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Carcinogenesis Advance Access originally published online on April 10, 2006
Carcinogenesis 2006 27(10):1980-1990; doi:10.1093/carcin/bgl034
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Diverse effects of zinc on NF-{kappa}B and AP-1 transcription factors: implications for prostate cancer progression

Robert G. Uzzo, Paul L. Crispen, Konstantin Golovine, Peter Makhov, Eric M. Horwitz and Vladimir M. Kolenko*

Department of Urological Oncology, Fox Chase Cancer Center Philadelphia, PA 19111, USA

*To whom correspondence and requests for reprints should be addressed at: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA. Tel: +1 215 728 5620; Fax: +1 215 728 2741; Email: Vladimir.Kolenko{at}fccc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nuclear factor-kappaB (NF-{kappa}B) and AP-1 nuclear transcriptional factors regulate expression of multiple genes involved in tumor growth, metastasis and angiogenesis; however, the relative contribution of each factor to cancer initiation and progression has not been established. Prostate carcinogenesis involves transformation of normal zinc-accumulating epithelial cells to malignant cells that do not accumulate zinc. Whereas activation of both NF-{kappa}B and AP-1 has been implicated in prostate cancer development and growth, we tested the relative effects of zinc supplementation on these important transcriptional factors. Herein, we demonstrate that physiological levels of zinc inhibit NF-{kappa}B but augment activities of AP-1 in DU-145 and PC-3 human prostate cancer cells. Additionally, we show that chelation of zinc with membrane-permeable zinc chelator, N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) abolishes this effect. We further propose a potential mechanism for this observation by demonstrating that zinc supplementation induces phosphorylation of the members of three major MAPK subfamilies regulating AP-1 and NF-{kappa}B activation (ERK 1/2, JNK and p38) while blocking TNF-{alpha}-mediated degradation of the inhibitory subunit I{kappa}B{alpha} and nuclear translocation of RelA in prostate cancer cells. VEGF, IL-6, IL-8 and MMP-9 are major pro-angiogenic and pro-metastatic molecules whose promoter regions contain binding sites for both NF-{kappa}B and AP-1. These cytokines have been associated with negative prognostic features in prostate cancer. We demonstrate that treatment of human prostate cancer cell lines with zinc reduces expression of VEGF, IL-6, IL-8 and MMP-9. We further show that zinc reduces expression of intercellular adhesion molecule-1 and functionally suppresses tumor cell invasiveness and adhesion. Therefore, the ability of zinc supplementation to inhibit NF-{kappa}B supercedes zinc-mediated activation of AP-1 family members. Upregulation of intracellular zinc levels may have important implications for inhibiting the angiogenic and metastatic potentials of malignant cells, predominantly through suppression of NF-{kappa}B signaling.

Abbreviations: AP-1, a dimeric complex that comprises members of the Jun (c-Jun, JunB and JunD)Fos (c-Fos, FosB, Fra-1 and Fra-2)ATF (activating transcription factor) and MAF (musculoaponeurotic fibrosarcoma) protein families; ICAM-1, intercellular adhesion molecule-1; IL-8/6, interleukin-8/6; MAPK, mitogen-activated protein kinases; MMP-9, matrix metalloproteinase-9; NF-{kappa}B, Nuclear transcriptional factor-kappaB; TNF-{alpha}, tumor necrosis factor; TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine; VEGF, vascular endothelial growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Substantial information exists implicating changes in zinc accumulation in the development and progression of prostatic malignancies (1–3). Normal human prostate accumulates the highest levels of zinc of any soft tissue in the body. This unique capability is retained in benign prostatic hyperplasia (BPH). In contrast, zinc levels in prostatic epithelial tumors are markedly diminished (4,5). The zinc content of normal prostate epithelial, BPH and cancerous tissues has been measured at 1018, 1142 and 146 µg/g dry tissue, respectively (5). Importantly, zinc accumulation diminishes early in the course of a prostatic malignancy and continues to decline during progression toward hormone-independent growth. Experimental support for this concept was provided by studies with malignant cell lines LNCaP and PC-3. LNCaP cells demonstrate low tumorigenicity as compared to the highly aggressive tumorigenic PC-3 cells. Correspondingly, LNCaP cells possess a high endogenous zinc level (248 ng/mg protein), as compared to much lower zinc levels (164 ng/mg protein) in PC-3 cells (6).

Nuclear transcriptional factors, nuclear factor-kappaB (NF-{kappa}B) and AP-1, contribute to the development and progression of prostatic malignancies by regulating the expression of genes involved in proliferation, apoptosis and angiogenesis as well as tumor invasion and metastasis (7–11). Experimental data demonstrate that thiol-reactive metals including zinc modulate activities of NF-{kappa}B and AP-1 (12–16). However, it is important to note that in most of these studies supra-physiological levels of zinc were employed, representing zinc concentrations which cells would never be exposed in situ. Plasma zinc levels are ~1 µg/ml, of which ~67% is mobile, transportable zinc available for uptake by cells (2). Moreover, recent findings regarding the effect of zinc on the status of transcriptional factors are not consistent. A zinc ionophore, pyrrolidine dithiocarbamate (PDTC) has been shown to have unique reciprocal activities in activating AP-1 and inhibiting NF-{kappa}B in endothelial cells. PDTC activation of AP-1, like its inhibition of NF-{kappa}B, was zinc-dependent. This is consistent with the contention that PDTC acts as a zinc ionophore and its reciprocal actions are mediated by zinc (13). Interestingly, PDTC as a single stimulus induced expression of the pro-angiogenic vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) genes in colon carcinoma cells (17). In contrast, other studies have revealed that zinc supplementation reduces DNA binding activity of both NF-{kappa}B and AP-1 transcriptional factors (18). Conversely, zinc chelation by N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) has been noted to increase NF-{kappa}B and AP-1 binding (19). Although the above studies have discrepant results, collectively they suggest that zinc levels can modulate the activity of transcriptional factors important to malignant transformation including NF-{kappa}B and AP-1.

VEGF, IL-8 and matrix metalloproteinase-9 (MMP-9) are three major pro-angiogenic and pro-metastatic molecules that have been associated with negative prognostic features in various types of cancer. Multiple studies demonstrate that expression of these genes is promoted by co-activation of NF-{kappa}B and AP-1 transcriptional factors (20–23). Suppression of NF-{kappa}B activity in human prostate cancer cells inhibits their tumorigenic and metastatic properties in nude mice by suppressing angiogenesis and invasion via downregulation of VEGF, IL-8 and MMP-9. Decreased expression of these molecules in vivo directly correlates with decreased neovascularization and fewer lymph node metastases (21). Inhibition of the expression of AP-1 component proteins and AP-1 activity also appears to reduce the invasiveness of a variety of cell types including murine and human squamous cell carcinomas (24–27). Recent studies demonstrate that the MEK inhibitor U0126 preferentially blocks AP-1 activity and expression of both IL-8 and VEGF, while a dominant negative inhibitor-{kappa}B blocks NF-{kappa}B activation and expression of IL-8 but not VEGF in human head and neck squamous carcinoma cells (23).

The Rel/NF-{kappa}B family of eukaryotic transcription factors is comprised of several structurally related proteins that form both homo- and heterodimers. The most common Rel/NF-{kappa}B dimer in mammals contains p50-RelA (p65) and is specifically called NF-{kappa}B. The activity of NF-{kappa}B is regulated by interaction with inhibitory I{kappa}B proteins, which block the ability of NF-{kappa}B to enter the nucleus and bind to DNA. Upon activation, I{kappa}B is phosphorylated and marked for ubiquitination and degradation by the proteosome-dependent pathway. This process allows translocation of active NF-{kappa}B complexes into the nucleus (28,29). AP-1 also is a dimeric complex that comprises members of the Jun (c-Jun, JunB and JunD), Fos (c-Fos, FosB, Fra-1 and Fra-2), ATF (activating transcription factor) and MAF (musculoaponeurotic fibrosarcoma) protein families. The main AP-1 proteins in mammalian cells are Fos and Jun, which form hetero- (Jun–Fos) or homodimers (Jun–Jun) (10,30). Several of the AP-1 proteins including c-Fos, FosB and c-Jun can efficiently transform cells in vitro while other proteins such as JunB have been shown to have tumor suppressive activity (10,31,32). Overexpression of Fra-1 induces transcription of multiple genes associated with tumor progression and promotes motility and invasiveness of mammary adenocarcinoma cells in vitro (33). Recent studies also demonstrate that constitutive activation of NF-{kappa}B, c-Jun, Fra-1 and JunD is associated with prostate cancer progression toward an androgen-independent phenotype (34,35). Both NF-{kappa}B and AP-1 have been shown to be constitutively activated in prostatic malignancies (7,8,35,36). NF-{kappa}B and AP-1 activation can be induced by multiple stimuli including tumor necrosis factor (TNF-{alpha}). Prostate cancer patients with elevated serum levels of TNF-{alpha} have a significantly higher mortality rate than those with undetectable serum TNF-{alpha} levels (30).

The present report reveals that zinc implements opposite effects on the status of AP-1 and NF-{kappa}B activities in PC-3 and DU-145 prostate cancer cells by activating AP-1 family members and inhibiting NF-{kappa}B DNA binding. The primary mechanism whereby zinc inhibits NF-{kappa}B binding appears to be downstream of mitogen-activated protein kinases (MAPKs) and related to its ability to inhibit I{kappa}B{alpha} degradation. We further demonstrate that the addition of zinc to prostate cancer cell cultures dramatically reduces IL-6, IL-8, VEGF and MMP-9 expression, similar to what is seen after pharmacological NF-{kappa}B inhibition, and functionally suppresses invasiveness and adherence of prostate cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells and materials
PC-3 and DU-145 human prostate cancer cell lines were obtained from ATCC (Rockville, MD) and maintained in RPMI 1640 medium (Bio-Whittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT), gentamicin (50 mg/l), sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM). For the actual experiments, cell culture medium has been replaced with RPMI 1640 medium containing 2% FCS. Experiments were performed under conditions indicated in the figure legends.

Antibodies and reagents
Antibodies to RelA, I{kappa}B{alpha} and Topo I were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to phospho-JNK, phospho-Erk 1/2 and phospho-p38 were obtained from Cell Signaling Technology (Beverly, MA). Antibody to actin, ZnSO4, pyrithione, TPEN, Calcein-AM and TNF-{alpha} were obtained from Sigma (St Louis, MO). Fibronectin and BAY11-7085 were obtained from Calbiochem (San Diego, CA). FluoZin-3 AM was obtained from Molecular Probes (Eugene, OR).

Measurement of AP-1 and NF-{kappa}B activities
Cells were pre-incubated with the indicated concentrations of zinc and/or pyrithione (4 µM) with or without TPEN (7.7 µM) for 30 min and cultured in either medium alone or in the presence of TNF-{alpha} (10 ng/ml) for 1.5 h. The activity of NF-{kappa}B (p65) and AP-1 (phospho-c-Jun, c-Fos, FosB, Fra-1, Fra-2, JunD and JunB) was examined in nuclear extracts by TransAM NF-{kappa}B (p65) and AP-1 family kits (Active Motif, Carlsbad, CA), respectively, in accordance with the protocols provided with the kits. TransAM kits contain 96-well plates to which oligonucleotides containing either an NF-{kappa}B consensus binding site or a TPA-responsive element (TRE) have been immobilized. The activated NF-{kappa}B or AP-1 dimers contained in nuclear extracts specifically bind to these oligonucleotides and are detected using specific antibodies. Nuclear extracts were prepared as described previously (37). Protein concentrations were measured with BCA protein assay reagents (Pierce, Rockford, IL).

Detection of intracellular zinc uptake
Cells were loaded with 5 µM FluoZin-3 AM at 37°C for 30 min in RPMI 1640 medium, washed twice with phosphate-buffered saline (PBS) and analyzed by flow cytometry. Analysis was performed using FACScan (Becton Dickinson, Franklin Lakes, NJ). Individual fluorescent populations were determined through the use of acquisition and analysis software (Cell Quest, Becton Dickinson).

Western blot analysis
Nuclear and cytoplasmic extracts and whole cell lysates were prepared as described previously (37). Protein concentrations were measured with BCA protein assay reagents (Pierce, Rockford, IL). Equivalent amounts of proteins (20 µg) were mixed in an equal volume of 2x Laemmli sample buffer, boiled and resolved by electrophoresis in 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE). The proteins were then transferred from the gel to a nitrocellulose membrane using an electroblotting apparatus (Bio-Rad) (15 V, 3 mA/cm2 for 24 min). Membranes were incubated in blocking solution containing 5% non-fat dry milk overnight to inhibit non-specific binding. The membranes were then incubated with specific antibody (1–3 µg/ml) for 2 h. After washing in Tris/0.1% Tween-20 for 30 min, membranes were incubated for another 30 min with horseradish peroxidase-conjugated secondary antibody. The membranes were then washed and developed with enhanced chemiluminescence (ECL; Western Blotting Kit, Amersham, Arlington Heights, IL).

Measurement of VEGF, IL-6, IL-8 and MMP-9 proteins
Cells were pre-incubated with indicated concentrations of zinc and/or pyrithione (4 µM) for 30 min followed by incubation with 10 ng/ml of TNF-{alpha} for 18 h. IL-6, IL-8, VEGF and MMP-9 levels in cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN).

Immunocytometry
Surface expression of intercellular adhesion molecule-1 (ICAM-1) and gp130 was determined by staining cells with either FITC-conjugated anti-ICAM-1 or PE-conjugated anti-gp130 antibodies (R&D Systems) for 30 min on ice. Stained cells were washed twice with PBS and analyzed by flow cytometry. Analysis was performed on the FACScan (Becton Dickinson). Individual fluorescent populations were determined through the use of acquisition and analysis software (Cell Quest, Becton Dickinson).

Measurement of cell death
Cells were harvested and stained with 1 µg/ml of propidium iodide (PI) followed by flow cytometry analysis.

Analysis of cancer cells invasiveness
Invasiveness was determined using BD Falcon HTS FluoroBlok system (BD Biosciences, Bedford, MA) in triplicate for each condition. PC-3 cells (2.5 x 103) were seeded in the upper compartment of the FluoroBlok chamber in the absence of serum with or without the indicated concentrations of zinc and/or pyrithione. Serum-containing medium in the lower compartment served as a chemoattractant. Cells were incubated at 37°C for 12 h and then stained with 2 µM of Calcein-AM. An intervening membrane was present to block fluorescence from labeled cells present in the top chamber of the insert system. Fluorescence from labeled cells present in the lower chamber of the insert system was detected using a microplate fluorimeter with excitation and emission wavelengths of 485 and 530 nm, respectively.

Adhesion assay
PC-3 cells were stained with Calcein-AM (2 µM), pre-incubated with indicated concentrations of zinc and/or pyrithione for 1 h and plated in triplicates onto 96-well plates (2.5 x 103 cells/well) pre-coated with fibronectin (50 µg/ml). Cells were allowed to attach at 37°C for 30 min. Wells were washed twice with PBS, and fluorescence was detected using a microplate fluorimeter with excitation and emission wavelengths of 485 and 530 nm, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Differential effect of zinc on the activity of AP-1 and NF-{kappa}B transcription factors in prostate cancer cells
Recent studies present discrepant data on the effect of zinc on the activation of NF-{kappa}B and AP-1 nuclear transcriptional factors (13,17–19). Since NF-{kappa}B and AP-1 upregulate expression of numerous genes involved in tumor development and progression, we compared the effect of physiological levels of zinc on the activation status of these important transcriptional factors in prostate cancer cells. Two androgen-independent prostate cancer cell lines, DU-145 and PC-3, were pre-treated with indicated concentrations of zinc, with or without 4 µM of the zinc ionophore pyrithione, and then stimulated with 10 ng/ml of TNF-{alpha}. Prostate cancer cells have lost their ability to accumulate zinc; therefore, pyrithione was used to facilitate zinc transport across cell membrane (38). Nuclear extracts were prepared from cells and analyzed for NF-{kappa}B (p65) and AP-1 (phospho-c-Jun) activities using TransAM assay. The findings presented in Figure 1A and B demonstrate that inhibition of NF-{kappa}B binding was observed with 0.25 µg/ml of zinc, whereas 0.5 µg/ml of zinc completely abolished TNF-{alpha} induced NF-{kappa}B activation in both cell lines. Next, we examined the effect of zinc on the status of AP-1 activity. As seen in Figure 1A and B, physiological concentrations of zinc (0.25–0.5 µg/ml) that were noted to inhibit NF-{kappa}B activity noticeably upregulated AP-1 activation in DU-145 and PC-3 cells. Importantly, without pyrithione, physiological concentrations of zinc had no effect on NF-{kappa}B or AP-1 activation in prostate cancer cells (data not shown). Pyrithione alone also had no significant effect on the status of either NF-{kappa}B or AP-1 activity (Figure 1A and B). The addition of the intracellular zinc chelator TPEN at equimolar concentration almost completely abolished the effect of zinc on the inhibition of NF-{kappa}B and the activation of AP-1. Experiments were performed with both DU-145 and PC-3 cell lines to demonstrate that the observed effects were not cell line specific.


Figure 1
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  		Fig. 1 Effect of zinc on NF-{kappa}B, AP-1 and MAPKs activities in prostate cancer cells. (A) DU-145 and (B) PC-3 cells were pre-incubated with indicated concentrations of zinc in the form of ZnSO4 and/or pyrithione (4 µM) with or without TPEN (7.7 µM) in triplicates for 30 min followed by incubation with 10 ng/ml of TNF-{alpha} for 1.5 h. NF-{kappa}B (p65) and AP-1 (phospho-c-Jun) activities were examined using TransAM assay as described in the Materials and methods section. Statistical analysis was performed by one-way ANOVA. *P < 0.05; statistically different compared with cells stimulated with TNF-{alpha} alone. Representative data from one of four experiments. (C) Zinc induces phosphorylation of MAPKs in PC-3 cells. Cells were treated with ZnSO4 (0.5 µg/ml) and pyrithione (4 µM) for the indicated periods of time. Cell lysates were subjected to SDS–PAGE, blotted and probed with antibodies against phosphorylated or total ERK 1/2, JNK or p38. (D) Zinc suppresses nuclear translocation of RelA and blocks degradation of I{kappa}B{alpha} in PC-3 cells. Cells were pre-incubated with ZnSO4 (0.5 µg/ml) and pyrithione (4 µM) for 30 min followed by incubation with 10 ng/ml of TNF-{alpha} for 1.5 h. RelA protein levels were determined by western blotting analysis with specific antibodies in nuclear extracts. Cytoplasmic extracts from the same samples were subjected to SDS–PAGE followed by western blotting with anti-I{kappa}B{alpha} antibody. Expression of Topo I (nuclear extract) and actin (cytoplasmic extract) was used to control equal protein loading. Representative data from one of five experiments.

 
The signaling cascade of the MAPK regulates the activation of various transcription factors. For this reason, we examined the effects of zinc on the phosphorylation states of p38, ERK 1/2 and JNK, which are the upstream modulators of NF-{kappa}B and AP-1 signaling (39–42). We observed that zinc induced rapid phosphorylation of all three MAPKs in PC-3 cells (Figure 1C). We subsequently examined the effects of zinc on TNF-{alpha}-induced nuclear accumulation of RelA and the degradation of the inhibitory subunit I{kappa}B{alpha}. In PC-3 cells pre-treated with zinc, the lack of nuclear accumulation of RelA corresponded with failure to degrade I{kappa}B{alpha} (Figure 1D). These findings indicate that inhibition of NF-{kappa}B activation by zinc occurs downstream from MAPKs.

We have also examined intracellular zinc uptake in DU-145 cells incubated with various concentrations of zinc with or without using the cell permeable zinc-specific indicator, FluoZin-3. The findings presented in Figure 2 demonstrate that in the absence of pyrithione there was no detectable zinc uptake in DU-145 cells. Pyrithione alone had no significant effect on zinc accumulation while concomitant administration of pyrithione and zinc resulted in a dose-dependent rise in the intracellular zinc levels (Figure 2).


Figure 2
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  		Fig. 2 Intracellular zinc accumulation in DU-145 cells. Cells were pre-incubated with indicated concentrations of zinc with or without 4 µM pyrithione in RPMI 1640 medium supplemented with 2% FCS for 30 min, washed twice in Ca2+ free PBS and incubated with cell-permeant AM ester form of fluorescent zinc indicator FluoZin-3 for additional 30 min. Fluorescence was examined by flow cytometry. x-axis represents fluorescence intensity, y-axis represents cell number. Representative data from one of three experiments.

 
To further explore the effect of zinc on AP-1 activity, we investigated DNA binding activities of individual members of the AP-1 family. As demonstrated in Figure 3, concomitant treatment with zinc and pyrithione induced notable levels of activation of all AP-1 family members tested in PC-3 cells. Zinc and pyrithione had no effect on the activation of the AP-1 family members when used separately (data not shown). Thus, our results demonstrate that the addition of physiological concentrations of zinc to cell culture medium exerts opposite effects on the activities of NF-{kappa}B and AP-1 in prostate cancer cells.


Figure 3
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  		Fig. 3 AP-1 family profiling upon zinc treatment in PC-3 prostate cancer cells. PC-3 cells were treated with zinc (0.5 µg/ml) in the form of ZnSO4 and pyrithione (4 µM) in triplicates for 1.5 h. DNA binding activities of AP-1 family members were examined using TransAM assay as described in the Materials and methods section. Statistical analysis was performed by one-way ANOVA. *P < 0.05; statistically different compared with cells cultured in medium alone. Representative data from one of three experiments.

 
Physiological concentrations of zinc decrease expression of proteins involved in tumor angiogenesis and invasion; VEGF, IL-8, MMP-9 and ICAM-1
Progressive growth and metastasis of prostate cancer is mediated by the secretion of angiogenic and metastatic factors, including VEGF, IL-8 and MMP-9. Expression of these molecules by prostate cancer cells has been shown to correlate with malignant potential (43–45). Our studies demonstrate that treatment with zinc increases AP-1 while inhibiting NF-{kappa}B (Figures 1 and 3). Since the human VEGF, IL-8 and MMP-9 promoters contain binding sites for both NF-{kappa}B and AP-1 transcription factors, ELISA was undertaken to determine the effect of physiological levels of zinc on the expression of these molecules in prostate cancer cells. Expression of VEGF, IL-8 and MMP-9 was examined in cell culture supernatants of PC-3 cells pre-incubated with various concentrations of zinc and stimulated with TNF-{alpha} for 18 h. As shown in Figure 4A, supernatants collected from PC-3 cells treated with 0.12 µg/ml of zinc showed a significantly decreased amount of secreted VEGF, IL-8 and MMP-9 when compared with supernatants obtained from cells cultured either in medium or in the presence of pyrithione alone. Lower levels of zinc (0.12 µg/ml) were required to produce a noticeable effect on the expression of VEGF and IL-8 molecules by PC-3 cells (Figure 4A) than to modulate the activities of NF-{kappa}B and AP-1 transcription factors (0.25 µg/ml) (Figure 1A and B). This finding may be explained by the fact that PC-3 cells had been cultured in the presence of zinc for different periods of time (18.5 versus 2 h). To provide a direct link between zinc-mediated NF-{kappa}B inhibition and decreased expression of VEGF, IL-8 and MMP-9 in prostate cancer cells, PC-3 cells were pre-incubated with the NF-{kappa}B inhibitor BAY 11-7085 before TNF-{alpha} stimulation in the absence of zinc. BAY 11-7085 is a specific and irreversible inhibitor of I{kappa}B{alpha} phosphorylation (46). Pre-treatment with BAY 11-7085 efficiently blocked TNF-{alpha}-induced expression of VEGF, IL-8 and MMP-9 proteins (Figure 4A). These data confirm the critical role of the NF-{kappa}B pathway in zinc-mediated inhibition of VEGF, IL-8 and MMP-9 expression in prostate cancer cells.


Figure 4
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  		Fig. 4 Physiological concentrations of zinc inhibit expression of VEGF, IL-8, MMP-9 and ICAM-1 in prostate cancer cells. (A) PC-3 cells were pre-incubated with indicated concentrations of zinc in the form of ZnSO4 and/or pyrithione (4 µM) or with BAY11-7085 (2.5 µM) in triplicates for 30 min followed by incubation with 10 ng/ml of TNF-{alpha} for 18 h. VEGF, IL-8 and MMP-9 levels in cell culture supernatants were determined by ELISA as described in the Materials and methods section. Statistical analysis was performed by one-way ANOVA. *P < 0.05; statistically different compared with cells stimulated with TNF-{alpha} alone. (B) PC-3 cells were pre-incubated with indicated concentrations of zinc in the form of ZnSO4 and/or pyrithione (4 µM) or with BAY11-7085 (2.5 µM) for 30 min followed by incubation with 10 ng/ml of TNF-{alpha} for 6 h. Expression of ICAM-1 was determined by immunostaining with anti-ICAM-1 antibody followed by flow cytometry analysis. Matched isotypic control was used for a particular subclass of Ig and system employed (dotted line). x-axis represents fluorescence intensity; y-axis represents cell number. Representative data from one of three experiments. (C) Effect of zinc on viability of PC-3 cells. Cells were cultured with indicated concentrations of zinc and/or pyrithione (4 µM) for 18 h, harvested and stained with PI followed by flow cytometry analysis. x-axis represents fluorescence intensity, y-axis represents cell number.

 
NF-{kappa}B and AP-1-regulation of the ICAM-1 (47,48) plays an important role in cell–cell and cell–extracellular matrix interactions, especially during tumor invasion. Increased expression of ICAM-1 correlates with increased metastatic potential of prostate cancer cells (44,49). ICAM-1 expression is greater for highly metastatic androgen-independent PC-3 cells compared with poorly metastatic androgen-dependent LNCaP cells (44). Moreover, enhanced expression of ICAM-1 in tumor cells correlates with the size of primary tumor and vascular density (49). Given these data, we examined the effect of physiological concentrations of zinc on the expression of ICAM-1 protein on PC-3 cells (Figure 4B). Immunostaining with anti-ICAM-1 antibody reveals that addition of zinc to the cell culture medium drastically inhibits TNF-{alpha}-mediated ICAM-1 upregulation on PC-3 cells. Similar results were obtained with the NF-{kappa}B inhibitor BAY 11-7085 (Figure 4B).

To rule out the possibility that zinc inhibits IL-8, VEGF, MMP-9 and ICAM-1 expression by affecting cell survival, we performed PI staining of PC-3 cells. Cell membranes in viable cells are impermeable to PI. In contrast, only dead cells whose plasma membrane integrity has been disrupted incorporate PI. Cells both suspended in the media and attached to the plate were harvested, stained with PI and analyzed by flow cytometry. As shown in Figure 4C, only a small number (10.1%) of prostate cancer cells treated with 0.5 µg/ml of zinc for 18 h had positive PI staining indicating 90% cell viability.

Zinc downregulates expression of IL-6 and gp130 subunit in prostate cancer cells
NF-{kappa}B inducible IL-6 is an important multifunctional cytokine involved in tumor progression towards androgen-independent growth and metastatic spread (50–52). IL-6 signaling in target tissues is mediated through a receptor composed of the ligand-binding (gp80, IL-6R-{alpha}) and signal-transducing (gp130, IL-6R-ß) subunits. Constitutive activation of gp130 leads to neuroendocrine differentiation in prostate cancer cells in vitro and in vivo and is associated with an increased risk of death (53). To determine whether increased zinc uptake is capable of suppressing IL-6-mediated signaling and therefore might have an impact on the development of androgen independence in prostate cancer cells, we examined the effect of zinc on the expression of IL-6 by PC-3 prostate cancer cells. As shown in Figure 5A, the addition of zinc significantly reduces the amount of IL-6 secreted by androgen-dependent PC-3 prostate cancer cells. Additionally, we examined expression of the gp130 subunit in the aliquots of the same cells. As demonstrated in Figure 5B, PC-3 cells constitutively express high levels of gp130 subunit, which was markedly downregulated in the presence of physiological zinc concentration. Of note, inhibition of NF-{kappa}B activation in PC-3 cells with BAY 11-7085 also resulted in decreased IL-6 production and gp130 expression (Figure 5 A and B).


Figure 5
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  		Fig. 5 Zinc downregulates expression of IL-6 and gp130 subunit in PC-3 prostate cancer cells. (A) PC-3 cells were pre-incubated with 0.25 µg/ml of zinc in the form of ZnSO4 and/or pyrithione (4 µM) or with BAY11-7085 (2.5 µM) in triplicates for 30 min followed by incubation with 10 ng/ml of TNF-{alpha} for 18 h. IL-6 levels in cell culture supernatants were determined by ELISA as described in the Materials and methods section. Statistical analysis was performed by one-way ANOVA. *P < 0.05; statistically different compared with cells stimulated with TNF-{alpha} alone. Representative data from one of three experiments. (B) Aliquots of the same cells were subjected to immunostaining with anti-gp130 antibody followed by flow cytometry analysis. Matched isotypic control was used for a particular subclass of Ig and system employed (dotted line). x-axis represents fluorescence intensity; y-axis represents cell number. Representative data from one of three experiments.

 
Effects of zinc on invasion and adhesion of PC-3 cells
Invasion through the extracellular matrix represents a critical step in tumor metastasis. Recent studies indicate that metastatic activity of prostate cancer cells correlates with expression of pro-angiogenic factors (44). Therefore, we anticipated that zinc-mediated inhibition of NF-{kappa}B activity and expression of VEGF, IL-8 and MMP-9 proteins would have a functional impact on the metastatic potential of tumor cells. The findings presented in Figure 6 demonstrate that in the presence of pyrithione, physiological concentrations of zinc (0.12–0.5 µg/ml) noted to suppress the expression of pro-angiogenic and pro-metastatic proteins drastically reduced invasiveness of highly invasive PC-3 cells, whereas pyrithione alone had no significant effect on cell invasion. We then examined the effect of zinc on PC-3 cells adhesion since cell migration and adhesion are interrelated processes responsible for the invasion and metastasis of cancer cells. As shown in Figure 7, there was a clear dose–response relationship of the inhibitory effects of zinc on adhesion of PC-3 cells to fibronectin. Physiological levels of zinc without pyrithione failed to inhibit invasiveness and adhesion of PC-3 cells (data not shown). These results suggest that restoration of physiologically normal levels of zinc may inhibit the tumorigenic phenotype of prostate cancer cells.


Figure 6
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  		Fig. 6 Physiological levels of zinc reduces invasiveness of PC-3 cells. Cells were seeded in the upper compartment of the FluoroBlok chamber in the absence of serum with indicated concentrations of zinc in the form of ZnSO4 and/or pyrithione (4 µM) in triplicate for 18 h. (A) Representative images of PC-3 cells treated under indicated conditions that invaded through the FluoroBlok membrane. Images have been captured using fluorescence microscope equipped with a digital camera. (B) Migration of PC-3 cells through the filters was quantified as described in the Materials and methods section. Statistical analysis was performed by one-way ANOVA. *P < 0.05; statistically different compared with the untreated control. Representative data from one of four experiments.

 

Figure 7
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  		Fig. 7 Effect of zinc on the adhesion of PC-3 cells. Adhesion of PC-3 cells was determined as described in the Materials and Methods section. (A) Representative images of attached PC-3 cells. Images have been captured using a fluorescence microscope equipped with a digital camera. (B) Adhesion capacity is depicted as tumor cell binding and related to 100% binding of non-treated control. Statistical analysis was performed by one-way ANOVA. *P < 0.05; statistically different compared with the untreated control. Representative data from one of four experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
NF-{kappa}B and AP-1, nuclear transcriptional factors, regulate the expression of multiple genes involved in tumor growth, metastasis and angiogenesis (14–21), and therefore represent promising therapeutic targets for cancer prevention and treatment. A number of studies have implicated zinc in the control of NF-{kappa}B and AP-1 activation (12–16,18,54,55). However, results of these studies regarding the outcome of zinc treatment on the status of NF-{kappa}B and AP-1 activities are inconsistent. Some reports suggest that zinc supplementation reduces the DNA binding activity of NF-{kappa}B and AP-1 (18) while zinc chelation increases NF-{kappa}B and AP-1 DNA binding (19). Conversely, other studies demonstrate that the addition of zinc exerts opposite effects on these transcription factors and increases DNA binding activity of NF-{kappa}B and AP-1 (13,56). Prostate carcinogenesis involves transformation of normal zinc-accumulating epithelial cells to malignant cells, which do not accumulate zinc (2,4,5). Therefore, in the present study we explored the role and mechanisms of physiological levels of zinc on activation of NF-{kappa}B and AP-1 transcriptional activity in human prostate cancer cells as well as the implications that modulation of both transcriptional factors have on tumor growth, adhesiveness and invasion. Our experiments reveal that physiological concentrations of zinc (0.25–0.5 µg/ml) noted to inhibit NF-{kappa}B activity promote AP-1 activation in androgen-independent DU-145 and PC-3 cells. These results are in agreement with recent findings demonstrating that zinc implements opposite effects on the activity of NF-{kappa}B and AP-1 transcription factors in bovine cerebral endothelial cells (13). In the present study, we also demonstrate that treatment of PC-3 cells with zinc induces prominent phosphorylation of the members of three major MAPK subfamilies regulating NF-{kappa}B and AP-1 activation, namely ERK 1/2, JNK and p38 (Figure 1C). In addition, our experiments show that zinc blocks TNF-{alpha}-mediated degradation of the inhibitory subunit I{kappa}B{alpha} and nuclear translocation of RelA (Figure 1D). Together, these data indicate that zinc induces NF-{kappa}B inhibition downstream from MAPKs, probably by blocking I{kappa}B kinase (IKK). Indeed, recent studies demonstrate that thiol-reactive metal compounds inhibit NF-{kappa}B activation by blocking IKK (57).

Increased constitutive activation of NF-{kappa}B and AP-1 transcription factors in prostate cancer cells is believed to be a major event contributing to malignant transformation and progression of the prostate cancer phenotype (8,21,35,58). Molecular mechanisms underlying tumor progression are not yet completely characterized. However, there is growing evidence that increased expression of genes related to angiogenesis and metastasis, e.g. VEGF, IL-8 and MMP-9, directly correlates with malignant potential (43–45). It is well established that the expression of angiogenic and metastatic molecules in cancer cells is largely controlled by NF-{kappa}B (21,59–61). Therefore, it is expected that as a result of its inhibitory effect on the status of NF-{kappa}B activity, zinc will also abolish expression of NF-{kappa}B-regulated genes. On the other hand, AP-1 is another nuclear transcription factor that is known to be important in the regulation of VEGF, IL-6, IL-8 and MMP-9 expression (20,22,23). Interestingly, although zinc activates AP-1 in prostate cancer cells, the expression of VEGF, IL-6, IL-8 and MMP-9 is suppressed in zinc treated cells (Figures 4 and 5). Thus, our data are in agreement with results of others demonstrating that NF-{kappa}B signaling blockade is sufficient to inhibit expression of pro-angiogenic molecules and therefore potentially decrease neoplastic angiogenesis (21). It is well established that certain concentrations of zinc may trigger loss of mitochondrial potential and induce apoptosis in prostate cancer cells (1). The concentrations of zinc utilized in our experiments did not significantly affect cell viability (Figure 4C). Thus, the suppressive effect of zinc on the angiogenic and metastatic potentials of cancer cells was not simply attributable to cell death but rather was mediated through the inhibition of specific pathways regulating progression of prostate cancer.

Prostate cancer cells have lost their ability to accumulate zinc. Therefore, the concomitant administration of pyrithione and zinc was required to induce adequate cellular zinc uptake in PC-3 cells. The exact mechanism underlying the relative zinc deficiency in prostate cancer cells compared to normal prostate cells is unknown. One regulatory systems of zinc accumulation altered in prostate cancer is the expression and/or function of zinc transport proteins. Zinc transporters are largely assigned to two metal-transporter families: the ZIP family which imports zinc and the ZnT family which functions in releasing zinc or sequestering zinc internally (62). Alterations in ZnT family member expression have been noted in prostate cancer cell lines and human prostate cancer specimens (63). Furthermore, a recent study revealed a strong association of prostate cancer in African-American men with downregulation of zinc uptake transporters, hZIP1 and hZIP2 (64).

The loss of the unique ability to retain normal intracellular levels of zinc may be an important factor in the development and progression of prostate cancer. Taken together, our data demonstrate that physiological levels of zinc have contrasting effects on NF-{kappa}B and AP-1 activities. However, the relative effects on NF-{kappa}B inhibition appear to supercede those of AP-1 family member activation based on our observation that zinc reduces the expression of the pro-angiogenic and pro-metastatic molecules VEGF, IL-6, IL-8, MMP-9 and ICAM-1, and functionally suppresses the tumorigenic potential of prostate cancer cells. These data may support the development of novel strategies for therapeutic interventions in prostate cancer.


   Acknowledgments
 
This work was supported in part by National Institutes of Health Grant RO1 CA108890 (to V.M.K.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 10, 2005; revised February 21, 2006; accepted March 27, 2006.


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