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The following are abstracts of three seminal papers that discuss the molecular biology of DIM in immune response enhancement. All papers are available in most biomedical libraries or electronic medical libraries on the Internet.

3,3′-Diindolylmethane stimulates murine immune function in vitro and in vivo,  Xue L, Pestka J, Maoxiang L, Firestone GL, Bjeldanes LF,  Journal of Nutritional Biochemistry, published online, 8-20-07. Department of Nutritional Sciences and Toxicology, University of California, Berkeley, 94720-3104, USA.

3,3′-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol, exhibits chemopreventive properties in animal models of cancer. Recent studies have shown that DIM stimulates interferon-gamma (IFN-γ) production and potentiates the IFN-γ signaling pathway in human breast cancer cells via a mechanism that includes increased expression of the IFN-γ receptor. The goal of this study was to test the hypothesis that DIM modulates the murine immune function. Specifically, the effects of DIM were evaluated in a panel of murine immune function tests that included splenocyte proliferation, reactive oxygen species (ROS) generation, cytokine production and resistance to viral infection. DIM was found to induce proliferation of splenocytes as well as augment mitogen- and interleukin (IL)-2-induced splenocyte proliferation. DIM also stimulated the production of ROS by murine peritoneal macrophage cultures. Oral administration of DIM, but not intraperitoneal injection, induced elevation of serum cytokines in mice, including IL-6, granulocyte colony-stimulating factor (G-CSF), IL-12 and IFN-γ. Finally, in a model of enteric virus infection, oral DIM administration to mice enhanced both clearance of reovirus from the GI tract and the subsequent mucosal IgA response. Thus, DIM is a potent stimulator of immune function. This property might contribute to the cancer inhibitory effects of this indole.

Activation and potentiation of interferon-gamma signaling by 3,3'-diindolylmethane in MCF-7 breast cancer cells. Riby JE, Xue L, Chatterji U, Bjeldanes EL, Firestone GL, Bjeldanes LF. Department of Nutritional Sciences and Toxicology, University of California, Berkeley, 94720-3104, USA. Molecular Pharmacology. 2006 Feb;69(2):430-9.

3,3'-Diindolylmethane (DIM), a natural autolytic product in plants of the Brassica genus, including broccoli, cauliflower, and Brussels sprouts, exhibits promising cancer protective activities, especially against mammary neoplasia in animal models. We observed previously that DIM induced a G(1) cell-cycle arrest and strong induction of cell-cycle inhibitor p21 expression and promoter activity in both estrogen-responsive and -independent breast cancer cell lines. We showed recently that DIM up-regulates the expression of interferon gamma (IFNgamma) in human MCF-7 breast cancer cells. This novel effect may contribute to the anticancer effects of DIM because IFNgamma plays an important role in preventing the development of primary and transplanted tumors. In this study, we observed that DIM activated the IFNgamma signaling pathway in human breast cancer cells. DIM activated the expression of the IFNgamma receptor (IFNGR1) and IFNgamma-responsive genes p56- and p69-oligoadenylate synthase (OAS). In cotreatments with IFNgamma, DIM produced an additive activation of endogenous p69-OAS and of an OAS-Luc reporter and a synergistic activation of a GAS-Luc reporter. DIM synergistically augmented the IFNgamma induced phosphorylation of signal transducer and activator of transcription factor 1, further evidence of DIM activation of the IFNgamma pathway. DIM and IFNgamma produced an additive inhibition of cell proliferation and a synergistic increase in levels of major histocompatibility complex class-1 (MHC-1) expression, accompanied by increased levels of mRNAs of MHC-1-associated proteins and transporters. These results reveal novel immune activating and potentiating activities of DIM in human tumor cells that may contribute to the established effectiveness of this dietary indole against various tumor types.
 

DIM stimulates IFNgamma gene expression in human breast cancer cells via the specific activation of JNK and p38 pathways. Xue L, Firestone GL, Bjeldanes LF. Department of Nutritional Sciences and Toxicology, University of California, 119 Morgan Hall, Berkeley, CA 94720-3104, USA. Oncogene. 2005 Mar 31;24(14):2343-53.

3,3'-Diindolylmethane (DIM) is a promising anticancer agent derived from Brassica vegetables, but the mechanisms of DIM action are largely unknown. We have shown that DIM can upregulate the expression and stimulate the secretion of interferon-gamma (IFNgamma) in the human MCF-7 breast cancer cell line. This novel effect may provide important clues to explain the anticancer effects of DIM because it is well known that IFNgamma plays an important role in preventing the development of primary and transplanted tumors. Utilizing promoter deletions, we show here that the region between -108 and -36 bp in the IFNgamma promoter, which contains two conserved and essential regulatory elements, is required for DIM-induced IFNgamma expression. DIM activates both JNK and p38 pathways, induces the phosphorylation of c-Jun and ATF-2, and increases the binding of the homodimer or heterodimer of c-Jun/ATF-2 to the proximal AP-1.CREB-ATF-binding element. Moreover, studies with specific enzyme inhibitors showed that up-stream Ca2+-dependent kinase(s) is required for the inducing effects of DIM in MCF-7 cells. These results establish that DIM-induced IFNgamma expression in human breast tumor cells is mediated by activation of both JNK and p38 pathways, which is ultimately dependent on intracellular calcium signaling.

The following papers provide additional scientific references regarding DIM and the other important ingredients in ActivaMune:

Diindolylmethane is a novel topoisomerase II alpha catalytic inhibitor that induces S-phase retardation and mitotic delay in human hepatoma HepG2 cells. Mol Pharmacol. 2006 Apr;69(4):1320-7. Gong Y, Firestone GL, Bjeldanes LF.Department of Nutritional Sciences and Toxicology, 119 Morgan Hall, University of California, Berkeley, CA 94720-3104, USA.

Epidemiological evidence suggests that high consumption of Brassica genus vegetables, such as broccoli, cabbage, and Brussels sprouts, is very effective in reducing the risks of several types of cancers. 3,3'-Diindolylmethane (DIM), one of the most abundant and biologically active dietary compounds derived from Brassica genus vegetables, displays remarkable antitumor activity against several experimental tumors. In the present study, we demonstrate for the first time that DIM is a novel catalytic topoisomerase IIalpha inhibitor. In supercoiled DNA relaxation assay and kinetoplast DNA decatenation assay, DIM strongly inhibited DNA topoisomerase IIalpha and also partially inhibited DNA topoisomerases I and IIbeta. DIM did not stabilize DNA cleavage complex and did not prevent etoposide-induced DNA cleavage complex formation. Further experiments showed that DIM inhibited topoisomerase IIalpha-catalyzed ATP hydrolysis, which is a necessary step for the enzyme turnover. In cultured human hepatoma HepG2 cells, DIM blocked DNA synthesis and mitosis in a concentration-dependent manner, which was consistent with the outcome of topoisomerase inhibition in these cell-cycle phases. Our results identified a new mode of action for this intriguing dietary component that might be exploited for therapeutic development.

3,3′-Diindolylmethane and Paclitaxel Act Synergistically to Promote Apoptosis in HER2/Neu Human Breast Cancer Cells. Journal of Surgical Research, 2006 May 15;132(2):208-13. K. McGuire, N. Ngoubilly, M. Neavyn, S. Lanza-Jacoby Department of Surgery, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

HER2/neu positive breast tumors are difficult to treat. About 25 to 30% of invasive breast tumors overexpress the HER2/neu oncogene. These tumors are aggressive and become resistant to chemotherapeutic drugs. 3'3'-diindolylmethane (DIM), the active metabolite of indole-3-carbinol, a naturally occurring compound found in cruciferous vegetables, has been found to have anti-cancer properties in both humans and animals. DIM has been shown to induce cell cycle arrest and apoptosis in animal breast cancer models. Because HER2/neu overexpression confers resistance to paclitaxel, and DIM has anti-tumor effects, we hypothesized that DIM will enhance the cytotoxic effects of paclitaxel, a common taxane drug, on human Her2/neu breast cancer cells by potentiating its effect on cell cycle and stimulating apoptosis. METHODS: The MDA-MB-435eB1 human Her2/neu breast cancer cells were treated with varying concentrations of DIM and paclitaxel. The cells were analyzed at different time points (24, 48, and 72 h). Proliferation was measured by a commercial cell proliferation assay (Promega Procheck Assay). Cell-cycle analysis and apoptosis were determined by flow cytometry. Western blot analysis was performed on to determine the effect of DIM and/or paclitaxel on the proteins involved in apoptosis, and epidermal growth factor-induced activation of HER2/neu and ERK1/2 signaling proteins. RESULTS: Both DIM and paclitaxel exhibited time and concentration dependent inhibition of cell proliferation. TUNEL assay indicated that the combination also increased the number of apoptotic cells more than either agent alone. The presence of cleaved poly (ADP-Ribose) polymerase (PARP) significantly increased in the combination treatment, whereas Bcl-2 is decreased. DIM alone decreased the activation of the Her2/neu receptor; the combination decreased the activation of ERK1/ERK2. CONCLUSIONS: DIM in combination with paclitaxel synergistically inhibits growth of Her2/neu human breast cancer cells through G2M phase cell-cycle arrest and induction of apoptosis/necrosis. The Her2/neu receptor and its downstream signaling protein ERK1/2 appear to be involved in DIM's affect on cell growth and differentiation, whereas apoptosis appears to be mediated through the mitochondrial pathway (Bcl-2/PARP). It appears DIM, a naturally occurring, nontoxic compound, may be a beneficial addition to a traditional (taxane-based) chemotherapy regimen.

Pilot study: effect of 3,3'-diindolylmethane supplements on urinary hormone metabolites in postmenopausal women with a history of early-stage breast cancer. Journal of Nutrition and Cancer. 2004;50(2):161-7. Dalessandri KM, Firestone GL, Fitch MD, Bradlow HL, Bjeldanes LF Department of Molecular and Cell Biology, University of California, Berkeley, 94720-3200, USA.

Dietary indoles, present in Brassica plants such as cabbage, broccoli, and Brussels sprouts, have been shown to provide potential protection against hormone-dependent cancers. 3,3'-Diindolylmethane (DIM) is under study as one of the main protective indole metabolites. Postmenopausal women aged 50-70 yr from Marin County, California, with a history of early-stage breast cancer, were screened for interest and eligibility in this pilot study on the effect of DIM supplements on urinary hormone metabolites. The treatment group received daily DIM (108 mg DIM/day) supplements for 30 days, and the control group received a placebo capsule daily for 30 days. Urinary metabolite analysis included 2-hydroxyestrone (2-OHE1), 16-alpha hydroxyestrone (16alpha-OHE1), DIM, estrone (El), estradiol(E2), estriol (E3), 6beta-hydroxycortisol (6beta-OHC), and cortisol in the first morning urine sample before intervention and 31 days after intervention. Nineteen women completed the study,for a total of 10 in the treatment group and 9 in the placebo group. DIM-treated subjects, relative to placebo, showed a significant increase in levels of2-OHE1 (P=0. 020), DIM (P =0. 045), and cortisol (P = 0.039), and increase of 47% in the 2-OHE1/16alpha-OHE1 ratio from 1.46 to 2.14 (P=0.059). In this pilot study, DIM increased the 2-hydroxylation of estrogen urinary metabolites.

Estrogen metabolism and risk of breast cancer: a prospective study of the 2:16alpha-hydroxyestrone ratio in premenopausal and postmenopausal women. Epidemiology. 2000 Nov;11(6):635-40. Muti P, Bradlow HL, Micheli A, Krogh V, Freudenheim JL, Schunemann HJ, Stanulla M, Yang J, Sepkovic DW, Trevisan M, Berrino F. Department of Social and Preventive Medicine, University at Buffalo, State University of New York at Buffalo, Buffalo, NY, USA, Epidemiology Division of the National Cancer Institute (Istituto Nazionale Tumori), Milan, Italy, Department of Pediatric Hematology and Oncology, Medical School of Hannover, Hannover, Germany.

Experimental and clinical evidence suggests that 16alpha-hydroxylated estrogen metabolites, biologically strong estrogens, are associated with breast cancer risk, while 2-hydroxylated metabolites, with lower estrogenic activity, are weakly related to this disease. This study analyzes the association of breast cancer risk with estrogen metabolism, expressed as the ratio of 2-hydroxyestrone to 16alpha-hydroxyestrone, in a prospective nested case-control study. Between 1987 and 1992, 10,786 women (ages 35-69 years) were recruited to a prospective study on breast cancer in Italy, the "Hormones and Diet in the Etiology of Breast Cancer" (ORDET) study. Women with a history of cancer and women on hormone therapy were excluded at baseline. At recruitment, overnight urine was collected from all participants and stored at -80 degrees C. After an average of 5.5 years of follow-up, 144 breast cancer cases and four matched controls for each case were identified among the participants of the cohort. Among premenopausal women, a higher ratio of 2-hydroxyestrone to 16alpha-hydroxyestrone at baseline was associated with a reduced risk of breast cancer: women in the highest quintile of the ratio had an adjusted odds ratio (OR) for breast cancer of 0.58 [95% confidence interval (CI) = 0.25-1.34]. The corresponding adjusted OR in postmenopausal women was 1.29 (95% CI = 0.53-3.10). Results of this prospective study support the hypothesis that the estrogen metabolism pathway favoring 2-hydroxylation over 16alpha-hydroxylation is associated with a reduced risk of invasive breast cancer risk in premenopausal women.

Plant-derived 3,3'-Diindolylmethane is a strong androgen antagonist in human prostate cancer cells. Journal of Biological Chemistry.  2003 June 6;278(23);21136-45. Le HT, Schaldach CM, Firestone GL, Bjeldanes LF. Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720-3104, USA.

3,3'-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a potential anticancer component of cruciferous vegetables. Our results indicate that DIM exhibits potent antiproliferative and antiandrogenic properties in androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA synthesis. These activities were not produced in androgen-independent PC-3 cells. Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM inhibited, in a concentration-dependent manner, the DHT-induced expression of a prostate-specific antigen promoter-regulated reporter gene construct in transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3 cells only when these cells were co-transfected with a wild-type androgen receptor expression plasmid. Using fluorescence imaging with green fluorescent protein androgen receptor and Western blot analysis, we demonstrated that DIM inhibited androgen-induced androgen receptor (AR) translocation into the nucleus. Results of receptor binding assays indicated further that DIM is a strong competitive inhibitor of DHT binding to the AR. Results of structural modeling studies showed that DIM is remarkably similar in conformational geometry and surface charge distribution to an established synthetic AR antagonist, although the atomic compositions of the two substances are quite different. To our knowledge, DIM is the first example of a pure androgen receptor antagonist from plants.

3,3'-Diindolylmethane (DIM) induces a G(1) cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of p21(WAF1/CIP1) expression. Carcinogenesis 2002 Aug;23(8):1297-305. Hong C, Kim HA, Firestone GL, Bjeldanes LF. Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720, USA.

3,3'-Diindolylmethane (DIM) is a promising cancer chemopreventive agent derived from Brassica food plants. To determine whether this natural indole has a direct growth inhibitory effect on human breast cancer cells, we examined the cell cycle regulatory effects of DIM in estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cell lines. Results of flow cytometry studies showed that DIM treatment produced a marked increase (from 51 to 79%) in the proportion of cells in the G(1) phase of the cell cycle, regardless of estrogen-receptor status. Analyses of G(1)-acting cell cycle components indicated that the enzymatic activity of cyclin-dependent kinase (CDK) 2 was also strongly reduced. Western blot analyses showed that, concurrent with the DIM-induced cell cycle arrest, DIM stimulated a rapid and pronounced increase in levels of the CDK inhibitor, p21(WAF1/CIP1) (p21). Northern blot analysis demonstrated that DIM increased p21 mRNA expression with a maximal 6-7-fold induction, and exposure to cycloheximide did not block the response. Similar increases in expression of p21 protein and mRNA were observed in both MCF-7 and MDA-MB-231 human breast cancer cells, suggesting that DIM induction of p21 expression is independent of estrogen-receptor signaling and p53. Transient transfection of 5'-deletion constructs of the p21 promoter demonstrated that the first 291 bp segment of the proximal promoter, which contains six promoter specific transcription factor 1 (Sp1) elements, maintained DIM responsiveness. Consistent with a role for Sp1 in this response, a reporter construct driven by three consensus Sp1 binding sites was responsive to DIM. In addition, electrophoretic mobility shift assays showed that DIM induced the binding of Sp1 and Sp3 to the consensus Sp1 responsive element. Thus, our observations have uncovered an antiproliferative pathway for DIM that implicates Sp1/Sp3-induced expression of p21 as a target for cell cycle control in human breast cancer cells.

Lycopene. Advances in Food and Nutrition Research 2006;51:99-164. Rao AV, Ray MR, Rao LG, Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.

Oxidative stress is now recognized as an important etiological factor in the causation of several chronic diseases including cancer, cardiovascular diseases, osteoporosis, and diabetes. Antioxidants play an important role in mitigating the damaging effects of oxidative stress on cells. Lycopene, a carotenoid antioxidant, has received considerable scientific interest in recent years. Epidemiological, tissue culture, and animal studies provide convincing evidence supporting the role of lycopene in the prevention of chronic diseases.

Combined lycopene and vitamin E treatment suppresses the growth of PC-346C human prostate cancer cells in nude mice. The Journal of Nutrition. 2006 May;136(5):1287-93. Limpens J, Shroder FH, de Ridder CM, Bolder CA, Wildhagen MF, Obermuller-Jevic UC, Kramer K., Van Weerden WM. Department of Urology, Erasmus MC, Rotterdam, The Netherlands.

Epidemiologic studies have repeatedly associated a high intake of lycopene and vitamin E with reduced prostate cancer risk. The present study examined the ability of the 2 compounds to reduce tumor growth and prostate-specific antigen (PSA) plasma levels in the PC-346C orthotopic mouse model of human prostate cancer. Three days after intraprostatic tumor injection, NMRI nu/nu mice were administered a daily oral dose of synthetic lycopene [5 or 50 mg/kg body weight (BW)], vitamin E in the form of alpha-tocopheryl acetate (5 or 50 mg/kg BW), a mixture of lycopene and vitamin E (5 mg/kg BW each), or vehicle. Intraprostatic tumor volume and plasma PSA concentrations were measured at regular intervals. Mice were killed when the tumor load exceeded 1000 mm(3) or on d 95 when the study was terminated. Prostate and liver were analyzed by HPLC for lycopene isomers and alpha- and gamma, delta-tocopherol concentrations. None of the single treatments significantly reduced tumor volume. In contrast, combined treatment with lycopene and vitamin E, at 5 mg/kg BW each, suppressed orthotopic growth of PC-346C prostate tumors by 73% at d 42 (P < 0.05) and increased median survival time by 40% from 47 to 66 d (P = 0.02). The PSA index (PSA:tumor volume ratio) did not differ between experimental groups, indicating that PSA levels were not selectively affected. Lycopene was detected only in mice supplemented with lycopene. As in humans, most tissue lycopene was in the cis-isomer conformation, whereas 77% trans-lycopene was used in the dosing material. Liver alpha-tocopherol concentrations were increased in mice supplemented with both 50 mg/kg (226%, P < 0.05) and 5 mg/kg vitamin E (41%, P < 0.05), whereas prostate alpha-tocopherol concentrations were increased only by the higher dose (83%, P < 0.05). Our data provide evidence that lycopene combined with vitamin E may inhibit the growth of prostate cancer and that PSA can serve as a biomarker of tumor response for this treatment regimen.

Tomato lycopene and its role in human health and chronic diseases. CMAJ 2000 Sep 19;163(6):739-44 Agarwal S., Rao AV., Department of Nutritional Sciences, Faculty of Medicine, University of Toronto.

Lycopene is a carotenoid that is present in tomatoes, processed tomato products and other fruits. It is one of the most potent antioxidants among dietary carotenoids. Dietary intake of tomatoes and tomato products containing lycopene has been shown to be associated with a decreased risk of chronic diseases, such as cancer and cardiovascular disease. Serum and tissue lycopene levels have been found to be inversely related to the incidence of several types of cancer, including breast cancer and prostate cancer. Although the antioxidant properties of lycopene are thought to be primarily responsible for its beneficial effects, evidence is accumulating to suggest that other mechanisms may also be involved. In this article we outline the possible mechanisms of action of lycopene and review the current understanding of its role in human health and disease prevention.

Combinations of Tomato and Broccoli Enhance Antitumor Activity in Dunning R3327-H Prostate Adenocarcinomas. Canene-Adams K, Lindshield B, Wang S, Jeffery E, Clinton S, Erdman J.,  Cancer Res 2007; 67: (2). January 15, 2007

The consumption of diets containing 5 to 10 servings of fruits and vegetables daily is the foundation of public health recommendations for cancer prevention, yet this concept has not been tested in experimental models of prostate cancer. We evaluated combinations of tomato and broccoli in the Dunning R3327-H prostate adenocarcinoma model. Male Copenhagen rats (n=206) were fed diets containing 10% tomato, 10% broccoli, 5% tomato plus 5% broccoli (5:5 combination), 10% tomato plus 10% broccoli (10:10 combination) powders for approximately 22 weeks starting 1 month prior to receiving s.c. tumor implants. We compared the effects of diet to surgical castration (2 weeks before termination) or finasteride (5 mg/kg body weight orally, 6 d/wk). Castration reduced prostate weights, tumor areas, and tumor weight (62%, P<0.001), whereas finasteride reduced prostate weights (P<0.0001), but had no effect on tumor area or weight. Tomato reduced tumor weight by 34% (P<0.05). Broccoli decreased tumor weights by 42% (P<0.01) whereas the 10:10 combination caused a 52% decrease (P<0.001). Tumor growth reductions were associated with reduced proliferation and increased apoptosis, as quantified by proliferating cell nuclear antigen immunohistochemistry and the ApopTag assay. The combination of tomato and broccoli was more effective at slowing tumor growth than either tomato or broccoli alone and supports the public health recommendations to increase the intake of a variety of plant components.

Selenomethionine induction of DNA repair response in human fibroblasts. Oncogene (2002) 21, 3663-3669. Seo Y, Sweeney C, and Smith M. Indiana University Cancer Center, Department of Microbiology, Walther Oncology Center, Indiana University School of Medicine and Walther Cancer Institute, Indianapolis, IN 46202, USA. Department of Medicine, Section of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

Selenium compounds have a long history in chemoprevention of mammary and colon cancers in rodent models. Selenium compounds are in current clinical trials, having shown promise in prevention of prostate and other human cancers. In human tissues, it has been estimated that each cell sustains approximately 10 000 potentially mutagenic (if not repaired) lesions per day due to endogenous DNA damage. Almost no studies have addressed the potential for selenium compounds to induce DNA repair, a potential mechanism for their cancer-preventive actions. We show that selenium in the form of selenomethionine induces a DNA repair response in normal human fibroblasts in vitro, and protects cells from DNA damage. We show a possible mechanism for the inducible DNA repair response, in which enhanced repair complex formation was observed in selenomethionine-treated cells.

Low serum selenium and total carotenoids predict mortality among older women living in the community. Journal of Nutrition. 2006 Jan;136(1):172-6. Ray AL, Semba RD, Walston J., Ferrucci L, Cappola AR, Ricks MO, Xue QL, Fried LP. The Johns Hopkins Medical Institutions, Baltimore, MD, USA.

Selenium and the carotenoids play an important role in antioxidant defenses and in the redox regulation involved in inflammation. We tested the hypothesis that low selenium and carotenoids predict mortality in older women living in the community. Women who were enrolled in the Women's Health and Aging Studies I and II in Baltimore, MD (n = 632; 70-79 y old) had serum selenium and carotenoids measured at baseline and were followed for mortality over 60 mo. Median (minimum, maximum) serum selenium and carotenoids were 1.53 (0.73, 2.51) micromol/L and 1.67 (0.13, 9.10) micromol/L; 14.1% of the women died. The 5 major causes of death were heart disease (32.6%), cancer (18.0%), stroke (9.0%), infection (6.7%), and chronic obstructive pulmonary disease (5.6%). Adjusting for age, education, smoking, BMI, poor appetite, and chronic diseases, higher serum selenium [hazard ratio (HR) 0.71, 95% CI 0.56-0.90/1 SD increase in log(e) selenium; P = 0.005] and higher serum total carotenoids (HR 0.77, 95% CI 0.64-0.84/1 SD increase in log(e) total carotenoids; P = 0.009) were associated with a lower risk of mortality. Women living in the community who have higher serum selenium and carotenoids are at a lower risk of death.

From dietary antioxidants to regulators in cellular signaling and gene regulation: Sulforaphane and selenium, partners in adaptive response and prevention of cancer. Free Radical Research. 2006 Aug;40(8):775-87. Brigelius-Flohe R., Banning A. German Institute of Human Nutrition, Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114 - 116, D-14558, Nuthetal, Germany.

The association of decreased cancer risk with intake of cruciferous vegetables and selenium is stronger than that reported for fruits and vegetables in general. An active constituent in cruciferae is sulforaphane. Chemopreventive effects of both, sulforaphane and selenium have been attributed to an antioxidant action which certainly is too simplicistic. Sulforaphane induces via activation of the Nrf2/Keap1 system phase 2 enzymes that protect against carcinogens and oxidants. Induced enzymes comprise the selenoproteins thioredoxin reductase-1 (TrxR1) and gastrointestinal glutathione peroxidase (GI-GPx, GPx2), which contain antioxidant response elements (ARE) in their promoter regions. Translational realisation of the enhanced transcripts depends on adequate selenium supply, which explains the synergism of Nrf2 activators and selenium. Regarding tumorigenesis the role of TrxR1 is ambiguous: it is essential for fast tumor cell growth but also diminishes vascularisation of tumors. The anticarcinogenic role of GI-GPx is evident from enhanced gastrointestinal tumor formation in gpx2/gpx1 double KO mice.

  Synergy between sulforaphane and selenium in the induction of thioredoxin reductase 1 requires both transcriptional and translational modulation. Carcinogenesis, 2003 Mar;24(3):497-503. Nutrition Division, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK. Zhang J, Svehikova V., Bao Y, Howie AF, Beckett GJ, Williamson G.

Selenium in the maintenance and therapy of HIV-infected patients. Chemical and Biological Intaractions. 1994 Jun;91(2-3):199-205. Schrauzer GN, Sacher J. University of California, San Diego, Department of Chemistry and Biochemistry, La Jolla 92093.

Due to its antiviral effects and its importance for all immunological functions, the administration of selenium is suggested as a supportive measure in early as well as in advanced stages of HIV-induced disease. Initial observations on the effects of selenium supplementation in HIV-infected patients indicate that selenium causes symptomatic improvements and possibly slows the course of the disease. As selenium inhibits reverse transcriptase activity in RNA-virus-infected animals, supplemental selenium could also prevent the replication of HIV and retard the development of AIDS in newly HIV-infected subjects. An adequate supply of selenium and of antioxidant vitamins is also proposed as a measure to reduce the probability of the placental transmission of HIV in pregnancy.

  Suppression of human immunodeficiency virus type 1 viral load with selenium supplementation: a randomized controlled trial. Archives of Internal Medicine. 2007 Jan 22;167(2):148-54. Hurwitz BE, Klaus JR, Lllabre MM, Gonzalez A, Lawrence PJ, Maher KJ, Greenson JM, Baum MK, Shor-Posner G, Skyler JS, Schneiderman N.

Despite findings that selenium supplementation may improve immune functioning, definitive evidence of its impact on human immunodeficiency virus (HIV) disease severity is lacking. METHODS: High selenium yeast supplementation (200 mug/d) was evaluated in a double-blind, randomized, placebo-controlled trial. Intention-to-treat analyses assessed the effect on HIV-1 viral load and CD4 count after 9 months of treatment. Unless otherwise indicated, values are presented as mean +/- SD. RESULTS: Of the 450 HIV-1-seropositive men and women who underwent screening, 262 initiated treatment and 174 completed the 9-month follow-up assessment. Mean adherence to study treatment was good (73.0% +/- 24.7%) with no related adverse events. The intention-to-treat analyses indicated that the mean change (Delta) in serum selenium concentration increased significantly in the selenium-treated group and not the placebo-treated group (Delta = 32.2 +/- 24.5 vs 0.5 +/- 8.8 microg/L; P<.001), and greater levels predicted decreased HIV-1 viral load (P<.02), which predicted increased CD4 count (P<.04). Findings remained significant after covarying age, sex, ethnicity, income, education, current and past cocaine and other drug use, HIV symptom classification, antiretroviral medication regimen and adherence, time since HIV diagnosis, and hepatitis C virus coinfection. Follow-up analyses evaluating treatment effectiveness indicated that the nonresponding selenium-treated subjects whose serum selenium change was less than or equal to 26.1 microg/L displayed poor treatment adherence (56.8% +/- 29.8%), HIV-1 viral load elevation (Delta = +0.29 +/- 1.1 log(10) units), and decreased CD4 count (Delta = -25.8 +/- 147.4 cells/microL). In contrast, selenium-treated subjects whose serum selenium increase was greater than 26.1 microg/L evidenced excellent treatment adherence (86.2% +/- 13.0%), no change in HIV-1 viral load (Delta = -0.04 +/- 0.7 log(10) units), and an increase in CD4 count (Delta = +27.9 +/- 150.2 cells/microL). CONCLUSIONS: Daily selenium supplementation can suppress the progression of HIV-1 viral burden and provide indirect improvement of CD4 count. The results support the use of selenium as a simple, inexpensive, and safe adjunct therapy in HIV spectrum disease.

  Study of prediagnostic selenium level in toenails and the risk of advanced prostate cancer. Yoshizawa K, Willett WC, Morris SJ, et al.  J Natl Cancer Inst. 1998;90(16):1219-1224. 

In a recent randomized intervention trial, the risk of prostate cancer for men receiving a daily supplement of 200 microg selenium was one third of that for men receiving placebo. By use of a nested case-control design within a prospective study, i.e., the Health Professionals Follow-Up Study, we investigated the association between risk of prostate cancer and prediagnostic level of selenium in toenails, a measure of long-term selenium intake. METHODS: In 1986, 51,529 male health professionals aged 40-75 years responded to a mailed questionnaire to form the prospective study. In 1987, 33,737 cohort members provided toenail clippings. In 1988, 1990, 1992, and 1994, follow-up questionnaires were mailed. From 1989 through 1994, 181 new cases of advanced prostate cancer were reported. Case and control subjects were matched by age, smoking status, and month of toenail return. Selenium levels were determined by neutron activation. All P values are two-sided. RESULTS: The selenium level in toenails varied substantially among men, with quintile medians ranging from 0.66 to 1.14 microg/g for control subjects. When matched case-control data were analyzed, higher selenium levels were associated with a reduced risk of advanced prostate cancer (odds ratio [OR] for comparison of highest to lowest quintile = 0.49; 95% confidence interval [CI] = 0.25-0.96; P for trend = .11). After additionally controlling for family history of prostate cancer, body mass index, calcium intake, lycopene intake, saturated fat intake, vasectomy, and geographical region, the OR was 0.35 (95% CI = 0.16-0.78; P for trend = .03). CONCLUSIONS: Our results support earlier findings that higher selenium intakes may reduce the risk of prostate cancer.

Supplementation with the carotenoids lutein or zeaxanthin improves human visual performance. Ophthalmic & Physiological Optics. Kvansakul J, Rodriguez-Carmona M., Edgar DF, Barker FM, Kapcke W., Schalch W., Barbur JL. Applied Vision Research Centre, Department of Optometry and Visual Science, City University, London, UK.

Macular pigment (MP) is found in diurnal primate species when vision spans a range of ambient illumination and is mediated by cone and rod photoreceptors. The exact role of MP remains to be determined. In this study we investigate two new hypotheses for possible MP functions. OBJECTIVE: As MP absorption coincides partly with that of rhodopsin, MP may reduce rod signal effectiveness in the mesopic range, thus extend the usefulness of cone-mediated vision into the mesopic range. Forward light scatter in the eye can reduce retinal image contrast. If blue light contributes significantly to intraocular scatter, selective blue light absorption by MP could reduce the effects of scatter. DESIGN: We investigated 34 subjects from a carotenoid supplementation trial. The measurements included high mesopic contrast acuity thresholds (CATs), macular pigment optical density (MPOD), wavefront aberrations, and scattered light. The measurements were made after 6 months of daily supplementation with zeaxanthin (Z), lutein (L), a combination of the two (C), or placebo (P), and again after a further 6 months of doubled supplementation. RESULTS: The data reveal a trend toward lower CATs in all groups supplemented, with a statistically significant improvement in the lutein group (p = 0.001), although there was no correlation with MPOD. Light scattering in the eye and the root-mean-square wavefront aberrations show decreasing trends as a result of supplementation, but no correlation with MPOD. CONCLUSIONS: The results suggest that supplementation with L or Z increases MPOD at the fovea and at 2.5 degrees , and that supplementation can improve CATs at high mesopic levels and hence visual performance at low illumination.

Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration. Eye Disease Case-Control Study Group. Journal of the American Medical Association.1994 Nov 9;272(18):1413-20. Seddon JM, Ajani UA, Sperduto RD, Hiller R, Blair N, Burton TC, Farber MD, Gragoudas ES, Haller J., Miller DT. Epidemiology Unit, Massachusetts Eye and Ear Infirmary, Boston 02114.

OBJECTIVE--To evaluate the relationships between dietary intake of carotenoids and vitamins A, C, and E and the risk of neovascular age-related macular degeneration (AMD), the leading cause of irreversible blindness among adults. DESIGN--The multicenter Eye Disease Case-Control Study. SETTING--Five ophthalmology centers in the United States. PATIENTS--A total of 356 case subjects who were diagnosed with the advanced stage of AMD within 1 year prior to their enrollment, aged 55 to 80 years, and residing near a participating clinical center. The 520 control subjects were from the same geographic areas as case subjects, had other ocular diseases, and were frequency-matched to cases according to age and sex. MAIN OUTCOME MEASURES--The relative risk for AMD was estimated according to dietary indicators of antioxidant status, controlling for smoking and other risk factors, by using multiple logistic-regression analyses. RESULTS--A higher dietary intake of carotenoids was associated with a lower risk for AMD. Adjusting for other risk factors for AMD, we found that those in the highest quintile of carotenoid intake had a 43% lower risk for AMD compared with those in the lowest quintile (odds ratio, 0.57; 95% confidence interval, 0.35 to 0.92; P for trend = .02). Among the specific carotenoids, lutein and zeaxanthin, which are primarily obtained from dark green, leafy vegetables, were most strongly associated with a reduced risk for AMD (P for trend = .001). Several food items rich in carotenoids were inversely associated with AMD. In particular, a higher frequency of intake of spinach or collard greens was associated with a substantially lower risk for AMD (P for trend < .001).

Vitamin E and immune response in the aged: molecular mechanisms and clinical implications. Immonology Reviews. 2005 June;205:269-84. Meydani SN, Han SN, Wu D. Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, USA

Nutritional status has been indicated as a contributing factor to age-related dysregulation of the immune response. Vitamin E, a lipid-soluble antioxidant vitamin, is important for normal function of the immune cells. The elderly are at a greater risk for vitamin E intake that is lower than recommended levels. Vitamin E supplementation above currently recommended levels has been shown to improve immune functions in the aged including delayed-type hypersensitivity skin response and antibody production in response to vaccination, which was shown to be mediated through increased production of interleukin (IL)-2, leading to enhanced proliferation of T cells, and through reduced production of prostaglandin E(2), a T-cell suppressive factor, as a result of a decreased peroxynitrite formation. Vitamin E increased both cell-dividing and IL-producing capacities of naive T cells, but not memory T cells. The vitamin E-induced enhancement of immune functions in the aged was associated with significant improvement in resistance to influenza infection in aged mice and a reduced risk of acquiring upper respiratory infections in nursing home residents.

Vitamin E supplementation reverses the age-associated decrease in effective immune synapse formation in CD4+ T cells. Annals of the New York Academy of Sciences. 2004 December; 1031:412-4. Ahmed T, Marko M, Wu D, Chung H, Huber B, Meydani SN. Nutritional Immunology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA.

Aging is associated with impairment of T cell function. We demonstrate here that age-associated declines in T cell signaling are due to the inability to form effective immune synapses at the site of the T cell receptor and antigen interaction. On the basis of our previous research with vitamin E (VE), we hypothesized that VE supplementation of old CD4(+) T cells enhances effective immune synapse formation through increased translocation of signaling proteins. Using confocal microscopy, we found that when exposed to antigen-presenting cells, CD4(+) T cells from old mice have a lower percentage of effective immune synapses compared to those from young mice. Furthermore, we show that in vitro and in vivo VE supplementation increases the percentage of old CD4(+) T cells capable of forming a functional immune synapse.

Carotenoid action on the immune response. Journal of Nutrition.2004 Jan;134(1):257S-261S. Chew BP, Park JS. Washington State University, Pullman, WA 99164-6351, USA.

Early studies demonstrating the ability of dietary carotenes to prevent infections have left open the possibility that the action of these carotenoids may be through their prior conversion to vitamin A. Subsequent studies to demonstrate the specific action of dietary carotenoids have used carotenoids without provitamin A activity such as lutein, canthaxanthin, lycopene and astaxanthin. In fact, these nonprovitamin A carotenoids were as active, and at times more active, than beta-carotene in enhancing cell-mediated and humoral immune response in animals and humans. Another approach to study the possible specific role of dietary carotenoids has used animals that are inefficient converters of carotenoids to vitamin A, for example the domestic cat. Results have similarly shown immuno-enhancement by nonprovitamin A carotenoids, based either on the relative activity or on the type of immune response affected compared to beta-carotene. Certain carotenoids, acting as antioxidants, can potentially reduce the toxic effects of reactive oxygen species (ROS). These ROS, and therefore carotenoids, have been implicated in the etiology of diseases such as cancer, cardiovascular and neurodegenerative diseases and aging. Recent studies on the role of carotenoids in gene regulation, apoptosis and angiogenesis have advanced our knowledge on the possible mechanism by which carotenoids regulate immune function and cancer.

Relationship between plasma carotenoids and prostate cancer. Nutritional Cancer. 2005;53(2):127-34. Hursting SD. Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

Carotenoids, particularly lycopene, are thought to decrease prostate cancer risk, but the relationship between plasma carotenoid concentrations and risk in various populations has not been well characterized. Comparing 118 non-Hispanic Caucasian men mainly from southeast Texas with nonmetastatic prostate cancer with 52 healthy men from the same area, we conducted a case-control analysis evaluating associations between risk and plasma levels of total carotenoids, beta-cryptoxanthin, alpha- and trans-beta-carotene, lutein and zeaxanthin, total lycopenes, trans-lycopene, total cis-lycopenes, and cis-lycopene isoforms 1, 2, 3, and 5. Risk for men with high plasma levels of alpha-carotene, trans-beta-carotene, beta-cryptoxanthin, and lutein and zeaxanthin was less than half that for those with lower levels. In contrast, we observed no significant associations for total lycopenes, all-trans-lycopene, and cis-lycopene isomer peaks 2, 3, and 5, although high levels of cis-lycopene isomer peak 1 were inversely associated with risk. Analysis of men with aggressive disease (Gleason scores of > or =7, n = 88) vs. less aggressive cases (Gleason scores of <7, n = 30) failed to reveal significant associations between carotenoid levels and the risk of diagnosis with aggressive disease. These findings suggest that, in these men, higher circulating levels of alpha-cryptoxanthin, alpha-carotene, trans-beta-carotene, and lutein and zeaxanthin may contribute to lower prostate cancer risk but not to disease progression.

Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. 2002 May 28;99(11):7610-5. Proclamation of the National Academy of Sciences. Fahey JW, Haristoy X, Dolan PM, Kensler TW, Sholtus I, Stephenson KK, Talalay P, Lozniewski A. Lewis B. and Dorothy Cullman Cancer Chemoprotection Center, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA.

Gastric infection with Helicobacter pylori is a cosmopolitan problem, and is especially common in developing regions where there is also a high prevalence of gastric cancer. These infections are known to cause gastritis and peptic ulcers, and dramatically enhance the risk of gastric cancer. Eradication of this organism is an important medical goal that is complicated by the development of resistance to conventional antimicrobial agents and by the persistence of a low level reservoir of H. pylori within gastric epithelial cells. Moreover, economic and practical problems preclude widespread and intensive use of antibiotics in most developing regions. We have found that sulforaphane [(-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane], an isothiocyanate abundant as its glucosinolate precursor in certain varieties of broccoli and broccoli sprouts, is a potent bacteriostatic agent against 3 reference strains and 45 clinical isolates of H. pylori [minimal inhibitory concentration (MIC) for 90% of the strains is <or=4 microg/ml], irrespective of their resistance to conventional antibiotics. Further, brief exposure to sulforaphane was bactericidal, and eliminated intracellular H. pylori from a human epithelial cell line (HEp-2). In complementary experiments, sulforaphane blocked benzo[a]pyrene-evoked forestomach tumors in ICR mice. This protection resulted from induction of phase 2 detoxication and antioxidant enzymes, and was abrogated in mice lacking the nrf2 gene, which regulates phase 2 enzymes. Thus, the dual actions of sulforaphane in inhibiting Helicobacter infections and blocking gastric tumor formation offer hope that these mechanisms might function synergistically to provide diet-based protection against gastric cancer in humans.

Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proclamations of the National Academy of Sciences. 1997 Sep 16;94(19):10367-72. Fahey JW, Zhang Y, Talalay P. Brassica Chemoprotection Laboratory and Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Induction of phase 2 detoxication enzymes [e.g., glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases] is a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity of electrophiles and reactive forms of oxygen. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of malignancies, it is of interest that a number of edible plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, edible plants belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (mostly in the form of their glucosinolate precursors) some of which (e.g., sulforaphane or 4-methylsulfinylbutyl isothiocyanate) are very potent inducers of phase 2 enzymes. Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers including broccoli and cauliflower contain 10-100 times higher levels of glucoraphanin (the glucosinolate of sulforaphane) than do the corresponding mature plants. Glucosinolates and isothiocyanates can be efficiently extracted from plants, without hydrolysis of glucosinolates by myrosinase, by homogenization in a mixture of equal volumes of dimethyl sulfoxide, dimethylformamide, and acetonitrile at -50 degrees C. Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin or sulforaphane as the principal enzyme inducer) were highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats.

Prospective Study of Fruit and Vegetable Intake and Risk of Prostate Cancer. Journal of the National Cancer Institute. 2007 Jul 24; (Epub ahead of print) Krish VA, Peters U, Mayne ST, Subar AF, Chatterjee N, Johnson CC, Hayes RB. Affiliations for authors: Research Unit, Division of Preventive Oncology, Cancer Care Ontario, Toronto, ON, Canada (VAK); Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT (VAK, STM); Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA (UP); Department of Epidemiology, University of Washington, Seattle, WA (UP); Divisions of Cancer Epidemiology and Genetics (NC, RBH) and Cancer Control and Population Sciences (AFS), National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD; Josephine Ford Cancer Center, Henry Ford Health System, Detroit, MI (CCJ).