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High intracellular Zn2+ ions modulate the VHR, ZAP-70 and ERK activities of LNCaP prostate cancer cells

Malignant prostate tissues have markedly reduced zinc (Zn2+) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn2+ level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn2+ (10 μg/ml) over 5 weeks. The intracellular Zn2+ level increased in the Zn2+-treated cells, and there was a marked increase in the presence of zincosomes, a Zn2+-specific intracellular organelle. The proliferation rate of the Zn2+-treated cells was markedly reduced. There was also a significant increase (36.6% ± 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn2+, reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn2+-treated LNCaP cell proliferation to a rate comparable to that of the non Zn2+-treated cells. These results highlight the importance of a high intracellular Zn2+ content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.


INTRODUCTION
Prostate cancer is one of the most common malignancies affecting males in Western countries [1]. The etiology of the disease is complex and it has a multitude of potential contributing factors including genetic and environmental factors [2]. One of the consistent markers of prostate malignancy is the significantly low Zn 2+ concentration in malignant prostate tissues [3]. It is presently unknown how prostate cells could lose the ability to accumulate high levels of intracellular Zn 2+ , and whether this occurs as a consequence of acquiring cancerous properties. In a number of earlier studies, Zn 2+ was reported to inhibit prostate cancer cell proliferation and invasion through several different pathways, including the induction of mitochondrial apoptogenesis [4,5], the suppression of the NF-κB signaling pathways [6], and the suppression of the invasive potentials of human prostate aminopeptidase N and urokinase-type plasminogen activator [7]. In other studies, Zn 2+ is shown to enhance telomerase activity [8] and antagonize the inhibitory effects of bisphosphonates on tumor cell invasion potentials [9]. The diverse effects of Zn 2+ on prostate cells reflect the diversity of the interaction of this ion with the various cellular kinases, phosphatases and transcription factors in the regulation of cellular processes [10]. These include the stimulation of protein tyrosine phosphorylation through the inhibition of phosphatases (PTPs) such as protein tyrosine phosphatase 1B (PTP1B), PTP1C (SHP-1) and vaccinia H1-related phosphatase (VHR) [11][12][13][14], and also the activation of the mitogen-activated protein kinase (MAPK) cascade [15][16][17][18][19]. These earlier findings strongly emphasize the potential importance of Zn 2+ regulation in the pathogenesis of prostate cancers. However, most of these earlier findings are derived from the short-term exposure of prostate cancer cells to Zn 2+ . These cellular responses may be transient responses and do not reflect the long-term effects of a high intracellular Zn 2+ content. In this study, we simulated the effects of progressive long-term intracellular Zn 2+ accumulation in prostate cells by prolonged culture of LNCaP prostate cancer cells in a growth medium supplemented with a supraphysiological concentration of Zn 2+ as a model for the identification of the cell signalling pathways affected by the accumulation of high intracellular Zn 2+ .

Cell cultures and treatments
Human prostate cancer cells with the LNCaP.FGC designation (American Type Culture Collection, Rockville, USA) were maintained in RPMI 1640 (Flowlab, Australia) supplemented with 10% Fetal Bovine Serum (FBS; Biowhittaker, Walkersville, USA). The cells were propagated in growth medium supplemented with Zn 2+ (1 μg/ml and 10 μg/ml) using ZnSO 4 for 5 passages in 5 weeks. Cell cultures supplemented with only the Zn 2+ diluent were propagated in parallel, and used as controls. Cells were also treated with 100 μM N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN; Sigma, St. Louis, USA) for 90 min at 37ºC to chelate Zn 2+ . In separate experiments, the Zn 2+ -supplemented cells were also treated with H 2 O 2 (200 μM), a known inhibitor of phosphatases, for 40 min at 37ºC for immunoblot analyses. The cell proliferation rate was measured over a period of 6 days in week 6 post-initiation of the Zn 2+ treatment using a Cell Titer 96 ® Non Radioactive Cell Proliferation Assay Kit (Promega, Madison, USA). Cells at week 5 post-initiation of the Zn 2+ treatment were used for all the other experiments.

Colorimetric Zn 2+ assay
The concentration of Zn 2+ in the LNCaP cells was determined using the watersoluble pyridylazo dye, 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropyl amino) phenol disodium salt dihydrate (5-Br-PAPS, Fluka, Japan) as described previously [20]. Briefly, cells were scraped from the tissue culture flasks, collected, and washed three times with ice-cold PBS. The cells were then sedimented by centrifugation at 800 × g for 5 min, resuspended in 10% trichloroacetic acid (TCA), and incubated on ice for 15 min. After another centrifugation at 4ºC for 10 min, the pellet was saved and the resulting supernatant was removed and incubated with 0.08 mM 5-Br-PAPS and 29 mM salicylaldoxime at room temperature for 10 min. The absorbance of the mixture was then measured at 570 nm. The TCA-precipitated proteins were solubilized with a buffer containing 8 M Urea, 4% CHAPS, 40 mM Tris and 2 mM Tributylphosphine (TBP). Following an overnight incubation, the protein suspension was centrifuged at 40,000 × g for 1 hour. The supernatant was collected and quantitated using the Micro BCA assay (Pierce, Illinois, USA). The total cellular Zn 2+ concentration was normalized to the cell protein concentration.

Cell cycle distribution analysis
Cells were trypsinized from tissue culture flasks and stained with propidium iodide using the CycleTEST™ PLUS DNA Reagent Kit strictly according to the manufacturer's protocol (BD Biosciences Pharmingen, San Diego, USA). Freshly stained cells were then subjected to flow cytometry analysis using the BD FACS Calibur (BD Biosciences, San Diego, USA). Prior to data acquisition, the linearity and resolution of the flow cytometer were verified and adjusted using DNA Quality Control Particles (BD Biosciences, San Diego, USA). Data acquisition and analysis were performed using BD CellQuest™ Pro Software (BD Biosciences, San Diego, USA) and ModFit LT 3.1 (Verity Software House, Topsham, USA).
Protein tyrosine kinase (PTK) and phosphatase (PTP) assays PTK and PTP assays were performed in separate experiments as described previously [12]. Cells were detached from the tissue culture flasks and washed three times with buffer A, consisting of 20 mM HEPES/NaOH and 20 mM MgCl 2 , pH 7.5. For the PTK assay, cells were resuspended in Buffer A, sonicated and incubated at room temperature for 30 min to allow dephosphorylation. Sodium orthovanadate (10 mM) was added to inhibit phosphatase activities. Protein phosphorylation was initiated by adding ATP (1 mM) to the lysates, and at selected time points, the cell lysates (100 μl) were aliquoted and separated by SDS-PAGE. For the PTP assay, cell lysates obtained following sonication in Buffer A at selected time points were immediately removed and separated by SDS-PAGE. The total tyrosine phosphorylated proteins were detected by immunoblotting using a antibody specific against phosphotyrosine.

Statistical analysis
Statistical analyses were performed using GraphPad Prism Version 4.03 (GraphPad Software Inc., San Diego, USA). The data was expressed as the mean ± standard error of the mean (SEM). Statistical differences were analyzed using Student's T-test or one-way ANOVA as indicated. A P-value < 0.05 was considered statistically significant.

Prolonged Zn 2+ supplementation restores a high intracellular Zn 2+ level to LNCaP prostate cancer cells
LNCaP prostate cancer cells were continuously cultured in a growth medium supplemented with 1 μg/ml and 10 μg/ml Zn 2+ as a means of restoring a high intracellular Zn 2+ level to these cells. At week 5 post-initiation of the Zn 2+ treatment, the Zn 2+ -supplemented LNCaP cells and cells treated with 1 μg/ml Zn 2+ in the presence of Zn 2+ pyrithione exhibited numerous intense fluorescing vesicular zincosome structures ( Fig. 1C, D, E) when stained with Zinquin, a Zn 2+ -specific fluorescent probe which stains for zincosomes in the cell cytoplasm. By contrast, PNT2 and LNCaP cells cultured in a growth medium without the additional Zn 2+ showed a much lower presence of zincosomes ( Fig. 1A, B). A dull homogenous fluorescence that was not representative of Zn 2+ -specific staining was observed in cells where Zn 2+ was chelated using TPEN (Fig. 1F). Zn 2+ in the presence of Zn 2+ pyrithione to enhance Zn 2+ uptake (E) and negative control LNCaP cells cultured in the growth medium supplemented with 1 μg/ml Zn 2+ in the presence of Zn 2+ pyrithione and then treated with TPEN to chelate the Zn 2+ (F). Arrows indicate the zincosomes. The cell nuclei were stained with propidium iodide and viewed under 100× magnification.
At the beginning of the study, the concentrations of Zn 2+ when measured using pyridylazo dye in the PNT2 normal prostate cells and in all the LNCaP cell cultures were respectively 7.8 nmol/mg and ~6.5 nmol/mg protein (Tab. 1). At week 5 post-initiation of the Zn 2+ -treatment, the concentration of Zn 2+ in the Zn 2+ -supplemented LNCaP cells increased to an average of ~10.0 nmol/mg protein, consistent with the increase in the number of zincosomes (Fig. 1B, C). By contrast, the Zn 2+ concentration in the control non-supplemented LNCaP cells remained at ~6.2 nmol/mg protein, and the cells showed a much lower number of zincosomes (Fig. 1A). These results suggested that high intracellular Zn 2+ was restored to the LNCaP cells propagated continuously in the growth medium supplemented with Zn 2+ . Values are means ± SD, n = 2. *significantly different, P < 0.05, b Cells cultured in the growth medium without additional Zn 2+ , c Cells cultured in the growth medium supplemented with 1 μg/ml Zn 2+ , d Cells cultured in the growth medium supplemented with 10 μg/ml Zn 2+ , nd, not determined

Prolonged Zn 2+ supplementation increases protein tyrosine phosphorylation activities in LNCaP cells
The effects of the restoration of a high intracellular Zn 2+ level to LNCaP prostate cancer cell signaling pathways were investigated by examining the effects on tyrosine phosphorylation activities. This was done by initially measuring the PTK and PTP activities in these cells. In the PTK assay, all the PTP activities in the cell lysates were first inhibited with sodium orthovanadate. From the assay, it was shown that protein tyrosine phosphorylation increased in both the Zn 2+supplemented and non-supplemented cell lysates when stimulated with ATP. However, the total tyrosine phosphorylated proteins in the lysates of cells supplemented with 1 μg/ml and with 10 μg/ml Zn 2+ were found to be significantly higher (22.9 ± 1.8% and 28.3 ± 2.3%, respectively; P < 0.05, Student's T-test) when compared to the non-supplemented cells at 5 min poststimulation with ATP ( Fig. 2A, lanes 2, 5 and 8; Fig. 2B, C). These findings suggest that in general, the tyrosine phosphorylation activities were higher in the Zn 2+ -supplemented LNCaP prostate cancer cells.

Prolonged Zn 2+ supplementation does not inhibit protein tyrosine dephosphorylation in LNCaP cells
When no ATP was added in the PTP assay, the intrinsic total tyrosine phosphorylated proteins level of cells supplemented with 10 μg/ml Zn 2+ were found to be 36.6 ± 6.4% and 55.2 ± 9.3% higher than those for the non- after Zn 2+ was chelated with TPEN (Fig. 4A, lane 5 and Fig. 4B). These reductions were lower when compared to the marked reduction (45, 64, 53 and 69%, respectively) observed in cells treated with H 2 O 2 , a known inhibitor of phosphatases (Fig. 4A, lane 4 and Fig. 4B). These findings suggested that the restoration of Zn 2+ to LNCaP cells following continuous supplementation with a supraphysiological concentration of Zn 2+ resulted in an increased abundance of LNCaP prostate cancer cell phosphatases, including VHR, PTP1B and PTP1C.

Prolonged Zn 2+ supplementation increases ZAP-70 kinase and ERK activities in LNCaP cells
The abundance of ZAP-70, a kinase that phosphorylates VHR and participates in the activation of ERK, increased by 32% in cells supplemented with 10 μg/ml Zn 2+ when compared to the non-supplemented control (Fig. 4C, lanes 1 and 3  and Fig. 4D). The abundance of ZAP-70 kinase in these cells decreased to 23% when the Zn 2+ was chelated with TPEN (Fig. 4C, lane 5 and Fig. 4D). This observation suggests that ZAP-70 kinase is responsive to changes in the intracellular Zn 2+ levels. The abundance of phosphorylated ERK1 (p-ERK1; 44 kDa) and p-ERK2 (42 kDa), which were the substrates for VHR dephosphorylation, also increased in LNCaP cells supplemented with Zn 2+ 1 μg/ml (36 and 2%, respectively; Fig. 4C, lane 2 and Fig. 4D) and with Zn 2+ 10 μg/ml (140 and 335%, respectively; Fig. 4C, lane 3 and Fig. 4D). By contrast, only the p-ERK1 protein band was detectable in the untreated control cells (Fig. 4C, lane 1). The abundance of p-ERK1 increased by 45% when the Zn 2+ was chelated with TPEN, but the abundance of p-ERK2 decreased by 38% relative to the non-chelated cells (Fig. 4C, lanes 3 and 5). The level of unphosphorylated ERK in all the cells remained unchanged before and after the Zn 2+ was chelated. By contrast, there was a marked reduction of the unphosphorylated ERK, p-ERK1 and p-ERK2 in the H 2 O 2 -treated cells (Fig. 4C,  lane 4; Fig. 4D). Taken together, these findings suggest that the sustained presence of high intracellular Zn 2+ in the LNCaP prostate cancer cells resulted in an elevation in the activities of ZAP-70 kinase and VHR phosphatase, and in the activation of the ERKs.

Prolonged Zn 2+ supplementation retards LNCaP cell proliferation
At week 5 post-initiation of the Zn 2+ treatment, slight increases in the population of cells in the G1/G0 and S phases in the Zn 2+ -supplemented cell cultures relative to the control non-supplemented cell culture were observed (Fig. 5). In the Zn 2+ -supplemented cell cultures, the percentage of cells in the G2/M phase markedly decreased from 8.8 ± 0.2% to 5.2 ± 0.1% following long-term culture in 10 μg/ml Zn 2+ (Fig. 5C). This indicated that continuous culture of the LNCaP prostate cancer cells in a supraphysiological concentration of Zn 2+ reduced the proportion of mitotic cells but was otherwise not cytotoxic. A significantly lower number of cells was obtained for the LNCaP prostate cancer cells maintained in long-term culture with 10 μg/ml Zn 2+ than for the cells supplemented with only 1 μg/ml Zn 2+ or without additional Zn 2+ when the cells were stimulated to proliferate by passaging the cells into fresh 10% FBS-supplemented growth medium on week six post-initiation of the Zn 2+ treatment ( Fig. 6A; P < 0.05, ANOVA). In these cells, the inhibition of cell proliferation was 42.2 ± 7.4% and 76.7 ± 2.0% on days 3 and 6, respectively (Fig. 6B). These results suggest that the continuous cultivation of LNCaP cells in 10 μg/ml Zn 2+ resulted in a marked retardation of the ability of the LNCaP cells to proliferate. The proliferation rate was restored when cells supplemented with 10 μg/ml Zn 2+ were treated with TPEN on day 6 when compared to the non-TPEN-treated Zn 2+ -supplemented cells and cells cultured without additional Zn 2+ ( Fig. 6C; P < 0.05, ANOVA). The cell proliferation rate of these cells increased by 94.5 ± 45.0% and 427.2 ± 55.1% and on day 3 and 6, respectively, when Zn 2+ was chelated from the cell cultures (Fig. 6D). These findings suggest that removing the high intracellular Zn 2+ from the cell cultures restored the LNCaP prostate cancer cells' cancerous proliferation rate.

DISCUSSION
The importance of Zn 2+ in prostate health was suggested by the findings that the Zn 2+ concentration is highest in the prostate gland but tends to be very low in cancerous prostates. Analyses of malignant prostate tissues showed a 60-70% reduction in Zn 2+ levels in comparison to those of the normal peripheral zone tissues [3]. A decrease in Zn 2+ uptake is also observed in the RWPE2 prostate cancer cell line when compared to its non-tumorigenic equivalent, RWPE1, even though initially, the normal RWPE1 cells had a slightly higher Zn 2+ concentration than the cancerous RWPE2 cells [21].
In this study, we simulated the effects of progressive long-term intracellular Zn 2+ accumulation in LNCaP prostate cancer cells. LNCaP cells were used because they have been shown to exhibit the characteristics of the lateral prostate cells of the rat, which is homologous to the lateral lobes of the peripheral zone of the human prostate [22], the dominant region for the origin of prostate malignancy. Furthermore, LNCaP cells, unlike the in situ prostate cancer cells, retain the ability to accumulate high Zn 2+ levels through a rapid-uptake Zn 2+ transporter like hZIP1 (SLC39A1) [23], and hence, would better facilitate a Zn 2+ uptake study. The results from this study showed that all the Zn 2+ -treated LNCaP cells exhibited the presence of a high number of zincosomes, which are membranebound vesicles with a high amount of labile Zn 2+ and a high total intracellular Zn 2+ concentration. This is in contrast to the much lesser number of zinquinstaining zincosomes and lower total intracellular Zn 2+ concentration in the non-Zn 2+ -treated LNCaP cells and the PNT2 prostate normal cells. The zincosomes were found to be present throughout the five-week treatment period, but absent when the cells were treated with TPEN, a known Zn 2+ chelator. These findings strongly suggest that it is possible to restore a high intracellular Zn 2+ content to prostate cancer cells and that it can be achieved in vitro by prolonged the culture of the cells in supraphysiological Zn 2+ concentrations. Restoring a high intracellular Zn 2+ level affected the LNCaP prostate cancer cell proliferation. This is consistent with the assertion that the high intracellular Zn 2+ level found in normal prostate tissues plays an important role in the regulation of the prostate cells [4][5][6]. It was shown here that the removal of Zn 2+ through chelation with TPEN restored the LNCaP cell proliferation rate, which further substantiates the importance of Zn 2+ in maintaining a healthy prostate. Similar effects were observed in the PC3 cell induced tumors in nude mice, where diet supplementation with Zn 2+ for 5 weeks resulted in an increased plasma Zn 2+ level and subsequent reduction of the tumor growth [24]. We showed that these effects could be achieved in LNCaP prostate cancer cells in vitro, and that enabled the identification of the signaling pathways affected by high intracellular Zn 2+ in prostate cancer cells. How Zn 2+ reduces the proportion of mitotic LNCaP cells or exerts its effects on the cell proliferation rate is not clearly understood. Even though the role of certain cell cycle components has not been excluded in this study, several earlier studies suggested that Zn 2+ affects the MAPK, NF-κB and PI3K pathways [25][26][27]. Zn 2+ also increases the phosphorylation of ERK1/2, JNK and p38 MAPK in prostate cancer cells and in other cell lines [6,16,17]. Furthermore, Zn 2+ mediates cell proliferation or cell death differentially though the ERK signaling pathway [28,29]. In this study, a high level of protein tyrosine phosphorylation activities is observed in LNCaP cells treated for a prolonged period with a supraphysiological concentration of Zn 2+ . The ZAP-70 kinase, VHR and ERK levels are also high in the Zn 2+ -treated cells. The increase in VHR is perhaps directly associated with the increase in ZAP-70 kinase, as the latter uses VHR as its phosphorylation substrate [30]. The ZAP-70/VHR/ERKassociated pathways could be the mechanism by which Zn 2+ exerts its effects on LNCaP prostate cancer cells. This suggestion is supported by the findings that TPEN treatment not only abrogates the inhibition of Zn 2+ -induced cell proliferation, but also reduces the abundance of ZAP-70 kinase and VHR. Alternatively, the increase in VHR could also be due to the accumulation of phosphorylated ERKs, although other MAP kinase phosphatases (MKPs) present in the cells may be competing to inactivate ERKs [31,32]. It is noted from this study that a decrease in VHR following the chelation of Zn 2+ from the cells using TPEN only increased the abundance of p-ERK1 and not p-ERK2. ERK1 could be a specific target of dephosphorylation by VHR in the Zn 2+ -treated LNCaP prostate cancer cells. How the ZAP-70/VHR/ERK-associated pathways modulate the prostate cancer cell growth is currently unclear, but an earlier study reported that ZAP-70 activates SLP76 and its associated proteins downstream for the activation of Ras and subsequently ERKs in T cells [33], and, in several studies, sustained activation of ERK has been reported to promote cell growth inhibition [34][35][36]. However, further study will be necessary to examine these possibilities in Zn 2+ -treated LNCaP prostate cancer cells.
In summary, the results of this study suggest that high intracellular Zn 2+ could be restored to prostate cancer cells in vitro by prolonged supplementation with high Zn 2+ . It is likely that high intracellular Zn 2+ affects the cells' proliferation rate through activation of the ERK via the VHR/ZAP-70 pathway.