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Circumstantial evidence indicates that zinc may have an important role in the prostate. Total zinc levels in the prostate are 10 times higher than in other soft tissues. Zinc concentrations in prostate epithethial cancer cells are decreased significantly. Zinc supplementation for prevention and treatment of prostate cancer in humans has yielded controversial results. No studies have been reported in animal models to show the effect of zinc supplementation on prevention of prostate cancer, thus far. In this study, we have examined the effect of zinc supplementation on development of prostate cancer in a TRAMP mouse model. Results from our study indicate that dietary zinc plays an important role in prostate carcinogenesis. Tumor weights were significantly higher when the dietary zinc intake was either deficient or high in comparison to normal zinc intake level, suggesting that an optimal dietary zinc intake may play a protective role against prostate cancer. Further, our studies also showed decreased insulin-like growth factor (IGF)-1 and IGF-1/IGF binding protein-3 ratio in normal zinc-supplemented animals, suggesting that zinc may modulate IGF-1 metabolism in relation to carcinogenesis. We conclude that optimal prostate zinc concentration has a protective role against cancer.
Zinc (Zn), a homeostatically regulated essential trace element, is required for the activities of approximately 300 enzymes, and over 2,000 transcription factors require zinc for their integrity.1 In humans, zinc is essential for optimal growth, development, immune and cognitive functions.1–4
The primary dietary sources of zinc are red meat, seafood, poultry, grains, and legumes. Dietary phytate, which is present in high quantities in cereal products and legumes, however, decreases availability of zinc.5 The first recommended daily intake of zinc (RDA) as published in 19746 was 15mg for men, and for women it was 12mg; however, in the revised RDA in 2001, it has been reduced to 11mg for men and to 8mg for non-pregnant, non-lactating women. The rationale for this downward revision is not well-justified and deserves further consideration. Zinc intake is decreased in many patients with cancer, especially those who are affected with head and neck cancer.7,8
The role of zinc has been implicated in the development and progression of prostate malignancy.9–14 The specialized normal prostate glandular epithelium produces and secretes high levels of citrate, and the accumulation of zinc in these cells is essential for their metabolic function.10,11 The levels of citrate and zinc are decreased in malignant prostate cells in comparison to normal cells.10,11 Decreased zinc concentration leads to increased M-aconitase activity, allowing citrate oxidation to proceed via the Krebs cycle and generate ATP, which results in increased bioenergetics efficiency of malignant cells. A genetic alteration in the expression of Zip-1, a zinc transporter, has been implicated with this metabolic transformation.12
Based on the clinical and experimental observations and inasmuch as zinc is also an anti-inflammatory agent,2 it has been suggested that zinc supplementation may be a useful agent in the prevention and treatment of prostate cancer.11,15 However, epidemiological studies thus far have shown divergent and conflicting results regarding the efficacy of zinc supplementation against prostate cancer.15
Our study was designed to evaluate the role of dietary zinc on the development of prostate cancer in the TRAMP mouse model. This is the first report of studies in an animal model to test the effect of zinc on development of prostate cancer.
Transgenic males for this study were routinely obtained as [TRAMP×C57BL/6] F1 or as [TRAMP×C57BL/6] F2 offspring. Identity of transgenic mice was established by the polymerase chain reaction-based DNA screening as reported by Gingrich et al.16
Zn-deficient and Zn-supplemented diets were purchased from Research Diets (New Brunswick, NJ, USA). Insulin-like growth factor (IGF)-1 and IGF binding protein (IGFBP)-3 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Diagnostic Systems Laboratories (Webster, TX, USA). Interleukin (IL)-1β and IL-6 ELISA kits were purchased from BD Biosciences (San Diego, CA, USA).
To perform this study, we used 8-week-old male TRAMP mice. The TRAMP mice provide an autochthonous spontaneous model system to study the molecular basis of transformation of normal prostate cells and the factors influencing the progression of prostate cancer. Eighteen TRAMP mice were divided into three groups: zinc-deficient (0.85ppm), optimal (normal) zinc (30ppm), and high zinc (150ppm). AIN-76A rodent diet modified to contain egg whites instead of casein was obtained from Research Diets Inc. (Table 1). Two hundred fifty grams of the respective diets was placed in each cage and replaced twice every week. Body weight and diet consumed by each group of animals were recorded up to 22 weeks. At 22 weeks of age blood and prostate tissues were obtained and weighed. Blood was withdrawn from the retro-orbital venous plexus of animals of each group at week 22. ELISA was performed for serum IL-1β, IL-6, IGF-1, and IGFBP-3 levels. All animals were sacrificed at 22 weeks of age by carbon dioxide inhalation followed by cervical dislocation, and the urogenital apparatus containing the prostate, bladder, and seminal vesicles was removed en bloc. Prostate tissues that also contained the tumors were dissected out under a dissecting stereomicroscope and weighed. All animal procedures were according to the guidelines established by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee.
A two-step sandwich-type enzyme immunoassay technique was used to quantitate IGF-I and IGFBP-3 levels in the serum of TRAMP mice by following the manufacturer's protocol. In brief, standards, controls, and unknown plasma samples were incubated with the appropriate specific antibody in microtitration wells. After incubation and washing, the wells were treated with a second detection antibody labeled with the enzyme horseradish peroxidase. After a second incubation and washing step, the wells were incubated with the substrate tetramethylbenzidine. An acidic stopping solution was then added, and the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement at 450 and 620nm. The absorbance measured was directly proportional to the concentration of the IGF-I or IGFBP-3 present. A set of standards was used to plot a standard curve of absorbance from which the concentrations in the unknowns were calculated.
Sandwich ELISA protocols were used to assay for IL-1β and IL-6 in the serum. In brief, in a 96-well plate coated with primary antibodies 5–25-μL aliquots of serum samples diluted to 50μL with phosphate-buffered saline were added, followed by overnight incubation. Unbound antibodies were removed by washing followed by incubation in second biotinylated antibody. Known amounts of controls and standards were assayed in parallel in the same plate. The plates were incubated at 23°C for 2 hours and subsequently washed three times with the ELISA buffer. The captured immune complexes were further incubated with streptavidin-horseradish peroxidase conjugate, washed, and colorimetrically developed with tetramethylbenzidine substrate for horseradish peroxidase, and absorbance measurements were taken at 450nm.
All results are expressed as mean±SD values, and P<.05 was considered significant. Significant mean differences among treated groups were determined for main effects by analysis of variance. When the F test from analysis of variance was significant, individual treatment means were tested for significant differences by post hoc analysis using Scheffe's or Tukey's test.
Prostate tissues along with tumors were dissected out from 22-week-old TRAMP mice and weighed. At this time point the mice had been on experimental diet for 14 weeks. As shown in Figure 3, prostate tumor weights were 39% higher in zinc-deficient mice receiving 0.8ppm Zn compared to normal mice (30ppm Zn), and this difference was statistically significant (P<.05). In contrast, prostate tumor weights in mice fed 150ppm Zn-supplemented diet were 17% higher compared to the deficient mice; however, this difference was not statistically significant. Prostate tumor weights were 3% higher in the 150ppm group compared with the normal (30ppm) Zn-supplemented mice. These results suggest that Zn at optimal levels is preventive, whereas at both deficient and higher levels it may enhance tumor growth.
Serum IGF-I levels were measured at 22 weeks in TRAMP mice fed Zn-deficient and Zn-supplemented diet. IGF-I levels were 86% higher (P<.01) in TRAMP mice fed 0.8ppm Zn-supplemented diets (deficient level) and 200% higher (P<.001) in TRAMP mice fed 150ppm Zn-supplemented diet compared to the normal zinc (30ppm) group (Fig. 4). In contrast, no significant changes were observed in serum IGFBP-3 levels in the three groups (Fig. 5). However, when data were plotted in terms of IGF-I/IGFBP-3 ratios (Fig. 6), a significant (P<.01) increase in the ratio was observed in mice fed zinc-deficient diet (0.8ppm) compared to the normal group (30ppm), and a significant increase in the ratio was observed in mice fed 150ppm Zn-supplemented diet in comparison to the normal and zinc-deficient groups.
No significant differences were observed in serum IL-1β and IL-6 among the different treatment groups. Mean±SD serum IL-6 zinc levels in the three groups were as follows: Zn-deficient, 22±9pg/mL; Zn-normal, 25±9pg/mL; and high Zn, 32±12.5pg/mL. No statistical significance was observed among the three groups. Mean±SD serum IL-1β levels in the three groups were as follows: Zn-deficient, 27±9.5pg/mL; Zn-normal, 25±9.5pg/mL; and high Zn, 21±9pg/mL. No statistical significance was observed among the three groups.
Normal human prostate accumulates the highest levels of zinc of any soft tissue in the body. This unique capacity is retained in benign prostate hypertrophy, but, in contrast, zinc concentrations in prostate epithelial cancer cells are decreased significantly.9–11 Zinc accumulation decreases early in the course of prostatic malignancy and continues to decline during progression towards hormone-independent growth.10,11
Nuclear factor κB (NF-κB) and activator protein-1 (AP-1) (Jun oncogene) activation contributes to development and progression of prostate cancer by regulating the expression of genes involved in proliferation, apoptosis, angiogenesis, tumor invasion, and metastasis.13,14 Vascular endothelial growth factor, IL-8, and matrix metalloproteinase-9 are three major pro-angiogenic and pro-metastatic molecules that have been associated with negative prognostic features in various malignancies.13,14 Expression of these genes is promoted by co-activation of NF-κB and AP-1.13,14 Decreased expression of NF-κB in human prostate cancer cells inhibits their tumorigenic and metastatic properties in nude mice by suppressing angiogenesis and invasion via down-regulation of vascular endothelial growth factor, IL-8, and matrix metalloproteinase-9.13
Both NF-κB and AP-1 are constitutively activated in prostatic cancer.13 Prostate cancer patients with increased levels of tumor necrosis factor (TNF)-α have a significantly higher mortality rate than those with undetectable TNF-α levels.13,14 TNF-α is a known inducer of NF-κB activation.13,14
Uzzo et al.13 have demonstrated that physiological levels of zinc inhibit NF-κB but augment activitation of AP-1 in DU-145 and PC-3 prostate cancer cells. Chelation of zinc with N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine abolished this effect. They also demonstrated that zinc induced phosphorylation of three mitogen-activated protein kinase subfamilies regulating AP-1 and NF-κB activation (extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38), while blocking TNF-α-mediated NF-κB activation. Zinc reduced expression of vacular endothelial growth factor, IL-6, IL-8, and matrix metalloproteinase-9 and also reduced expression of intercellular adhesion molecule-1, which functionally suppresses tumor cell invasiveness and adhesion.13 Their studies thus showed that the ability of zinc supplementation to inhibit NF-κB supersedes zinc-mediated activation of AP-1 family members and that suppression of NF-κB signaling may have important implications for inhibiting the angiogenic and metastatic potential of malignant prostate cells.13
Our studies have shown that zinc is both an antioxidant and an anti-inflammatory agent.2,17,18 Zinc supplementation to humans decreases the gene expression and production of pro-inflammatory cytokines and decreases oxidative stress markers.17,18 In HL-60cells (a human promyeloycytic leukemia cell line), zinc deficiency increased the levels of TNF-α, IL-1β, and IL-8 cytokines and mRNA.17,18 In these cells, zinc induced A20, a zinc finger protein that inhibited NF-κB activation via the TNF receptor-associated factor pathway, and thus decreased gene expression of pro-inflammatory cytokines.17,18 These effects of zinc thus make it a very suitable molecule for trial as a chemoprevention agent for prostate cancer. Our studies in the animal model as presented here support this suggestion.
Solute carrier family 39 (Zn transporter [ZnT]) (hZIP)-1 gene expression and transporter protein were down-regulated and the cellular zinc level was decreased in adenocarcinoma cells and in prostate intra-epithelial neoplastic loci, in comparison to normal peripheral zone glandular epithelium and in benign hyperplastic glands.12 This study suggests that hZIP-1 is a critical early event in the development of prostate cancer. Besides down-regulation of hZIP-1, abnormalities in other transporters, such as solute carrier family 30 ZnT-4, hZIP-2, and hZIP-3, have also been reported.19,20
hZIP-1, hZIP-2, and hZIP-3 are important zinc uptake transporters involved in the unique ability of prostate cells to accumulate high levels of cellular zinc.12,19,20 hZIP-1 is important for transfer of zinc from circulation to prostate, and hZIP-2 and hZIP-3 are involved in retention of zinc in the cellular compartment. The down-regulation of all three transporters in the malignant cells is consistent with the loss of zinc accumulation in those cells. These observations support the concept that Zip1, Zip2, and Zip3 function as tumor suppressor genes and that zinc is a tumor suppressor agent.12,19
Expression of hZnT-4 was decreased in benign prostatic hyperplasia and tumor samples compared to normal tissue. These studies showed that zinc homeostasis in normal prostate tissue results from an interplay of multiple transporters and that decreased hZnT-4 expression is associated with prostate tissue abnormalities independent of total cellular zinc content.20
In recent epidemiologic studies, relatively high plasma IGF-1 and low IGFBP-3 levels have been independently associated with greater risk of prostate carcinoma.21–24 IGFs have mitogenesis and anti-apoptotic effects on normal and transformed prostate epithelial cells.23,24 Therefore IGFs may be important in the development and progression of prostate cancer. IGFBPs have opposing actions in part by binding IGF-1 but also by direct inhibiting effects on target cells. In our study, we observed that in animals that received 30ppm zinc, IGF-1 levels and IGF-1/IGFBP ratios were significantly decreased in comparison to the deficient group. Thus zinc seems to have an additional effect via its action on IGF-1.
Recent studies indicate that IGF-1 stimulates IL-8 expression through the mitogen-activated protein kinase kinase-extracellular signal-regulated kinase pathway in DU-145 cells partly by augmenting transcriptional activity.25 It remains to be established, however, whether IL-8 mediates certain effects of IGF-1 on prostate cancer cells.
The association between supplemental zinc intake and prostate cancer risk among 46,974 new subjects in the United States participating in the Health Professionals Follow-up Study was examined.15 During 14 years of follow-up, 2,901 new cases of prostate cancer were diagnosed, of which 434 cases were diagnosed as advanced cancer. Supplemental zinc intake at doses of up to 100mg/day was not associated with prostate cancer risk. However, compared with nonusers, men who consumed more than 100mg/day supplemental zinc had a relative risk of advanced prostate cancer of 2.29 (95% confidence interval=1.06 to 4.95; P trend=.003), and men who took supplement for 10 or more years had a relative risk of 2.37 (95% confidence interval=1.42 to 3.95; P trend <.001). The authors suggested that chronic supplementation of excess zinc may play a role in prostate carcinogenesis, although they did not rule out other related factors such as calcium intake or dietary intake of some other confounding elements.15
Zinc has long been associated with prostate health. The observed associations may reflect the effects of self-excess supplementation of zinc for long-standing prostate symptoms. Whether or not this resulted in decreased medical surveillance, which ultimately resulted in late detection of prostate cancer and thus a greater possibility of advanced prostate cancer in these men, remains to be documented. Daily supplementation of zinc in doses higher than 100mg is likely to cause significant interactions with other essential elements, and the deficiency of copper may result.
Zinc is known to antagonize the catalytic properties of the redox-active transition metals (Fe and Cu) with respect to their abilities to promote formation of OH from H2O2 and superoxide.26 Accumulation of copper may enhance generation of reactive oxygen species.27 Decline in the oxidative stress in animals fed zinc could be either due to activation of antioxidant systems such as reduced glutathione and antioxidant enzymes or decrease in copper due to high levels of zinc. Inasmuch as in our studies the normal level of zinc had a preventive role in prostate cancer and the high level resulted in increased tumor growth, it is unlikely that change in the copper level played any preventive role in prostate cancer.
Although evidence from the reviewed epidemiologic studies, together with laboratory and clinical studies, is suggestive of a role for prostatic inflammation in the etiology of prostate cancer,28,29 additional large, prospective studies are necessary to address methodological limitations of existing studies and to investigate a broader range of potential sources of intraprostatic inflammation.
This work was done at Wayne State University School of Medicine, Detroit, MI, USA and the University of Wisconsin Medical Science Center, Madison, WI, USA. Research was supported in part by the National Institutes of Health (grants 5 R01 A150698-04 and R01 CA120451) and Labcatal Laboratories, Paris, France.
All authors have confirmed that no competing financial interests exist.