be complemented with additional biomarkers of oxidative damage to more clearly describe the efficacy of these markers as surrogate endpoints in cancer prevention research. [Supported by National Institutes of Health, National Cancer Institute R25 Cancer Prevention and Control Postdoctoral Fellowship Training Grant.]

Inositol Hexaphosphate May Exert Its Antiproliferative Effects by Affecting the Proteins Involved in the Regulation of the G₁/S Transition of the Cell Cycle. I. Vucenik, K. Tantivejkul, L. M. Anderson and D. Ramljak. University of Maryland, Baltimore, MD and National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD.

Previously, we showed that antiproliferative effects of IP₆ in human breast cancer cell lines MCF-7 and MDA-MB 231 may be mediated through activation of protein kinase C (PKC) . In another study, IP₆ caused a growth arrest in G₁ phase of the cell cycle in the same cells. Here, we investigated the mechanisms of action of inositol hexaphosphate (IP₆) responsible for cell cycle arrest in the G₁ phase of the cell cycle, potentially as a result of PKC activation. The status of cell cycle proteins involved in control of G₁/S transition of the cell cycle in both cell lines was analyzed by immunoblotting. Treatment with IP₆ at 2 mmol/L for 24 h caused a reduction in the protein expression of cyclins D1 and E and cyclin-dependent kinase 2 (Cdk2) and Cdk4 only in MCF-7 cells. However, in both cell lines, IP₆ induced a significant increase in protein levels of Cdk inhibitor p27 with no detectable effects on p21. Also, IP₆ markedly reduced the expression of retinoblastoma protein (pRb) in both cell lines. Although the MCF-7 and MDA-MB-231 cells differ in the status of estrogen receptor (ER), p53 and Ras, IP₆ exhibited similar effects on p27 and pRb in both. Our results indicate that effects of IP₆ on the expression of cyclins and Cdks are ER, p53 and Ras dependent whereas the status of p27 and pRb is independent of ER, p53 and Ras. Furthermore, these changes in both p27 and pRb could be the results of previously detected IP₆-mediated activation of PKC . On the basis of our results, it seems that although the antiproliferative effects of IP₆ involve rather complex molecular events, IP₆ could potentially have positive application in a treatment of breast cancers that are ER-negative, p53-mutated or overexpressing the oncogenic Ras.

UCLA Pilot/Feasibility Study of Nutrition Intervention for Treatment of Prostate Cancer. I. Yip, V.L.W. Go, W. Aronson, H. J. Wang, R. Elashoff and D. Heber. Center for Human Nutrition and Department of Urology, UCLA School of Medicine, Los Angeles, CA.

Specific nutrition intervention may be used as an adjuvant therapy for the treatment of prostate cancer. This pilot study was originally designed to test whether an intensive dietary therapy can delay prostate cancer progression. Forty patients with adenocarcinoma of the prostate who had undergone radical prostatectomy and who had undetectable prostate-specific antigen postoperatively were randomly assigned to a treatment or a control group. The treatment group received a low-fat (15% of total energy), high-fiber (18 g/1000 kcal) diet supplemented with 40 g soy protein. At baseline and 12 mo, subjects were given a food frequency questionnaire, and urinary isoflavones excretion and serum lipids level were measured. At baseline there were no differences in age, weight, body mass index, percent dietary fat intake, soluble and insoluble fiber intake, serum lipids and urinary genistein and daidzein excretion between the two groups. At 12 mo the intervention group showed a significant reduction (P < 0.05) in intake of percent dietary fat, total fat intake (by weight), dietary cholesterol, monounsaturated fatty acids and polyunsaturated fatty acids (PUFA) as compared with the control group. Vitamin C intake was increased significantly in the intervention group (P < 0.05). A significant increase in urinary excretion of daidzein and genistein occurred in the intervention group, which indicated compliance regarding taking the soy protein supplement. A significant reduction in serum total cholesterol in the intervention group (P < 0.05) was also observed. These subjects will be followed for 4 y. This study will provide us with important information on the effects of intensive dietary intervention on prostate cancer progression.

Phytoestrogen Concentrations in the Serums of Japanese Women and Men. M. S. Morton,* O. Arisaka, A. Miyake, L. D. Morgan and B.A.J. Evans. *Bioclinical Services, Cardiff, United Kingdom; Juntendo University School of Medicine, Tokyo, Japan and Department of Child Health, University of Wales College of Medicine, Cardiff, United Kingdom.

The incidences of breast and prostate cancer are very much lower in Japanese women and men, respectively, than in women and men from the developed Western countries. Soya, consumed daily in large amounts in Japan, contains genistin and daidzin, glycoside conjugates of the isoflavones genistein and daidzein. On consumption of soya the glycosides are metabolized by gut bacteria to their respective aglycones. Daidzein can be further metabolized to the isoflavan equol. These isoflavonic phytoestrogens have been proposed to be cancer-protective agents in Japanese and other Asian populations. This study reports the concentrations of the isoflavonoids daidzein, genistein and equol in the serum of Japanese women (n = 125) and men (n = 102) as determined by a robust gas chromatography–mass spectrometry method. The women were aged 40–89 y (mean 66.9 y) and the men were aged 40–85 y (mean 64 y). Comparison data for British women (n = 60) and men (n = 50) are also presented. The mean serum levels of daidzein and genistein in Japanese women were 62.6 ng/mL (range 0–611 ng/mL) and 135.6 ng/mL (range 0–1133 ng/mL), respectively. The mean equol concentration was13.8 ng/mL (range 0–241 ng/mL) with just 32.5% of the women producing equol concentrations > 5 ng/mL. In British women the mean concentrations of daidzein and genistein were 3.7 and 8.1 ng/mL, respectively. The mean concentrations of genistein and daidzein in the serums from Japanese men were 133.2 ng/mL (range 0–1160 ng/mL) and 71.1 ng/mL (range 0–577 ng/mL), respectively. The mean concentration of the isoflavan equol was 23.8 ng/mL (range 0–461 ng/mL) and 57 (56%) of these samples had equol concentrations > 5 ng/mL. For the British men in this study, the mean concentration of genistein was 9.2 ng/mL (range 0–31.8 ng/mL) and for daidzein was 4.63 ng/mL (range 0–16.4 ng/mL). It is clear from this study that Japanese women and men have much higher levels of genistein and daidzein in their serum than do British women and men, and it is possible that a life-long exposure to these soya-derived phytoestrogens may in part be responsible for the lower incidences of breast and prostate cancer observed in Japanese and other Asian populations.

Genistein and Daidzein—Two Soy-Derived Isoflavones—Inhibit Angiogenesis. Karine Bonjean, Roberto Locigno, Laetitia Devy, Olivier Heymans, Jean-Michel Foidart and Vincent Castronovo. Metastases Research Laboratory and Development and Tumor Biology Laboratory, University of Liège, Belgium.

Genistein and daidzein are the most abundant isoflavonoids in soybeans, soy meal and tofu. These two molecules exhibit anticancer chemopreventive activities. The significantly lower incidence of breast and prostate cancer in populations consuming high amounts of food containing genistein and daidzein has been attributed to their ability to bind to the estrogen receptor and compete with endogenous estrogens. Furthermore, genistein is an inhibitor of tyrosine kinase and decreases breast cancer cell proliferation. A few reports also suggest that genistein is inhibits endothelial cell proliferation and could potentially inhibit angiogenesis. We examined the effect of genistein and daidzein on both the cell cycle and apoptosis of human umbilical vein endothelial cells (HUVEC). We demonstrate that these two molecules inhibit, in a dose-dependent fashion, HUVEC proliferation by blocking the cell cycle at the phases G2/M and G1 and also significantly induce their apoptosis. Genistein and daidzein abolish the sprouting of capillaries from rat aortic rings and inhibit in vivo angiogenesis in the chicken chorioallantoic membrane assay. Interestingly, the combination of both isoflavonoids increases the antiangiogenic effect of these compounds. Our data demonstrate that genistein and daidzein are angiogenesis inhibitors that could be included in the antiangiogenic arsenal currently being developed to fight cancer and other angiogenesis-dependent diseases.

The Specific Role of Genistein in Reducing Hormonal and Proliferative Risk Parameters in Prostate Cancer. N. B. Kumar, J. Pow-Sang, K. Besterman-Dahan and A. Cantor. H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL.

OBJECTIVE. Our objective was to evaluate the effectiveness of a dietary supplement of the isoflavone genistein in producing a change in hormonal and proliferative risk parameters implicated in the promotion of prostate cancer. METHODS. Twenty of 66 patients with prostate cancer grade 1–2, aged 50–80 y, were admitted to the study and randomly assigned to either an experimental group supplemented with soy (60 mg genistein/d) or a control group consuming a placebo for 12 wk. Changes in proliferative and hormonal biomarkers were analyzed at baseline and postintervention. RESULTS. Twenty subjects have completed the study. Preliminary observations indicate a decrease in proliferation (a 6-point increase in the ratio of the percentage of free to total prostate-specific antigen in 47% of the subjects, a decrease in 29% and no change in 24%). Free testosterone decreased > 10 points in 53% of the subjects, increased in 37% and did not change in 10%. CONCLUSIONS. These data suggest that increased genistein intake in patients with early-stage prostate cancer resulted in reduced proliferation of prostatic cells and an alteration in the concentrations of sex hormone implicated in prostate cancer promotion or inhibition. [Supported by American Institute of Cancer Research 99–99BO30.]

Effects of Dietary Soy and CLA on the Growth of Dunning R-3327-AT-1 Prostate Tumor Cells Inoculated into Male Rats. L. A. Cohen, Z. Zhao, B. Pittman and J. Scimeca. The American Health Foundation, Valhalla, NY, and Kraft-General Foods, Glenview, IL.

Conjugated linoleic acid (CLA), a family of positional and geometric isomers of linoleic acid with double bonds in positions 9 and 10 or 11 and 12 (cis or trans), has been shown to confer protection in a number of tumor models. Similarly, soy isoflavones have been reported to act as chemoprotective agents in tumor models and have been proposed to account for the low rates of prostate cancer typical of Asian countries. The mechanisms of action of these agents are unclear at present, but in structure and activity studies they appear to have different molecular targets. Hence, these agents may be more effective given in combination than given individually. The AT-1 line used in this study is a rapidly growing androgen-independent rat prostate cancer cell line that provides a useful model of advanced prostate cancer. The protocol involved 12 experimental groups arranged in a 3 x 4 design. A soy protein isolate rich in genistein and daidzein replaced casein in the AIN-93M diet at 0%, 5%, 10% and 20% by weight and CLA was added as a supplement at 0%, 0.5% and 1% by weight. Cultured AT-1 cells were inoculated subcutaneously into male Copenhagen rats at 0.8 x10⁶ cells/rat. Rats were then placed on experimental diets for 35 d and tumor growth monitored twice weekly. Soy protein isolate and CLA, alone or in combination, did not inhibit the in vivo growth of AT-1 tumor cells. Moreover, at the highest combined concentrations (20% soy protein isolate + 1% CLA) there was a statistically significant increase in tumor volume over that of controls, suggesting a functional antagonism between these two chemopreventive agents. Our results cast doubt on the usefulness of either CLA or soy isoflavones as dietary supplements in controlling the growth of advanced prostate cancer.

cDNA Microarray Analyses of Biochanin A–Induced Changes in Gene Expression in Prostate Cancer (LNCaP) Cells. Lori Rice,* Von G. Samedi, Theresa A. Medrano, Carol A. Sweeney,* Anne Stenstrom and Kathleen T. Shiverick. *Department of Surgery, Division of Urology and Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL.

BACKGROUND. Recent evidence suggests that soy-derived isoflavones inhibit the growth of some types of tumors, including prostate adenocarcinoma, via various pathways including modulation of cell cycle regulators. However, the underlying mechanisms have not been determined. Identifying isoflavone-induced changes in the expressed genome may improve our understanding of how cancer cell proliferation is disrupted. This study describes the effects of the isoflavone biochanin A (a precursor of genistein) on gene expression in human prostate cancer LNCaP cells. METHODS. LNCaP cells were exposed to various doses of biochanin A to determine cytostatic and cytotoxic concentrations. Regulation of gene expression in LNCaP cells exposed to a cytostatic dose of biochanin A was determined by using flow cytometry, Western immunoblotting and cDNA microarray technology. RESULTS. LNCaP cells incubated with biochanin A exhibit a dose-dependent inhibition of proliferation and [H]thymidine incorporation. An eightfold increase in apoptosis in cells treated with a cytostatic dose of biochanin A was observed in cells stained with hemolysin and eosin. Flow cytometry analyses indicate arrest at the G₁ phase of the cell cycle. Although there was little effect on p21, biochanin A produced a 95% decrease in cyclin B protein. cDNA microarray analyses of LNCaP cells treated with cytostatic doses of biochanin A revealed 31 down-regulated genes and expressed sequence tags (ESTs), with 6 dropping below the assay detection limits. Fifteen genes and ESTs were up-regulated, including 9 that were undetectable in control cells. Some of the regulated genes are known to be involved with cell cycle regulation. CONCLUSIONS. The results suggest that biochanin A inhibits prostate cancer cell growth through induction of cell cycle arrest and apoptosis. Biochanin A–regulated genes indicate possible pathways of action and substantiate proliferation and cell cycle assay results that show G₁ arrest. Therefore, microarray analyses proved useful in elucidating mechanisms affected by isoflavone treatment. Studies to characterize biochanin A–regulated genes are underway in our laboratory. [Supported by American Institute for Cancer Research 99A109.]

Presurgical Fat Restriction and Flaxseed Supplementation in Men with Prostate Cancer: Effects on PSA, Hormonal Factors and Proliferation Rates. W. Demark-Wahnefried,* D. T. Price,* T. Polascik,* E. E. Anderson,* P. J. Walther,* and D. F. Paulson,* M. Gannon and R. Vollmer. *Duke University Medical Center and Durham VA Medical Center, Durham, NC.

Previous research in both animals and humans suggests that dietary fat and fiber affect the hormonal and eicosanoid milieu and thus may influence the progression of hormonally linked cancers. We undertook a pilot study to explore whether a flaxseed-supplemented (30 g/d), fat-restricted (20% of energy) diet could affect indexes of prostatic neoplasia in men with early stage prostate cancer. Men adhered to diets for 3–7 wk before prostatectomy. Data now exist on 25 subjects and suggest that this dietary regimen is associated with significant decreases in total testosterone (422 ± 122 to 360 ± 128 ng/dL; P = 0.002), free androgen index (36.3 ± 18.9 to 29.3 ± 16.8%; P = 0.01) and total cholesterol (201 ± 39 to174 ± 42 mg/dL; P < 0.0001). No change was seen in insulin-like growth factor 1 levels. Although no differences were observed in prostate-specific antigen (PSA) levels overall from baseline to follow-up, when men with Gleason sums 7 were excluded from the analysis, a trend toward decreased PSA was noted (7.0 ± 4.7 to 5.5 ± 3.9 ng/dL; P = 0.05). Histological sections from the first 12 diet-treated patients compared with historic controls (matched on age, race, PSA at diagnosis and biopsy results, i.e., Gleason sum, disease laterality and percentage of positive cores) showed a trend toward decreased proliferation (MIB-1). No differences were observed with regard to apoptotic indexes (i.e., TUNEL, bcl-2, p53). These data provide evidence that a flaxseed-supplemented, fat-restricted diet may have a biological effect on prostate cancer that may be mediated through a hormonal mechanism. Continued study is warranted to determine the efficacy of this dietary regimen as either a complementary or preventive therapy.

Role of Antisense Oligonucleotides to the Liver Fatty Acid Binding Protein in Breast and Prostate Cancer Cells. Rasha Hammamieh, Rina Das and Marti Jett. Walter Reed Army Institute of Research, Silver Spring, MD.

Many studies over the past few years have shown a relationship between dietary fat intake and increased incidence and growth of hormonally regulated cancers, such as breast and prostate cancer. Although one thorough study (Holmes et al. 1999 ) did not find a direct correlation between dietary fat and breast cancer, there is a general consensus that the issue is complicated and correlations may depend on individual aspects of lipid metabolism, such as those established in heart disease. Dietary fatty acids such as arachidonic acid are readily metabolized into potent bioactive lipids that have been identified as stimulators of proliferation in breast and prostate cancer cells. The role of lipids and arachidonic acid in prostate tumorigenesis may be mediated by small intracellular proteins called fatty acid binding proteins (FABPs). Our results show that inhibitors of some arachidonate metabolism pathways blocked cell proliferation in DU-145 and MCF-7 cell lines. Furthermore, these inhibitors induced apoptosis in both prostate and breast cancer cells, suggesting an important role for this fatty acid. Using reverse-transcription polymerase chain reaction, we showed that tumor FABPs (in intestine and liver) were 3–14-fold elevated in prostate cancer cells compared with normal prostate cells. Adipose-FABP and epidermal-FABP, however, were down-regulated in cancer cells. To establish the role of L-FABP in prostate cancer cells, we designed a specific antisense oligonucleotide for L-FABP. By labeling the antisense oligonucleotide with a fluorescent probe, we demonstrated that it was taken up into the cell. We have shown that the antisense oligonucleotide of L-FABP was able to inhibit cell proliferation in DU-145 and MCF-7 cell lines. Such data support the contention that certain FABPs correlate with tumorigenicity of the prostate gland. This study will provide the basis for a better understanding of the direct role of fatty acids and bioactive lipids and FABPs in the development and progression of prostate cancer.

Peroxisome Proliferator-Activated Receptor Gamma in Prostate Cancer Progression. Leanne Nause, Patricia Sheppard, Jacquie Schwartz and Janice G. Dodd. Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada.

Large geographical variations exist in the incidence of clinical prostate cancer whereas the incidence microfocal disease is essentially constant worldwide. Dietary fat is implicated in this marked difference in the progression of latent cancers. A putative effector is the peroxisome proliferator–activated receptor gamma (PPAR), a nuclear transcription factor that is elevated in human prostate cancers. Activation of PPAR in late-stage human prostate cancer cell lines and tumor tissue leads to cell death; however, the role of PPAR in early progression of prostatic intraepithelial neoplasia (PIN) to invasive carcinoma is unknown and difficult to study in human subjects. To address early events in tumorigenesis, we have established a transgenic mouse model for prostate cancer (designated LPB-Tag) in which progression of PIN-like lesions can be assessed by histology and known biomarkers, including secreted proteins that reflect the differentiation state of the prostatic epithelium. As in human prostate tumors, PPAR expression in the LPB-Tag tumor model is highly elevated relative to normal prostate tissue. Both PPAR1 and 2 isoforms are present; PPAR2 increases markedly with tumor progression, suggesting a mechanism for altered gene expression in the tumors. PPAR levels are enhanced after androgen ablation and suppressed by androgen replacement in both normal prostate and tumors. Oral administration of thiazolidinediaone to activate PPAR alters the isoform profile of PPAR and tumor growth and differentiation. These preliminary data suggest that progression of premalignant lesions to invasive cancer may be altered by interventions targeted to PPAR. Even modest changes in the rate of progression could result in substantial reductions in the incidence of clinically relevant prostate cancer.

Lyprinol, a Lipid Extract from the New Zealand Green-Lipped Mussel, Is a Lipoxygenase Inhibitor That Induces Apoptosis in Human Tumor Cells. C. L. Zhang,* W. H. Betts,* F. Chai and P. D. Zalewski. *Rheumatology Unit and Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, Adelaide, South Australia.

Metabolites of the lipoxygenase (LOX) pathway play a role in tumor growth. 5-Hydroxyeicosatetraenoic acid (5-HETE) from the 5-LOX pathway affects tumor growth and suppresses tumor cell programmed cell death (apoptosis) whereas 12-HETE from the 12-LOX pathway plays a role in tumor metastasis. Lyprinol, a lipid extracted from the New Zealand green-lipped mussel, which is readily available as a nutritional supplement, has been widely used to treat inflammatory conditions such as rheumatoid arthritis and asthma. In this study we demonstrate that Lyprinol is a potent inhibitor of the human neutrophil 5-LOX pathway and platelet 12-LOX pathway. This finding suggests that Lyprinol may have a role in the treatment of cancer via its effects on these pathways. In preliminary studies using cultured human bronchioepithelial carcinoma cells (NCI-H292) and colorectal cancer cells (LIM1215), Lyprinol induced significant apoptosis as measured by increased caspase-3 activity and DNA fragmentation. Phase-contrast microscopy and Hoechst dye staining also revealed typical morphological changes associated with apoptosis, such as cell membrane blebbing and shrinkage, cell rounding and detachment and chromatin condensation. In cultured human prostate cancer cells (hormone-dependent LnCaP and hormone-independent PC-3 cells), we have demonstrated that Lyprinol (10–50 µg/mL) induced dose- and time-dependent cell death, as measured by lactate dehydrogenase release. It also induced similar morphological apoptotic changes to those seen with another 5-LOX inhibitor, MK886. As measured by a DNA fragmentation enzyme-linked immunosorbent assay, Lyprinol (25 µg/mL) was equipotent to MK886 (10 µmol/L). These effects were seen as early as 1 h after Lyprinol addition. In view of the effectiveness of Lyprinol in the treatment of inflammation and the pronounced lack of side effects, our results suggest a potential role for this marine-derived lipid in the treatment of human cancers.

Role of 12-Lipoxgygenase-Mediated Arachidonic Acid Metabolism in Human Prostate Tumor Growth, Angiogenesis and Metastasis. Daotai Nie,* Yuchyu Chen,* Keqin Tang,* Gilda G Hillman,* David Grignon and Kenneth V Honn*. Departments of *Radiation Oncology and Pathology, Wayne State University and Karmanos Cancer Institute, Detroit, MI.

Cellular metabolism of free fatty acids can generate a number of bioactive lipids. 12(S)-Hydroxyeicosatetraenoic acid (HETE), an eicosanoid with potent biological activities including promoting tumor progression, is formed through 12-lipoxygenase (LOX)–mediated arachidonic acid metabolism. Previously, we found that platelet-type 12-LOX is expressed in human prostate cancer and that its level of expression is positively correlated with tumor stage (Gao et al. 1995 ). When overexpressed in human prostate cancer PC-3 cells, 12-LOX stimulated prostate tumor growth by increasing the angiogenic potential of cancer cells (Nie et al. 1998 ). Here we present evidence indicating that the expression of vascular endothelial growth factor (VEGF), a putative angiogenic factor, is regulated by 12-LOX and 12(S)-HETE in PC-3 cells. In 12-LOX–transfected PC-3 cells, VEGF production in culture media was increased threefold over that from vector controls. Northern blot analysis found an increase in VEGF mRNA levels in 12-LOX–transfected PC-3 cells. Using VEGF promoter luciferase constructs, we found a 10-fold increase in VEGF promoter activity in 12-LOX–transfected PC-3 cells. Inhibition of 12-LOX activity with nordihydroguaiaretic acid, a general LOX inhibitor, and baicalein, a specific 12-LOX inhibitor, reduced VEGF production in PC-3 cells. Neutralization of VEGF function by a function-blocking antibody significantly decreased the ability of 12-LOX–transfected PC-3 cells to stimulate endothelial cell migration, suggesting that the increased VEGF production in 12-LOX–transfected PC-3 cells accounts, at least partially, for the increased angiogenic potential of 12-LOX–transfected PC-3 cells. We also present data indicating that 12-LOX enhances the metastatic potential of human prostate cancer cells and that a 12-LOX inhibitor, BMD188, reduced the growth of prostate tumors in a severe combined immunodeficient (SCID) mice human bone model. Our data provide insights into the multifaceted effects of arachidonic acid metabolites such as 12(S)-HETE on tumor progression.

A Nutritional Supplement Fortified with Fish Oil Reverses Weight Loss in Patients with Advanced Pancreatic Cancer. K. Ried,* K. Mayer,* K. Fearon and A. Voss*. *Ross Products Division, Columbus, OH, Department of Surgery, Royal Infirmary of Edinburgh, Scotland.

The aim of the present study was to see whether combining eicosapentaenoic acid (EPA) with a nutritional supplement might lead to net weight gain in patients with advanced pancreatic cancer. Twenty patients were asked to consume 2 cans daily of a nutritional supplement fortified with fish oil in addition to their normal food intake. Each can contained 310 kcal, 16 g protein and 1.09 g EPA. Weight, body composition, dietary intake, resting energy expenditure (REE) and performance status were measured at baseline and after 3 wk. No intervention-related adverse events were observed apart from steatorrhea, which responded to pancreatic enzyme replacement. Patients consumed a median 1.9 cans/d. All patients were losing weight at baseline at a median rate of 2.9 kg/mo (interquartile range 3.9–2.1 kg/mo). After administration of the supplement, patients had significant weight gain at 3 (median 1 kg, range 0–2 kg, P = 0.024) and 7 wk (median 2.5 kg, range 0.1–4.6 kg, P = 0.028). There was no change in percentage of total body water, suggesting that weight gain was not due to the accumulation of water. Analysis of body composition revealed a significant gain in lean body mass and no change in fat mass after 3 and 7 wk. Dietary intake increased significantly by >370 kcal/d (P = 0.0016). Although overall REE remained unchanged (P = 0.18), REE per kg body weight and per kg lean body mass fell significantly (P < 0.03). Performance status was significantly improved at 3 and 7 wk (P < 0.05). In contrast to previous studies of conventional oral nutritional supplements in weight-losing cancer patients that have shown no benefit, this study suggests that a nutritional supplement fortified with fish oil may reverse cachexia in advanced pancreatic cancer.

Preliminary Results of a Phase I Clinical Trial of Dietary Methionine Restriction for Adults with Refractory Solid Tumors. Daniel E. Epner* and Sydney Morrow. *Department of Medicine, Division of Oncology, Baylor College of Medicine and Houston VA Medical Center and Nutrition and Food Service, Houston VA Medical Center, Houston, TX.

Dietary methionine restriction inhibits growth of a variety of human tumor xenografts by causing cancer cells to arrest predominantly in the G2 phase of the cell cycle and to eventually undergo apoptosis. In contrast, methionine restriction is well tolerated by normal host tissues for prolonged periods. On the basis of these preclinical observations, we began a Phase I clinical trial of dietary methionine restriction for adults with refractory solid tumors at Baylor College of Medicine in April 1999. Seven patients have been enrolled so far. Patients in the trial have been prescribed 0.8 g methionine-free protein · kg^-1 · d^-1 and have received no cancer treatments other than the dietary modification. Dietary methionine restriction has reduced plasma methionine levels by 80% without affecting levels of other amino acids or albumin. The observed reduction in plasma methionine levels is therapeutically relevant, because similar reductions inhibit growth of human cancer cells in culture. The fact that albumin levels have remained stable indicates that the experimental diet does not indiscriminately block protein synthesis. Side effects include weight loss of approximately 0.5 kg/wk and mild fatigue, both of which are reversible. Although the trial was designed primarily to assess safety, three of the seven patients treated so far have shown objective responses. One patient with hormone-independent prostate cancer experienced a 25% reduction in serum prostate-specific antigen after 12 wk of the trial. Two other patients showed radiographic evidence of tumor regression. Although preliminary, these results strongly suggest that dietary methionine restriction has great promise as a novel treatment strategy for advanced cancer, either alone or in combination with other treatments. [Supported by Ross Products Division, Abbott Laboratories; the General Clinical Research Center at Baylor College of Medicine and the Houston VA Medical Center.]

Inhibition of AP-1/DNA Binding by Dietary Energy Restriction in Nuclear Extracts of Acetone- and TPA-treated SENCAR Mouse Epidermis Is Reversed by Adrenalectomy. Joseph Przybyszewski, Weiqun Wang, Angela Au, Jeanne Stewart and Diane F. Birt. Department of Food Science and Human Nutrition, Iowa State University Ames, IA.

Dietary energy restriction has been shown to significantly inhibit mouse skin carcinogenesis (Birt et al. 1991 ); however, the molecular mechanisms for the prevention of carcinogenesis by energy restriction are largely unknown. Pashko and Schwartz (1992) demonstrated that the adrenal gland was important for the inhibition of papilloma formation in food-restricted mice treated with dimethyl-benz[a]anthracene and 12-O' tetradecanoyl phorbol-13-acetate (TPA). Our study measured the binding of transcription factor activator protein-1 (AP-1) to its consensus DNA sequence by the electrophoretic mobility shift assay in epidermal nuclear extracts of acetone- vs. TPA-treated, adrenalectomized vs. intact and energy-restricted vs. energy-nonrestricted SENCAR mice. To examine the effect of the glucocorticoid hormone corticosterone (CCS) on AP-1/DNA binding, CCS was added to the drinking water of adrenalectomized mice and measured in blood plasma of killed mice. This study demonstrated that 1) overall, TPA produced a significant twofold induction in AP-1/DNA binding over acetone controls (P < 0.0001), 2) dietary energy restriction significantly reduced both basal (acetone) and TPA-induced AP-1/DNA binding in nonenergy-restricted mice (P < 0.05), 3) adrenalectomy significantly reduced TPA-induced AP-1/DNA binding in nonenergy-restricted mice (P < 0.05) but significantly increased TPA-induced AP-1/DNA binding in energy-restricted mice (P < 0.05), 4) supplementation of corticosterone to adrenalectomized energy-restricted mice reduces the AP-1/DNA binding level to near that of sham-operated mice and 5) adrenalectomy significantly reduced CCS blood levels over that of sham-operated mice (P < 0.05) whereas CCS supplementation restored CCS levels in adrenalectomized mice. Thus, dietary energy restriction may inhibit cancer mechanistically by reducing overall AP-1 transcription through a process that is mediated in part by the glucocorticoid hormone. [Funded by grants from the American Institute for Cancer Research and National Institutes of Health.]

Dietary Energy Restriction Inhibits Estrogen-Induced Mammary Carcinogenesis in the Female ACI Rat. D.M.E. Harvell,* T. E. Strecker,* B. Xie,* K. L. Pennington,* R. D. McComb and J. D. Shull*. *Eppley Institute for Research in Cancer, Department of Pathology and Microbiology, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.

The female ACI rat is a unique and physiologically relevant animal model for the study of breast cancer etiology in that it is highly susceptible to the development of estrogen-induced mammary cancer but rarely develops mammary cancer spontaneously. Data from our laboratory indicate that the genetic basis of the susceptibility of the ACI rat to estrogen-induced mammary cancer development is distinct from the more widely used dimethyl-benz[a]anthracene–induced mammary cancer model. It is well established that dietary energy restriction inhibits carcinogen-induced mammary carcinogenesis in rodents. However, the effect of dietary energy restriction on estrogen-induced mammary cancer development has yet to be determined. The purpose of the present study was to investigate whether dietary energy restriction inhibits mammary carcinogenesis induced by 17beta-estradiol (E₂) in female ACI rats. To test this hypothesis, we examined the incidence of mammary tumors and precancerous lesions in E₂-treated female ACI rats fed a 40% energy-restricted diet. The effects of dietary energy restriction and estrogen on mammary tumor latency, multiplicity and size were also examined. To address the mechanisms through which dietary energy restriction might exert its inhibitory effects on E₂-induced mammary cancer development, we examined progesterone receptor expression and cell proliferation in the mammary epithelium of E₂-treated rats fed the energy-restricted diet. The data presented indicate that dietary energy restriction inhibits E₂-induced mammary cancer development in female ACI rats apparently by inhibiting the progression of precursor lesions into carcinomas. The potential involvement of progesterone receptor expression and mammary cell proliferation in the ability of dietary energy restriction to inhibit E₂-induced mammary carcinogenesis is also discussed. [Supported by grant 97A146 from the American Institute for Cancer Research and grants CA68529 and CA77876 from National Institutes of Health.]

Calcium Intake and Prostate Cancer Risk in Men from the CLUE II Cohort, Washington County, Maryland. C. J. Kavanaugh* and E. A. Platz for CLUE II Investigators. *National Cancer Institute, Bethesda, MD, and School of Public Health and Hygiene, Johns Hopkins University, Baltimore, MD.

BACKGROUND. Prostate cancer is one of the leading causes of cancer death in men in the United States. Few risk factors have been identified for prostate cancer and most of these—such as age, race and family history—are not modifiable. However, calcium intake has been associated with increased risk of prostate cancer in several epidemiological studies and thus could be a modifiable dietary risk factor. Because calcium consumption is being promoted for its health benefits for osteoporosis, it is an important public health issue to determine whether increased calcium intake is associated with any adverse health effects. METHODS. We prospectively evaluated calcium intake and prostate cancer risk from 1989 to 1999 in 5719 men aged 35–94 y from the Washington County, Maryland, CLUE II cohort. Intake of dairy foods, calcium supplements and dairy calcium were calculated by using a baseline food frequency questionnaire. Two hundred and thirty-nine incident prostate cancer cases were confirmed by using the Maryland and Washington County tumor registries. We estimated relative risk (RR) and 95% confidence interval (CI) for prostate cancer associated with quintiles of dairy product intake from logistic regression models controlling for age, current smoking status, daily servings of tomato products (measure of lycopene) and body mass index at age 21. RESULTS. Compared with the bottom quintile (Q1) of dairy calcium consumption, the adjusted RRs were 1.17 (95% CI 0.76–1.80) for Q2, 1.37 (95% CI 0.90–2.08) for Q3, 1.70 (95% CI 1.12–2.57) for Q4 and 1.22 (95% CI 0.77–1.94) for Q5 adjusted for age, smoking status, tomato servings and body mass index at age 21. CONCLUSION. These data do not support a large increase in risk of prostate cancer among men with higher intake of dairy calcium.

Reduction in DNA Damage in Prostate of Elderly Dogs Supplemented with Selenomethionine. S. Shen,* D. M. Cooley,* L. T. Glickman,* * C. Oteham,* G. F.Combs JR and D. J. Waters*^,. *Departments of Veterinary Clinical Sciences and **Veterinary Pathobiology, Purdue University, West Lafayette, IN; Division of Nutritional Sciences, Cornell University, Ithaca, NY and Gerald P. Murphy Cancer Foundation, Seattle, WA.

During the next 12 y, the National Cancer Institute–sponsored PCPT-2 SELECT trial will study > 32,000 men to determine whether daily supplementation with selenium with and without vitamin E decreases the incidence of prostate cancer. However, the molecular mechanisms by which supranutritional selenium modulates key events in multistep prostate carcinogenesis remains unclear. We used the dog model to test the hypothesis that supranutritional selenium exerts a DNA damage–sparing effect on the aged prostate. Elderly (9–10.5-y) male beagle dogs were randomly assigned to groups: no treatment (sexually intact controls; n = 4 dogs), selenomethionine at 3 µg · kg^-1 · d^-1 (n = 5 dogs) and selenomethionine at 6 µg · kg^-1 · d^-1 (n = 5 dogs). After 7 mo of treatment, dogs were killed and single-cell gel electrophoresis (alkaline comet assay) was used to measure basal levels of DNA damage in freshly harvested prostate tissue. Mean (± SD) percentage of cells with extensively damaged DNA was compared between treatment groups by using ANOVA. Dogs in the low- and high-dose selenium groups had significantly reduced basal levels of DNA damage compared with age-matched controls. Mean (± SD) percentage of cells with extensive DNA damage was 81% ± 3% and 54% ± 5% for control dogs and selenium-supplemented dogs, respectively (P < 0.0001). There was no significant difference in mean serum glutathione peroxidase activity between the control and selenium-supplemented groups. These data show that supranutritional selenium exerts a DNA damage–sparing effect within the aged prostate. This model enables us to study the mechanism of selenium supplementation in a model of selenium adequacy, which is the case for almost all healthy Americans, including those who will participate in the SELECT trial. [Supported in part by a grant from the USAMRMC Prostate Cancer Research Program (PC-970492 awarded to DJW).]

Estrogen Effects on Iron Uptake by Human Mammary Cells Lines. J. Shawn Jones, Mary L. Thomas and Joachim G. Liehr*. University of Texas Medical Branch at Galveston and *Stehlin Foundation, Houston, TX.

Iron is an essential nutrient in the metabolic processes and the proliferation of cells, but iron overload has been associated with increased risk for cancer. We showed that feeding a diet high in iron doubled the incidence of estrogen-induced kidney tumors in hamsters (Wyllie and Liehr 1998). Estrogen also induced an increase in iron accumulation in hamster kidney (unpublished data). We hypothesize that estrogen-induced iron accumulation precedes estrogen-induced tumor formation. Thus, cells susceptible to development of estrogen-induced tumors will also exhibit estrogen-stimulated iron accumulation. The excess iron may cause free radical–mediated cell damage and contribute to tumor formation. We are investigating this hypothesis by studying estrogen-induced iron accumulation in two human breast cell lines: MCF7 tumor and MCF10A nontumor cells. Cells were treated with estradiol (E₂) at 1 pmol/L to 1 µmol/L for 1, 3 or 7 d. For the final 24 h of each treatment period, ⁵⁹Fe was present in the medium. The cells were lysed and ⁵⁹Fe was counted. Values were normalized to total protein. Baseline ⁵⁹Fe uptake differed between the cell lines and was passage-dependent in MCF10A cells. E₂ increased ⁵⁹Fe uptake in MCF7 breast cancer cells (but not MCF10A cells) with maximal effects at 7 d. E₂ effects were blocked by the estrogen-receptor antagonist ICI 182780. These data demonstrate that regulation of iron uptake is altered in MCF7 cancer cells and that E₂ further modifies iron uptake in these cells. Altered iron metabolism may lead to perpetual free radical–mediated mutagenesis followed by carcinogenesis, metastasis and drug resistance, indicating a role for iron in hormone-associated cancers such as breast cancer. [Supported National Institutes of Health, National Cancer Institute CA 74971.] Wyllie and Liehr. 1998 Carcinogenesis 19:1285–1290.

Changes in Body Composition, Metabolic Rates, Energy Intake and Physical Activity in the Year after the Diagnosis of Breast Cancer. Comparing Patients Who Receive Adjuvant Chemotherapy with Those Who Receive Localized Treatment. W. Demark-Wahnefried,* B. Peterson,* P. K. Marcom,* E. P. Winer, N. Aziz and B. K. Rimer. *Duke University Medical Center, Durham, NC; Dana Farber Medical Center, Boston, MA and National Cancer Institute, Bethesda, MD.

For >20 y, weight gain has been a commonly reported side effect of adjuvant chemotherapy (CT) for breast cancer. To determine causes for this energy imbalance, we undertook a study that assessed body weight and composition (via dual energy X-ray absorptiometry [DXA]), resting metabolic rate (RMR; via indirect calorimetry), energy intake (via 2-d 24-h dietary recalls) and physical activity (Stanford 5-City Physical Activity Questionnaires) throughout the year after the diagnosis of Stage I or II breast cancer in premenopausal patients who received CT compared with patients who received localized treatment (LT; surgery with or without radiation). Body weight and RMRs were measured at baseline and at 2, 6 and 12 mo; DXA was performed at baseline and at 6 and 12 mo; energy intake was assessed semimonthly and physical activity was assessed weekly. Analyses to determine differences in linear trends over time were performed (two-tailed tests; = 0.05). Preliminary analyses on complete data from 53 participants (n = 36 in the CT group, n = 17 in the LT group) suggest that there are significant gains in body weight by patients who receive CT compared with patients who receive LT (2.7 ± 4.5 vs. 1.0 ± 1.4 kg/y; P = 0.04). Furthermore, percent body fat increased from 33.6% ± 8.6% at baseline to 35.8% ± 9.0% at 1 y in the CT group whereas no change was noted in the LT group (i.e., 28.6% ± 6.7% to 28.5% ± 7.0%) (age- and race-controlled P-value = 0.001). Significant differences between groups also were seen with regard to lean body mass (P = 0.002), especially in the leg region (P = 0.01), where the CT group experienced losses and the LT group experienced slight gains. Decreases in physical activity were observed from baseline to follow-up in the AC group (506 ± 101 vs. 458 ± 114 kcal/d; P = 0.017), however, were not seen in the LT group. No differences over time or between groups were noted with regard to RMR or energy intake. These data support the need for early interventions focused on increased physical activity in order to reduce gains in weight and percent body fat by young women with breast cancer.

Expression and Function of Thromboxane A₂ Synthase in Human Prostate Carcinoma. Daotai Nie,* Alex Zacharek,* Li Li,* Mario Lamberti,* Keqin Tang,* Gilda G Hillman,* David Grignon and Kenneth V Honn*. *Departments Radiation Oncology and Pathology, Wayne State University School of Medicine and Karmanos Cancer Institute, Detroit, MI.

Thromboxane (TX) synthase metabolizes a cyclooxygenase (COX) product, prostanglandin H₂, into thromboxane A₂ (TXA₂), which is a potent modulator of platelet aggregation. Here we report that human prostate cancer (PCa) cells express TX synthase and that this enzyme is involved in PCa cell motility and tumor growth in vivo. Immunohistochemical analysis of PCa specimens revealed that 58% of cases (n = 24) have moderate or strong expression of TX synthase. In human PCa cell lines, PC-3, PC-3M and ML-2 cells expressed higher levels of TX synthase than did normal prostate epithelial cells or other PCa cell lines such as DU145. TXA₂ production was reduced by 86% in PC-3 cells when they were treated with carboxyheptal imidazole (CI), an inhibitor of TX synthase, at 10 µmol/L. Treatment of PC-3 cells with a COX-1–specific inhibitor, piroxicam, reduced TXA₂ synthesis by 45–50% whereas the COX-2–specific inhibitor NS398 reduced TXA₂ production by 75–80%, suggesting that TX synthase activity is dependent on COX-2 and to a lesser extent COX-1 to supply the substrate prostanglandin H₂. Inhibition of TX synthase activity with CI significantly reduced PC-3 cell migration on fibronectin in vitro and the growth of tumors derived from PC-3 cells in vivo. Our data suggest that human PCa cells express TX synthase and that this enzyme may modulate PCa growth and progression.

How Much Colorectal Cancer Could Be Prevented by Lowering Meat Consumption? A Meta-analysis of Epidemiological Studies. T. Norat, A. Lukanova, P. Ferrari and E. Riboli. International Agency for Research on Cancer, Lyon, France.

BACKGROUND. Cancer of the colon and rectum is the fourth most common cancer in terms of both incidence and mortality throughout the world. Several dietary and lifestyle factors can possibly modify colorectal cancer risk. The epidemiological evidence reviewed in 1997 (World Cancer Research Fund Panel) supported the conclusion that red meat intake probably increases colorectal cancer risk and processed meat possibly increases it. Some hypothesis about the mechanisms have been proposed, such as the facilitating effect of fat on bile acid production and the formation of carcinogens when meat is cooked or processed. In the present study, a meta-analysis was carried out to summarize the published data on the relationship between total meat, red meat and processed meat and colorectal cancer risk. METHODS. The inclusion criteria of studies were case-control and cohort studies on total meat, red meat or processed meat and colorectal cancer risk that were published in English between 1973 and 1999, were referenced in Medline and reported data necessary for the statistical analysis. The results were summarized in two ways: average relative risk and relative risk per gram of intake estimated from the dose-response linear trend. Random and fixed effect models were used. The proportion of colorectal cancer risk that can be attributed to meat consumption worldwide was estimated by using incidence figures from different geographical areas and meat intakes derived from FAO food balance sheets and national nutritional surveys. The proportion of cancer incidence that could potentially be avoided by a change in meat consumption pattern was estimated by assuming different levels of reduction in meat consumption. RESULTS. Thirteen cohort and 33 case-control studies on meat consumption and colorectal cancer risk were published between 1973 and 1999. Twenty-six studies on meat consumption, 21 on red meat and 24 on processed meat met the inclusion criteria. Summary relative risk (RR) estimates and their 95% confidence intervals based on a random effect model for highest consumption compared with lowest consumption category as reported in the original papers were total meat: RR = 1.16 (95% CI: 1.07–1.25), red meat: RR = 1.30 (95% CI: 1.21–1.38) and processed meat: RR = 1.21 (95% CI: 1.13–1.31). The slope of the linear dose-response relationship was estimated from 19 studies on total meat, 18 on red meat and 16 on processed meat consumption that reported the information necessary to do the analysis. The relative risks per 80 g/d are total meat: RR = 1.18, red meat: RR = 1.25 and processed meat: RR = 1.67. Based on the above risk estimates and the current consumption levels, the estimated worldwide population-attributable fraction for red meat consumption is 11%. A hypothetical reduction of average red meat consumption to 10 g/d would lead to an 8% reduction of colorectal cancer incidence in the world. However, in regions characterized by a high intake of red meat, such as Australia, New Zealand, North America and South America, both the population-attributable fraction and the potentially preventable fraction are substantially higher, on the order of 20% and 18%, respectively. These results corroborate the conclusions previously reached by both the World Cancer Research Fund Panel and the UK Department of Health reports regarding the association between meat and colorectal cancer risk and suggest that the effect of reducing average red meat consumption to the equivalent of one serving per week is nonnegligible in populations with Western lifestyle whereas it is almost trivial in Africa and most Asian countries, where red meat consumption has been traditionally low.

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