Skip Navigation


Carcinogenesis Advance Access originally published online on February 28, 2008
Carcinogenesis 2008 29(9):1692-1700; doi:10.1093/carcin/bgn027
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Figure 1
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/9/1692    most recent
bgn027v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dubi, N.
Right arrow Articles by Hershfinkel, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dubi, N.
Right arrow Articles by Hershfinkel, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Extracellular zinc and zinc-citrate, acting through a putative zinc-sensing receptor, regulate growth and survival of prostate cancer cells

Noga Dubi, Larisa Gheber1,2, Daniel Fishman, Israel Sekler3 and Michal Hershfinkel*

Department of Morphology
1 Department of Clinical Biochemistry
2 Department of Chemistry, Zlotowski Center for Neuroscience, Faculty of Health Sciences, Ben-Gurion University, PO Box 653, Beer Sheva 84105, Israel
3 Department of Physiology, Zlotowski Center for Neuroscience, Faculty of Health Sciences, Ben-Gurion University, PO Box 653, Beer Sheva 84105, Israel

* To whom correspondence should be addressed. Tel: +972 8 6477318; Fax: +972 8 6477627; Email: hmichal{at}bgu.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Prostate Zn2+ concentrations are among the highest in the body, and a marked decrease in the level of this ion is observed in prostate cancer. Extracellular Zn2+ is known to regulate cell survival and proliferation in numerous tissues. In spite of this, a signaling role for extracellular Zn2+ in prostate cancer has not been established. In the present study, we demonstrate that prostate metastatic cells are impermeable to Zn2+, but extracellular Zn2+ triggers a metabotropic Ca2+ rise that is also apparent in the presence of citrate. Employing fluorescent imaging, we measured this activity in androgen-insensitive metastatic human cell lines, PC-3 and DU-145, and in mouse prostate tumor TRAMP-1 cells but not in androgen-sensitive LNCaP cells. The Ca2+ response was inhibited by G{alpha}q and phospholipase C (PLC) inhibitors as well as by intracellular Ca2+ store depletion, indicating that it is mediated by a Gq-coupled receptor that activates the inositol phosphate (IP3) pathway consistent with the previously identified zinc-sensing receptor (ZnR). Zn2+-dependent extracellular signal-regulated kinase and AKT activation, as well as enhanced Zn2+-dependent cell growth and survival, were observed in PC-3 cells that exhibit ZnR activity, but not in a ZnR activity-deficient PC-3 subline. Interestingly, application of Zn2+-citrate (Zn2+Cit), at physiological concentrations, was followed by a profound functional desensitization of extracellular Zn2+-dependent signaling and attenuation of Zn2+-dependent cell growth. Our results indicate that extracellular Zn2+ and Zn2+Cit, by triggering or desensitizing ZnR activity, distinctly regulate prostate cancer cell growth. Thus, therapeutic strategies based either on Zn2+ chelation or administration of Zn2+Cit may be effective in attenuating prostate tumor growth.

Abbreviations: ATP, adenosine triphosphate; CaEDTA, calcium ethylenediamine tetra-acetic acid; ERK, extracellular signal-regulated kinase; GPCR, G-protein-coupled receptor; IP3, inositol phosphate; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide-3 kinase; PKC, protein kinase C; TG, thapsigargin; ZnR, zinc-sensing receptor; Zn2+Cit, Zn2+-citrate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Zn2+ homeostasis in the prostate is more dynamic than in other human tissues, as prostate cells accumulate this ion and secrete it into the prostatic and seminal fluid. Zn2+ is secreted with citrate into the prostatic fluid (1), where it is found at high, i.e. millimolar, concentrations (2). The concentration of ‘free Zn2+’ in this organ is, however, lower than these nominal values (3) since it is mostly complexed to citrate. The accumulation of Zn2+ in prostate cells is facilitated by the activity of Zn2+ transporters from the ZIP family, which mediate vectorial transport of this ion (4,5). By inhibiting mitochondrial aconitase, the major oxidizing enzyme of citrate, intracellular Zn2+ contributes to citrate accumulation in the prostate cells that is then secreted into the seminal fluid (6,7). The inhibition of mitochondrial aconitase significantly lowers adenosine triphosphate (ATP) production, leading to bioenergetic deficiency and attenuation of non-neoplastic prostate cell growth (2). It has been also shown that mitochondrial Zn2+ accumulation is involved in the apoptotic pathways in prostate epithelial cells (8,9). Thus, intracellular Zn2+ accumulation is expected to attenuate prostate cell growth.

That Zn2+ plays a role in prostate tumorigenesis is implied by the fact that Zn2+-accumulating cells were found primarily in the dorsolateral lobe in a rat model, where prostate cancer is also most common in human (10,11). Furthermore, during prostate cancer development a 10-fold decrease in both Zn2+ and citrate concentrations are observed in the prostate epithelium (2). Decreased intracellular Zn2+ has been associated with reduced expression of the Zn2+ transporters, ZIP, that mediate influx of Zn2+ into prostate epithelial cells (12). It has been suggested that because of the lower Zn2+ concentration in malignant cells, ATP production is more efficient than in the non-malignant cells, thereby facilitating enhanced proliferation of the malignant cells. While metabolic and signaling roles for intracellular Zn2+ have been suggested (2,6,13), the possible contribution of extracellular Zn2+, or the Zn2+-citrate (Zn2+Cit) complex, to prostate cell growth is still unknown.

Extracellular Zn2+ promotes cell growth by activating and regulating major signaling pathways in numerous cell types. Zn2+, for example, activates the mitogen-activated protein kinase (MAPK) pathway, leading to enhanced proliferation of the colonocytic tumor cell line, HT-29, and NIH3T3 fibroblasts (14–16). Application of Zn2+ also activates the phosphoinositide-3 kinase (PI3K) in human bronchial epithelial cells (17,18) and leads to enhanced survival of fibroblasts (15,19) and neurons (20). Supplementation of Zn2+ also reduces the cellular accumulation of the tumor suppressor p53 in breast cancer cells (21).

Our previous work showed that extracellular Zn2+ induces cellular signaling via a G-protein-coupled receptor (GPCR), termed zinc-sensing receptor (ZnR) (22). We subsequently demonstrated Zn2+ sensing activity in a variety of epithelial cells (14,22,23) with affinity in the 10–100 µM range, i.e. within the range of its physiological concentration in those tissues. ZnR triggers activation of the inositol phosphate (IP3) pathway upon a rise in extracellular Zn2+. The subsequent Ca2+ release in HT-29 colonocytes activates extracellular signal-regulated kinase (ERK) and PI3K pathways and enhances the activity of the Na+/H+ exchanger, NHE1, which regulates cell volume and survival (14). Desensitization of GPCRs is also a protective mechanism against prolonged and potentially harmful activation by their ligands. The ZnR is functionally desensitized following exposure to extracellular Zn2+ (14,23). Desensitization of ZnR in HT-29 colonocytes abolished the Zn2+-dependent activation of the IP3 and the MAPK pathways. Thus, ZnR is emerging as a major link between changes in extracellular Zn2+ and signaling related to cell growth.

In the present study, we have examined the signaling role of extracellular Zn2+ in prostate cancer cells. We demonstrate that a rise in extracellular Zn2+ concentration induces ZnR activity and triggers signaling pathways that facilitate cell growth. Prolonged exposure to Zn2+ subsequently desensitizes ZnR activity, suggesting that in normal prostate tissue, where Zn2+ concentration is high, ZnR is inactive. Based on our data, we propose that in the neoplastic prostate and in peripheral tissues where metastatic cells encounter substantially lower Zn2+ concentrations, ZnR is activated by transient increases in Zn2+ concentration, thereby promoting prostate tumor cell growth.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell culture
PC-3, DU-145, TRAMP-1 and LNCaP cells were obtained from the American Type Culture Collection; PC-3B was identified from a PC-3 line (American Type Culture Collection, Manassas, VA) cultured in the laboratory of Prof. Yossi Levy at Ben-Gurion University. All cells were cultured in RPMI 1640, supplemented with 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), 1 mM sodium pyruvate, 10% fetal bovine serum, 4 mM L-glutamine and 0.5 µg/ml penicillin–streptomycin (24). Cells were seeded on glass coverslips, one day prior to fluorescent imaging experiments.

Fluorescent calcium imaging
Cells grown on coverslips were loaded for 30 min at room temperature with 5 µM Fura-2 AM (TEF Labs, Austin, TX) in Ringer’s solution (in millimolar): 120 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgCl2, 10 HEPES, 10 glucose, pH 7.4, containing 0.1% bovine serum albumin. Ca2+ imaging was carried out as described previously (22). The ratio of the fluorescent signal (R = F340/F380) was normalized to an initial baseline averaged over first 10 points acquired (Rb) and presented as percentage of the baseline (R/Rb x 100). For Zn2+ imaging, cells were loaded as described with 5 µM Newport Green and the fluorescent signal was monitored using 488 nm wavelength for excitation (25). The fluorescent signal was normalized as described for Fura-2. In all experiments, representative graphs of 50–80 cells from at least three independent experiments are shown.

Immunoblot analysis
Cells were harvested immediately following treatment (unless otherwise indicated) into lysis buffer on ice in the presence of protease inhibitor mixture (Complete, Roche Applied Science, Mannheim, Germany) and centrifuged for 30 min (14 000 r.p.m.), as described previously (14). Supernatants (cytosolic fraction) were collected, sodium dodecyl sulfate sample buffer was added and the samples were boiled for 5 min and then frozen at –80°C until used. Cell samples containing the cytosolic fraction (20 µg) were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblotting. Antibodies against doubly phosphorylated ERK1/2 and total ERK1/2 or phosphorylated AKT and total AKT (Sigma Rehovot, Israel and Cell Signaling, Danvers, MA) were detected digitally using ChemImager 5 (Alpha-Innotech, Israel Labtrade), and blots were quantified using this software. Phospho-ERK1/2 or AKT levels were normalized to total ERK1/2 or AKT protein, respectively. Phosphorylation of ERK1/2 or AKT is presented as percentage of the effect triggered by application of 100 µM Zn2+ unless otherwise indicated.

Cell proliferation assay
The quantitative sulphorhodamine B colorimetric assay (26) was used to determine cell proliferation. Cells (5000 cells per well) were seeded in 96-well plates. Cells were incubated daily (5 days) for 10 min in Ca2+-free Ringer's solution supplemented with 100 µM ZnSO4 or with the Zn2+ chelator calcium ethylenediamine tetra-acetic acid (CaEDTA) (100 µM). Cells were then reintroduced into RPMI medium containing 1% serum and CaEDTA. ZnR desensitization was induced by pretreating cells daily with 100 µM ZnSO4 in Ca2+-containing Ringer's solution for 60 min followed by 3x wash with Zn2+-free Ringer's solution. Cells were then stained with sulphorhodamine B and optical density was measured at 492 and 620 nm using enzyme-linked immunosorbent assay plate reader. Statistical analysis was performed on the averaged cell number (±SEM) of at least three independent experiments.

Cell viability assay
Cells (70 000) were seeded in six-well plates. Cells treated with Zn2+, or controls, were centrifuged to sediment all cells to the bottom of the plate and trypan blue (0.4%, Sigma) was added (dilution 1:20). Three images were acquired from each plate, using an upright Olympus microscope (x10 objective) equipped with a SPOT digital camera. Cell counting was performed blind and averaged over three independent experiments.

Statistical analysis
Each bar graph represents an average ± SEM of at least three independent experiments. Statistical analysis was performed using paired Student’s t-test, comparing each treatment to addition of Zn2+ unless otherwise mentioned: *P < 0.05; **P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Since previous studies have shown a decrease in the expression of the ZIP transporters and intracellular Zn2+ during prostate cancer (12), we sought to determine whether changes in Zn2+ concentration activate cellular signaling. We first monitored Zn2+ permeation rates in the androgen-insensitive, metastatic prostate cancer cell line, PC-3, using the intracellular Zn2+-specific dye, Newport Green. PC-3 cells loaded with Newport Green were monitored while superfusing the cells with Zn2+-containing Ringer's solution (Figure 1A). No increase in fluorescence was monitored when Zn2+ (200 µM) was applied, whereas an increase in fluorescence was observed following addition of Zn2+ (200 µM) in the presence of the Zn2+ ionophore, pyrithione (5 µM). This indicates that PC-3 cells are highly impermeable to Zn2+ and is in agreement with previous studies (2,6,12,13).


Figure 1
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Extracellular Zn2+-dependent intracellular Ca2+ release is mediated by the IP3 pathway in the androgen-independent human prostate cancer cell line, PC-3. (A) PC-3 cells, loaded with Newport Green, were superfused with Ringer's solution containing 200 µM Zn2+, and subsequently Zn2+ (200 µM) was added in the presence of pyrithione (5 µM), at the times indicated by the arrows. (B) PC-3 cells, loaded with Fura-2, were superfused with Ringer's solution and 10–100 µM Zn2+ were applied at the indicated time (marked by the arrow). (C) PC-3 cells were superfused with Ca2+ free or Ca2+-containing Ringer's solution and Zn2+ was applied as indicated (arrow). (D) PC-3 cells were treated with the sarcoendoplasmic reticulum calcium ATPase inhibitor TG (0.5 µM) and the purinergic P2Y agonist UTP (uridine triphosphate, 100 µM) in Ca2+-free Ringer's to induce depletion of the Ca2+ stores. Subsequently Zn2+ was added. (E) Zn2+ was added to PC-3 cells pretreated with the PLC inhibitor U73122 [GenBank] (1 µM). (F) Zn2+ was applied to PC-3 cells and the Ca2+ response was observed, and Zn2+ was reapplied in the presence of the G{alpha}q inhibitor, YM-254890 (1 µM). A response to reapplication of Zn2+, at the same concentrations, in control PC-3 cells is shown in the insert.

 
We then asked if extracellular Zn2+ induces intracellular Ca2+ signaling. Changes in intracellular Ca2+ triggered by Zn2+ were monitored in PC-3 cells loaded with Fura-2. Application of 10–100 µM Zn2+ was followed by a rise in fluorescence that was already apparent with the addition of 10 µM Zn2+ and increased by 6-fold with 100 µM (Figure 1B). To determine if the Ca2+ rise resulted from Ca2+ influx from the extracellular medium or from Ca2+ released from intracellular pools, PC-3 cells were superfused with Ca2+-containing or Ca2+-free Ringer's solution. The increase in fluorescence triggered by 100 µM Zn2+ was independent of extracellular Ca2+ (Figure 1C), indicating that it did not result from Ca2+ permeation. To further study the role of the intracellular Ca2+ stores, Zn2+ was applied after depletion of intracellular Ca2+ stores with the sarcoendoplasmic reticulum calcium ATPase inhibitor, thapsigargin (TG, 0.5 µM) and the purinergic P2Y agonist (uridine triphosphate, 100 µM) in Ca2+-free Ringer's solution. Following store depletion, when cytoplasmic Ca2+ was at the basal level, application of Zn2+ failed to elicit an increase in Fura-2 fluorescence (Figure 1D). This indicates that the observed rise in fluorescence is related to Ca2+ release from TG-sensitive endoplasmic reticulum stores. Since Fura-2 is also a high affinity Zn2+ probe (Kd for Zn2+ ~2 nM), the lack of response following store depletion is a further proof that Zn2+ does not permeate into the cells.

We next asked if the IP3 pathway mediates the Zn2+-dependent Ca2+ response in PC-3 cells, similar to the ZnR (22). Application of the phospholipase C (PLC) inhibitor, U73122 [GenBank] (1 µM), was followed by inhibition of the Zn2+-dependent Ca2+ response (Figure 1E). To assess the role of G{alpha}q in linking Zn2+ to activation of the IP3 pathway, cells were incubated with a G{alpha}q inhibitor, YM-254890 [1 µM (27)], and were subsequently superfused with Zn2+. The Zn2+-dependent Ca2+ rise was completely inhibited by the application of YM-254890 (Figure 1F). Analysis of dose dependence of the Zn2+-dependent Ca2+ response yielded a K0.5 of 200 ± 35 µM for extracellular Zn2+ (supplementary Figure 1 is available at Carcinogenesis Online), indicating that although the ZnR response is strongly activated by low ‘free Zn2+’ concentrations found in the prostate, it is activated by a very broad range of Zn2+ concentrations. Thus, our results strongly indicate that PC-3 cells possess a G{alpha}q-coupled Zn2+ sensing receptor that mediates activation of the IP3 pathway to trigger intracellular Ca2+ signaling. A similar Zn2+-dependent signaling pathway, triggered by a putative ZnR, was described previously by us (14,22,23).

To determine if extracellular Zn2+-dependent signaling is also found in other prostate cancer cells, we studied Zn2+-dependent Ca2+ release in the androgen-insensitive human prostate cancer cells, DU-145, and in TRAMP-1 cells, derived from mouse prostate tumor (28). In DU-145 cells, application of 300 µM Zn2+ triggered an intracellular Ca2+ rise (Figure 2A). Yet, application of Zn2+ (300 µM) following depletion of intracellular Ca2+ stores, using ATP (100 µM) and TG (1 µM) in Ca2+-free Ringer's solution, failed to elicit an intracellular Ca2+ rise. In TRAMP-1 cells, application of 100 µM Zn2+ triggered an intracellular Ca2+ rise (Figure 2B) that was diminished by the G{alpha}q inhibitor, YM-254890 (1 µM). Finally, application of 100 µM (data not shown) or 300 µM Zn2+ to the androgen-sensitive LNCaP, prostate cancer cell line (Figure 2C) did not trigger a Ca2+ rise, although application of TG (1 µM) triggered a Ca2+ rise, suggesting that Ca2+ stores were not depleted. These results indicate that ZnR activity is triggered in the androgen-insensitive PC-3 and DU-145, as well as in TRAMP-1 cells, but not in the androgen-sensitive LNCaP cells.


Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. The androgen-independent DU-145 cells as well as the TRAMP-1 mouse prostate tumor cells show Zn2+-dependent Ca2+ response, which is not apparent in the human androgen-sensitive LNCaP cells. (A) Zn2+ was applied to DU-145 cells loaded with Fura-2. Two consecutive Ca2+ responses triggered by Zn2+ are shown. Subsequently, ATP (100 µM) and TG (1 µM) were added to induce store depletion and Zn2+ was then reapplied. No apparent Zn2+-dependent response is shown after store depletion. (B) TRAMP-1 cells were loaded with Fura-2 and Zn2+ was added in Ringer's solution to control cells or cells pretreated with the G{alpha}q inhibitor, YM254890 (1 µM). Following treatment with the G{alpha}q inhibitor, there is no apparent Zn2+-dependent Ca2+ release. (C) Zn2+ was applied to LNCaP cells, followed by application of TG, which triggered the release of Ca2+ from intracellular stores indicating that the latter are intact.

 
Desensitization of Zn2+-dependent Ca2+ signaling
We have previously demonstrated that the ZnR is prone to desensitization by prolonged exposure to Zn2+ (14,23). We therefore asked if desensitization plays a role in Zn2+-dependent signaling also in prostate cells. To test this, PC-3 cells were treated with 100 µM Zn2+ for 60 min in Ca2+-containing Ringer's solution, washed in Zn2+-free Ringer's (5 min) and subsequently Zn2+ was reapplied while monitoring intracellular Ca2+. Pretreatment with Zn2+ resulted in complete inhibition of the Zn2+-dependent Ca2+ response (Figure 3A), indicating that it is very effectively desensitized by Zn2+. In the prostate, Zn2+ is present in very high concentrations (i.e. 6 mM), but is largely complexed with citrate (10 mM), which effectively lowers the concentration of free Zn2 (3). It was of interest, therefore, to determine if Zn2+Cit, in this physiological milieu, modulates intracellular Ca2+ signaling. We accomplished this by monitoring the Ca2+ response of PC-3 cells following application of Zn2+Cit (6 mM Zn2+ and 10 mM citrate). As shown in Figure 3B, no intracellular Ca2+ rise was observed following application of Zn2+Cit at these concentrations. The free Zn2+ concentration using this Zn2+Cit ratio is estimated to be 20 µM (WebMax software), a concentration that is potentially sufficient to trigger Zn2+-dependent signaling (see Figure 1B). Modulating the concentrations of Zn2+ and citrate, leading to a larger excess of free Zn2+, resulted in a smaller Ca2+ response than that triggered by the same concentration of Zn2+ alone (Figure 3B and C). This suggests that the Zn2+-dependent signaling was partially attenuated by Zn2+Cit. We then asked if Zn2+Cit, at physiological concentrations, will desensitize the Zn2+-dependent Ca2+ response. The desensitization paradigm described for Zn2+ was performed using Zn2+Cit at the physiological concentration (6 mM Zn2+ and 10 mM citrate). Pre-exposure of PC-3 cells to Zn2+Cit was followed by profound attenuation of the Ca2+ response to extracellular application of 100 µM Zn2+ (Figure 3D). In contrast, when cells were preincubated with citrate (10 mM), in the absence of Zn2+, subsequent application of Zn2+ triggered a robust Ca2+ response, thereby attesting to a specific role for Zn2+Cit in desensitization of Zn2+-dependent signaling in PC-3 cells (see Discussion).


Figure 3
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Extracellular Zn2+-dependent Ca2+ signal is desensitized by either Zn2+ or Zn2+Cit, although the latter does not activate ZnR. (A) PC-3 cells were pretreated with 100 µM Zn2+ in Ca2+-containing Ringer's solution (desensitized) or Ringer's solution alone (control) for 60 min. Then, Zn2+ was reapplied (arrow) while monitoring intracellular Ca2+ response. (B) Comparison of the Ca2+ response triggered by application of 6 mM Zn2+ in the presence of 10 mM citrate (Zn2+Cit, 20 µM free Zn2+, black), 4.6 mM Zn2+ in the presence of 5 mM citrate (Zn2+Cit, 100 µM free Zn2+, black) or Zn2+ (100 µM, gray) alone. (C) The rate of Ca2+ response to the indicated concentrations of Zn2+ in the presence of 5 mM citrate. (D) PC-3 cells were pretreated with Zn2+Cit or with 10 mM citrate alone (Cit) for 60 min. Intracellular Ca2+ was monitored and Zn2+ (100 µM) was reapplied to the cells at the indicated time (arrow).

 
Role of Zn2+ in activation of MAPK and PI3K in prostate cancer cells
We then sought to determine whether in Zn2+-dependent PC-3 cells, extracellular Zn2+ regulates the MAPK and PI3K pathways linked to proliferation and survival of prostate cancer cells (29). Addition of 100 µM Zn2+ for 10 min to PC-3 cells induced phosphorylation of both ERK1/2 and AKT, whereas control cells treated with the extracellular Zn2+ chelator, CaEDTA (100 µM), exhibited very low phosphorylation (Figure 4A and B). No Zn2+ permeation was observed in PC-3 cells, using this paradigm (see Figure 1A), indicating that phosphorylation of ERK1/2 was trigged by extracellular Zn2+. We found that extracellular Zn2+ triggered prolonged phosphorylation of both ERK1/2 and AKT, which lasted at least 3 h. Rates of Zn2+-dependent activation of ERK1/2 and AKT were different as maximal phosphorylation of ERK1/2 was apparent immediately after extracellular Zn2+ application, whereas phosphorylation of AKT increased during the first 1.5 h period following application of Zn2+ (100 µM).


Figure 4
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Extracellular Zn2+-dependent phosphorylation of ERK1/2 and AKT is mediated by the ZnR signaling pathway in PC-3 cells. (A) PC-3 cells were treated with Zn2+ (100 µM), and at the indicated times, cytosolic extracts were prepared and subjected to immunoblotting using phospho-ERK1/2 (p-ERK1/2) and total ERK (t-ERK) antibodies. Control cells were treated with CaEDTA (100 µM). Bottom: densitometry of pERK1/2 normalized to total ERK1/2 is shown as percentage of Zn2+-dependent activation. (B) Time dependence of AKT phosphorylation and densitometry monitored with phospho-AKT (p-AKT) and total AKT (t-AKT) antibodies using the paradigm described in A. (C) Zn2+ was applied to PC-3 cells pretreated with the MEK1/2 inhibitor, U0126 (1 µM), G{alpha}q inhibitor, YM-254890 (1 µM), the PKC inhibitor bisindolylmaleimide (BI) (2 nM), the PI3K inhibitor wortmannin (100 nM) or coapplication of bisindolylmaleimide (2 nM) and wortmannin (100 nM). Cytosolic extracts were subjected to p-ERK1/2 analysis as in A. (D) p-AKT analysis was performed on cells treated with the same inhibitors as in C or coapplication of bisindolylmaleimide (2 nM) and U0126 (1 µM).

 
We next addressed the role of the IP3 pathway and possible cross talk between the MAPK and PI3K in mediating Zn2+-dependent signaling. Zn2+-dependent phosphorylation of ERK1/2 was completely blocked by the application of the MEK1/2 inhibitor, U0126 (1 µM), indicating that Zn2+-dependent ERK1/2 phosphorylation is mediated via MEK1/2 (Figure 4C). The application of the PI3K inhibitor, wortmannin (100 nM), inhibited the Zn2+-dependent AKT phosphorylation, indicating that it is mediated through the PI3K pathway (Figure 4D). Following application of the G{alpha}q inhibitor, YM-254890 (1 µM, Figure 4C and D), Zn2+-dependent ERK1/2 and AKT phosphorylation was inhibited by 30 and 50%, respectively. This suggests that the IP3 pathway is involved, but is not the sole pathway, in mediating Zn2+-dependent activation of ERK1/2 and AKT. Because protein kinase C (PKC) is activated by Ca2+ and diacylglycerol (30), both generated by the Zn2+-dependent signaling pathway, we asked if PKC is also participating in Zn2+-dependent activation of the MAPK and PI3K pathways (31). The application of the PKC inhibitor bisindolylmaleimide (2 nM) attenuated ERK1/2 and AKT phosphorylation by ~50%, suggesting that PKC activation is also partially mediating Zn2+-dependent activation of both pathways (Figure 4C and D). Since cross talk between the MAPK and PI3K pathways was shown previously, we next examined whether their interaction affects Zn2+-dependent signaling in PC-3 cells. Application of the PI3K inhibitor, wortmannin (100 nM), attenuated ERK1/2 phosphorylation by ~60%, whereas the MEK inhibitor, U0126 (1 µM), inhibited AKT phosphorylation by ~50% (Figure 4C and D). Finally, coapplication of bisindolylmaleimide with wortmannin or U0126 completely abolished the Zn2+-dependent phosphorylation of ERK1/2 or AKT, respectively (Figure 4C and D). These results indicate that PKC and cross talk between the MAPK and PI3K pathways are the major mediators of the Zn2+-dependent phosphorylation of ERK1/2 and AKT in prostate cancer cells.

The role of extracellular Zn2+-dependent signaling in prostate cell growth
Because of the strong activation of MAPK and PI3K pathways by extracellular Zn2+, we asked if it accelerates cell growth. Prolonged exposure to Zn2+ was shown previously to reduce prostate cell proliferation and induce cell death (32). Yet, prolonged exposure is expected to induce desensitization of intracellular signaling (Figure 3). We therefore employed a paradigm in which cells were only briefly exposed to Zn2+. This paradigm simulates more accurately the physiological environment in which most of the extracellular Zn2+ is bound, though it may produce a brief rise in free Zn2+. To assess the effect of extracellular, Zn2+-dependent signaling on cell growth, PC-3 cells were treated daily for 10 min with Zn2+-containing (10 or 100 µM) Ringer's solution and cell number was monitored using sulphorhodamine B stain. Zn2+-dependent intracellular signaling was fully activated under these conditions (see insert in Figure 5A), yet desensitization or Zn2+ permeation was not apparent. To eliminate residual Zn2+, Ringer's solution containing the Zn2+ chelator, CaEDTA (100 µM, 10 min), was used as an additional control. Cells were grown in serum-free medium to minimize the interference of Zn2+ contamination or serum-derived growth factors. As shown in Figure 5A, the growth rate of cells treated with nominally Zn2+-free solution (control) or CaEDTA was attenuated. The brief daily exposure to 100 µM Zn2+ enhanced PC-3 proliferation, already on the third day of treatment, by ~30% compared with control cells (Figure 5A). By the fifth day, the number of Zn2+-treated cells increased further and was 2-fold higher than the controls. The Zn2+-dependent enhancement of cell growth was dose dependent, as application of 10 µM Zn2+ induced only about a 35% increase in cells by the fifth day.


Figure 5
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Extracellular Zn2+-dependent cell growth is attenuated by ZnR desensitization. (A) PC-3 cells were treated daily for 10 min with Ringer's solution containing 10 (triangles) or 100 µM Zn2+ (circles); 100 µM CaEDTA (diamonds) or with Ringer's alone (control, open squares). Cell number was monitored using the sulphorhodamine B colorimetric assay during five consecutive days. The respective Zn2+-dependent Ca2+ response of the cells is shown in the insert. (B) PC-3 cells were pretreated, daily, with Zn2+Cit (6 mM Zn2+, 10 mM citrate) to desensitize ZnR (see Figure 3). Control cells were pretreated with citrate alone as marked. Then Zn2+ (open circles and filled black squares), or CaEDTA (filled circles), was applied, daily, and cell number monitored as in A. Following Zn2+Cit pretreatment, at concentration which induces ZnR desensitization, the Zn2+-dependent cell growth was abolished. Note that following Zn2+Cit pretreatment the growth curves in the presence of Zn2+ or CaEDTA (black squares or filled circles) are overlapping. (C) PC-3 cells, treated as in A, were stained using trypan blue on the fourth day. Averaged number of trypan blue-stained cells in the field of view (x10), in each treatment, is shown together with a representative light microscope image.

 
To determine the effect of desensitization on Zn2+-dependent proliferation, PC-3 cells were treated daily with Zn2+Cit (using the same experimental paradigm described in Figure 3), followed by a short exposure to Zn2+ (100 µM). Application of citrate alone (see Figure 3) served as a control. The number of control cells treated with citrate alone and then with Zn2+ increased by 4-fold on the fifth day (Figure 5B). In contrast, pre-exposure of PC-3 cells to Zn2+Cit completely attenuated Zn2+-dependent cell growth (Figure 5B). In fact, the number of cells pretreated with Zn2+Cit decreased by the fifth day, similar to the effect of CaEDTA. These results indicate that extracellular Zn2+ promotes cell growth suppressed by Zn2+Cit.

We next asked whether extracellular Zn2+ also has a prosurvival effect. PC-3 cells were treated daily for 4 days with Zn2+, the Zn2+ chelator CaEDTA or Ringer's solution (control) and subjected to trypan blue staining (Figure 5C). Brief daily exposure to Zn2+ resulted in a significant reduction in the number of trypan blue stained (i.e. dead) cells. Zn2+ chelation with CaEDTA resulted in a 10-fold increase in trypan blue staining compared with control cells. These results suggest that the effects of extracellular Zn2+ on cell growth are also mediated by attenuation of cell death.

Zn2+-dependent enhancement of cell growth is not observed in prostate cancer cells which are deficient in ZnR activity
Several sublines of PC-3 cells, differing in their metastatic potential and their response to chemotherapeutic drugs, have been described and attributed to their relatively high genetic instability and phenotypic drift (33,34). We therefore screened PC-3 lines from several sources, aiming to identify one not exhibiting Zn2+-dependent Ca2+ release to further link Zn2+-induced intracellular signaling and prostate cancer cell growth. A comparison between the Ca2+ rise triggered by extracellular Zn2+ in our original PC-3 cells and a second subline is shown in Figure 6A. Application of 300 µM Zn2+ to a subline, PC-3B, triggered a Ca2+ rise with a rate that was only ~10% of that observed in the original PC-3 cells. This attenuated Zn2+-dependent Ca2+ rise suggests that PC-3B cells lack ZnR activity. To determine if the IP3 pathway is functional in PC-3B cells, we tested if another Gq-protein-coupled receptor could trigger a Ca2+ rise in these cells. A pronounced Ca2+ rise was observed in the PC-3B cells following application of ATP (100 µM, Figure 6B), which induces a purinergic response, indicating that the metabotropic pathway in these cells is intact. Both PC-3 and PC-3B cells express the cytokeratins 8/18 and neuron-specific enolase, which are commonly used as markers for this cell type [(33), data not shown]. These results indicate that PC-3 and PC-3B sublines differ in their ZnR activity, providing a useful experimental tool to address the specific role of this receptor in prostate cancer cells.


Figure 6
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Attenuation of Zn2+-dependent signaling and cell growth in PC-3B, a subline, which exhibits reduced Zn2+-dependent Ca2+ response. (A) The intracellular Ca2+ response of a PC-3 subline (PC-3B) to extracellular Zn2+ is shown in comparison to PC-3 cells. (B) Application of ATP to PC-3B cells triggered a Ca2+ response. (C) PC-3B cells were treated with Zn2+ (300 µM) or phorbol 12–myristate 13–acetate (PMA) (250 nM) and subjected to immunoblotting with pERK1/2 antibody and densitometry analysis (left). AKT phosphorylation was monitored by immunoblotting using pAKT antibody and densitometry analysis (right) in PC-3B cells following application of Zn2+ (300 µM) or ATP (100 µM). (D) PC-3B cells were treated daily for 10 min with Ringer's solution containing 10 or 100 µM Zn2+, 100 µM CaEDTA, or with Ringer's alone (control). Cell number was monitored using the sulphorhodamine B colorimetric assay during five consecutive days. For comparison, the effect of 100 µM Zn2+ on cell number in the PC-3 cells is shown (same as in Figure 5A).

 
To determine the specific role of ZnR signaling in linking changes in extracellular Zn2+ and the activation of MAPK and PI3K pathways, we compared the effects of extracellular Zn2+ on ERK1/2 and AKT phosphorylation in the ZnR-deficient subline, PC-3B. Zn2+-dependent phosphorylation of ERK1/2 in the PC-3B subline was insignificant compared with control, untreated cells (Figure 6C) and much smaller than the Zn2+-dependent activation observed in the PC-3 cells (Figure 4A). Application of phorbol 12–myristate 13–acetate (250 nM), a known activator of MAPK in prostate cells (35), resulted in a 10-fold increase in ERK1/2 phosphorylation in PC-3B cells, indicating that the MAPK pathway is functional in these cells. Basal phosphorylation of AKT in PC-3B cells, in the absence of Zn2+, was higher than in the PC-3 cells (Figure 6C, note the left lane of the immunoblot as well as the scale of the densitometry as compared with Figure 4B). No significant Zn2+-dependent phosphorylation of AKT was apparent, whereas ATP (100 µM) induced a rise in AKT phosphorylation by ~7-fold, indicating the PI3K pathway is intact in the PC-3B cells. Thus, our results indicate that extracellular Zn2+-dependent activation of the MAPK and PI3K pathways is observed only in PC-3 cells exhibiting Zn2+-dependent metabotropic signaling.

To assess the role of ZnR activity in enhancing cell growth, we compared cell proliferation in the PC-3B and PC-3 cells. The PC-3B cells exhibited a growth rate that was ~2-fold slower than that of the PC-3 cells (control cells in Figures 5A and 6D, respectively). In contrast to PC-3 cells, daily application of Zn2+ to the PC-3B cells failed to significantly enhance cell growth over 5 days of treatment (Figure 6D). This suggests that Zn2+-dependent cell growth is apparent only in PC-3 cells which exhibit Zn2+-dependent metabotropic signaling.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
Prostate cancer is the second most common malignancy in men in Western countries and a major cause of death. While in the early stage, prostate cancer is treatable by hormones, the advanced stage is androgen insensitive and an effective therapeutic approach is still unavailable. Thus, novel molecular targets to facilitate treatment for this cancer are urgently sought. Among the most intriguing but less understood changes occurring in prostate cancer transformation is a 10-fold decrease in the concentration of Zn2+. In recent years, this ion has emerged as an important signaling agent, regulating downstream pathways involved in cell proliferation and survival (14,36). However, the signaling pathways activated by extracellular Zn2+ in healthy and malignant prostate tissue remain unknown.

In the present work, we identified extracellular, Zn2+-dependent metabotropic Ca2+ release in androgen-independent PC-3 and DU-145 prostate cancer cell lines and in the mouse prostate tumor TRAMP-1 cell line, but not in the androgen-sensitive LNCaP cells. Interestingly, the androgen-insensitive PC-3 cells exhibited two sublines that showed distinct ZnR activity. Our results, therefore, do not support a clear link between androgen sensitivity and extracellular Zn2+ response. Previous work, however, has suggested that changes in androgen sensitivity of LNCap cells may be followed by changes in intracellular Zn2+, Zn2+-buffering proteins, such as Zn2+ transporters and metallothioneins, as well as cell growth (37,38). This is consistent with other reports of decreased Zn2+ permeability in androgen-insensitive cells and malignant tissue, linked to a reduced expression of ZIP1 (12,39). Additional studies are needed to determine if a link exists between Zn2+ signaling, intracellular Zn2+ concentration and androgen sensitivity.

In the present report, we have shown that extracellular Zn2+ triggers the IP3 pathway in PC-3 cells, leading to release of intracellular Ca2+. This Ca2+ rise is mediated by a Gq-protein and activation of PLC and is abolished following depletion of TG-sensitive Ca2+ stores. This subsequently leads to activation of MAPK and PI3K. This signaling pathway was previously attributed to a GPCR, termed ZnR (14,22,23). Our data further indicate that a brief rise in extracellular Zn2+ plays a role in promoting prostate cancer cell growth only in those cells exhibiting ZnR activity (i.e. PC-3 but not PC-3B cells). The results presented here indicate that prostate cancer cells are largely impermeable to Zn2+, in agreement with previous studies (2,6,12,13). Although the apparent K0.5 of the prostate ZnR is 200 µM, our results indicate that Zn2+-dependent signaling in prostate cancer cells is activated, and induces enhanced proliferation, already at 10 µM. It is further important to note that even in the presence of citrate, Zn2+ can trigger a ZnR response at slightly higher free Zn2+ concentrations. Interestingly, Zn2+Cit complex led to functional desensitization of ZnR-mediated, intracellular Ca2+ rise. Furthermore, Zn2+Cit-induced desensitization was followed by attenuation of Zn2+-dependent cell growth. The concentrations of both Zn2+ and citrate used in our experiments are similar to those found in the non-neoplastic prostate, suggesting that ZnR is quiescent in the prostate tissue. One of the hallmarks of prostate cancer progression is a dramatic decrease in Zn2+ and citrate concentrations that lower the buffer capacity for free Zn2+ in the prostate (40). Furthermore, because of the relatively low affinity of Zn2+ to citrate, even a modest change in citrate concentration may dramatically alter the concentration of free Zn2+, thereby triggering a transient rise in Zn2+ and activation of the Ca2+ response. This suggests that a scenario in which ZnR is transiently activated in the prostate by a rise in extracellular Zn2+ may also be considered. This further suggests that desensitization by Zn2+Cit plays an important role in suppressing tumor growth. The role of ZnR signaling would be expected to be more pronounced following metastasis of prostate tumor cells into peripheral tissues in which the extracellular Zn2+Cit concentration is negligible, minimizing ZnR desensitization. Several plausible mechanisms may trigger a transient rise in Zn2+ concentration in the peripheral tissues. Among these, is the tissue destruction mediated by tumor invasion which can lead to release of Zn2+ from injured cells. In addition, NO and oxidative signaling mediated by the tumor cells may induce release of Zn2+ from metalloproteins (41). Finally, in bone, arguably the most common and devastating target of prostate metastatic cells, the high concentrations of Zn2+ released during bone destruction occurring in metastatic prostate cancer could potentially activate the ZnR (42). Consistent with this hypothesis is the finding that ZnR activity was particularly intense in the metastatic prostate cancer cell lines. Therefore, extracellular Zn2+ signaling in prostate cancer represents an important pathway linking changes in Zn2+ to prostate tumor cell growth and survival.

The identification of a PC-3 subline, PC-3B, that is deficient in extracellular Zn2+-dependent signaling, provides an important tool to assess the role of the Zn2+-dependent metabotropic response in prostate cancer. The spontaneous formation of PC-3 subline with distinct morphological and functional characteristics is common and is attributed to their genetic instability (33,34). It would be of interest to further characterize these sublines, for example, to determine if the difference in the response to extracellular Zn2+ is also accompanied by a change in the expression pattern of other zinc transporters involved in prostate cancer (5,12).

The Zn2+Cit complex, at concentrations that did not trigger ZnR activity, induced a profound functional desensitization of the Zn2+-dependent activity. Desensitization by Zn2+Cit was similar to the desensitization induced by Zn2+ alone, suggesting that the Zn2+Cit complex may allosterically interact with the ZnR. Although the molecular basis for this effect is still unknown, it is reminiscent of the constitutive desensitization encountered in other GPCRs (43). For example, such desensitization, without prior activation of the intracellular signaling, has been attributed to activation of GPCR kinases. Constitutive desensitization has been also described for the serotonin receptor, 5-hydroxytryptamine (44), a member of the metabotropic glutamate receptor family.

The MAPK- and PI3K-signaling pathways participate in a wide variety of physiological processes and have been shown to induce proliferation of prostate cancer cells (29,45). The PI3K pathway is prominent in prostate cancer, where constitutively activated PI3K has been correlated with tumor cell survival (46). Activation of PI3K has also been suggested to downregulate the androgen receptor, thereby inducing the shift toward advanced stages of this cancer (47). Furthermore, PI3K activation was reported to induce chemoresistance of prostate cancer cells (48). We have shown that the Zn2+-dependent MAPK and PI3K activation is correlated with enhancement of cell growth in PC-3 cells which exhibit ZnR activity. Our results indicate that extracellular Zn2+ is inducing the activation of MAPK at least partially via a signaling pathway involving PI3 and PKC. A derivative of prostaglandin also activated this signaling pathway to trigger MAPK activation in PC-3 cells (49). It was recently shown that MAPK activation is triggered by Zn2+ in prostate cancer cells (13); this effect, however, was produced only after addition of a Zn2+ ionophore. It is possible that in the presence of the ionophore, the activation of MAPK was also mediated via a distinct pathway. The ERK pathway has been linked to both proliferation and to attenuation of cell growth. While it was previously suggested that the rise in intracellular Zn2+, in the presence of the Zn2+ ionophore, inhibited nuclear factor-kappa B activity and suppressed the invasiveness of PC-3 cells (13), our work indicates that extracellular Zn2+ enhanced cell growth. These distinct effects of extracellular or intracellular Zn2+ on cell fate are consistent with previous studies (36,50,51). Activation of AKT by extracellular Zn2+ via the ZnR was largely mediated by the IP3-dependent pathway, as the Gq and PKC inhibitors attenuated AKT phophorylation. Previous work has suggested that the Ca2+-independent PKC{delta} is a major inhibitor of AKT which leads to apoptosis (52). Our results, therefore, indicate that another isoenzyme of PKC is activated by extracellular Zn2+ leading to cell survival. In support of this conclusion, the Ca2+-dependent PKC{alpha} was shown previously to activate ERK1/2 via transactivation by epidermal growth factor receptor in PC-3 cells (53). The prolonged 3 h period that the phosphorylation of AKT persists following a short exposure to Zn2+ underscores the role of Zn2+ in activating signaling, leading to enhanced tumor cell growth. Finally, attenuation of the Zn2+-dependent phosphorylation of ERK1/2 by a PI3K inhibitor, and a similar effect of a MEK inhibitor on PI3K activation, indicate that these pathways interact to promote Zn2+-dependent cell proliferation, similar to the effect previously observed in Zn2+-dependent signaling in colon cells (14,54). Taken together, our results indicate that Zn2+ activates several signaling pathways that interact to enhance tumor progression. The lack of a Zn2+-dependent phosphorylation of the MAPK and PI3K in ZnR-deficient, PC-3B cells, indicates that activation of both pathways is primarily mediated by the ZnR.

In conclusion, our results show that extracellular Zn2+ acts as a signaling agent via the ZnR in prostate cancer cells. Our results further identify a unique role for Zn2+Cit as an effective suppressor of extracellular Zn2+ signaling. The finding that a rise in extracellular Zn2+ significantly enhances prostate cancer cell proliferation and survival suggests that blocking extracellular Zn2+ signaling can be a safe and effective therapeutic strategy to attenuate prostate cancer cell growth and tumor progression.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Israeli Science Foundation (585/05); Binational Science Foundation (2003201) and German Israeli Foundation, grant number 912-90.11/2006 to M.H.


   Acknowledgments
 
We thank Astellas Pharma for generously providing the G{alpha}q inhibitor YM-254890 and Prof. Yossi Levy for fruitful discussions and critical reading of the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

  1. Sorensen MB, et al. Ultrastructural localization of zinc ions in the rat prostate: an autometallographic study. Prostate (1997) 31:125–130.[CrossRef][Web of Science][Medline]
  2. Costello LC, et al. The clinical relevance of the metabolism of prostate cancer; zinc and tumor suppression: connecting the dots. Mol. Cancer (2006) 5:17.[CrossRef][Medline]
  3. Costello LC, et al. The intermediary metabolism of the prostate: a key to understanding the pathogenesis and progression of prostate malignancy. Oncology (2000) 59:269–82.[CrossRef][Web of Science][Medline]
  4. Costello LC, et al. Evidence for a zinc uptake transporter in human prostate cancer cells which is regulated by prolactin and testosterone. J. Biol. Chem (1999) 274:17499–17504.[Abstract/Free Full Text]
  5. Franklin RB, et al. Human ZIP1 is a major zinc uptake transporter for the accumulation of zinc in prostate cells. J. Inorg. Biochem (2003) 96:435–442.[CrossRef][Web of Science][Medline]
  6. Singh KK, et al. Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues. Mol. Cancer (2006) 5:14.[CrossRef][Medline]
  7. Tsui KH, et al. Zinc blocks gene expression of mitochondrial aconitase in human prostatic carcinoma cells. Int. J. Cancer (2006) 118:609–615.[CrossRef][Web of Science][Medline]
  8. Costello LC, et al. Novel role of zinc in the regulation of prostate citrate metabolism and its implications in prostate cancer. Prostate (1998) 35:285–296.[CrossRef][Web of Science][Medline]
  9. Untergasser G, et al. High levels of zinc ions induce loss of mitochondrial potential and degradation of antiapoptotic Bcl-2 protein in in vitro cultivated human prostate epithelial cells. Biochem. Biophys. Res. Commun (2000) 279:607–614.[CrossRef][Web of Science][Medline]
  10. Wong YC, et al. Changes in serum and tissue zinc levels in sex hormone-induced prostatic carcinogenesis in the noble rat. Tumour Biol (2000) 21:328–336.[CrossRef][Medline]
  11. Magda D, et al. Motexafin gadolinium disrupts zinc metabolism in human cancer cell lines. Cancer Res (2005) 65:3837–3845.[Abstract/Free Full Text]
  12. Huang L, et al. Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression. Cancer Cell Int (2006) 6:10.[CrossRef][Medline]
  13. Uzzo RG, et al. Diverse effects of zinc on NF-kappaB and AP-1 transcription factors: implications for prostate cancer progression. Carcinogenesis (2006) 27:1980–1990.[Abstract/Free Full Text]
  14. Azriel-Tamir H, et al. Extracellular zinc triggers ERK-dependent activation of Na+/H+ exchange in colonocytes mediated by the zinc-sensing receptor. J. Biol. Chem (2004) 279:51804–51816.[Abstract/Free Full Text]
  15. Huang JS, et al. Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts. Eur. J. Biochem (1999) 266:943–951.[Web of Science][Medline]
  16. Kiss Z, et al. Bombesin and zinc enhance the synergistic mitogenic effects of insulin and phosphocholine by a MAP kinase-dependent mechanism in Swiss 3T3 cells. FEBS Lett (1997) 415:71–74.[CrossRef][Web of Science][Medline]
  17. Wu W, et al. p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression. Am. J. Physiol. Lung Cell Mol. Physiol (2005) 289:L883–L889.[Abstract/Free Full Text]
  18. Kim YM, et al. Zn2+-induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol (2006) 290:L1028–L1035.[Abstract/Free Full Text]
  19. Kim S, et al. Extracellular zinc activates p70 S6 kinase through the phosphatidylinositol 3-kinase signaling pathway. J. Biol. Chem (2000) 275:25979–25984.[Abstract/Free Full Text]
  20. An WL, et al. Zinc-induced anti-apoptotic effects in SH-SY5Y neuroblastoma cells via the extracellular signal-regulated kinase 1/2. Brain Res. Mol. Brain Res (2005) 135:40–47.[CrossRef][Medline]
  21. Ostrakhovitch EA, et al. Inhibition of extracellular signal regulated kinase (ERK) leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells: the lack of effect of ERK in p53 mediated copper induced apoptosis. J. Cell Biochem (2005) 95:1120–1134.[CrossRef][Web of Science][Medline]
  22. Hershfinkel M, et al. A zinc-sensing receptor triggers the release of intracellular Ca2+ and regulates ion transport. Proc. Natl Acad. Sci. USA (2001) 98:11749–11754.[Abstract/Free Full Text]
  23. Sharir H, et al. The extracellular zinc-sensing receptor mediates intercellular communication by inducing ATP release. Biochem. Biophys. Res. Commun (2005) 332:845–852.[CrossRef][Web of Science][Medline]
  24. Milon B, et al. Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells. Biochim. Biophys. Acta (2006) 1758:1696–1701.[Medline]
  25. Priel T, et al. Zinc influx and physiological consequences in the beta-insulinoma cell line, Min6. Biochem. Biophys. Res. Commun (2006) 346:205–212.[CrossRef][Web of Science][Medline]
  26. Skehan P, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst (1990) 82:1107–1112.[Abstract/Free Full Text]
  27. Taniguchi M, et al. YM-254890 analogues, novel cyclic depsipeptides with Galpha(q/11) inhibitory activity from Chromobacterium sp. QS3666. Bioorg. Med. Chem (2004) 12:3125–3133.[Medline]
  28. Narayanan BA, et al. Adenocarcina of the mouse prostate growth inhibition by celecoxib: downregulation of transcription factors involved in COX-2 inhibition. Prostate (2006) 66:257–265.[CrossRef][Web of Science][Medline]
  29. McCubrey JA, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim. Biophys. Acta (2006) 1773:1263–1284.
  30. Hassan S, et al. Involvement of MAP-kinase, PI3-kinase and EGF-receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells. Regul. Pept (2004) 120:155–166.[CrossRef][Web of Science][Medline]
  31. Gavrielides MV, et al. Protein kinase C and prostate carcinogenesis: targeting the cell cycle and apoptotic mechanisms. Curr. Drug Targets (2004) 5:431–443.[CrossRef][Web of Science][Medline]
  32. Feng P, et al. Direct effect of zinc on mitochondrial apoptogenesis in prostate cells. Prostate (2002) 52:311–318.[CrossRef][Web of Science][Medline]
  33. van Bokhoven A, et al. Molecular characterization of human prostate carcinoma cell lines. Prostate (2003) 57:205–225.[CrossRef][Web of Science][Medline]
  34. van Bokhoven A, et al. Widely used prostate carcinoma cell lines share common origins. Prostate (2001) 47:36–51.[CrossRef][Medline]
  35. Yin L, et al. Phorbol ester-induced apoptosis of C4-2 cells requires both a unique and a redundant protein kinase C signaling pathway. J. Biol. Chem (2005) 280:5533–5541.[Abstract/Free Full Text]
  36. Park KS, et al. The ERK pathway involves positive and negative regulations of HT29 colorectal cancer cell growth by extracellular zinc. Am. J. Physiol. Gastrointest. Liver Physiol (2003) 285:G1181–G1188.[Abstract/Free Full Text]
  37. Iguchi K, et al. Zinc and metallothionein levels and expression of zinc transporters in androgen-independent subline of LNCaP cells. J. Androl (2004) 25:154–161.[Abstract/Free Full Text]
  38. Iguchi K, et al. Isolation and characterization of LNCaP sublines differing in hormone sensitivity. J. Androl (2007) 28:670–678.[Abstract/Free Full Text]
  39. Liang JY, et al. Inhibitory effect of zinc on human prostatic carcinoma cell growth. Prostate (1999) 40:200–207.[CrossRef][Web of Science][Medline]
  40. Franklin RB, et al. Zinc and zinc transporters in normal prostate and the pathogenesis of prostate cancer. Front. Biosci (2005) 10:2230–2239.[Web of Science][Medline]
  41. Aizenman E, et al. Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release. J. Neurochem (2000) 75:1878–1888.[CrossRef][Web of Science][Medline]
  42. Ovesen J, et al. Autometallographic tracing of zinc ions in growing bone. J. Musculoskelet. Neuronal Interact (2004) 4:428–435.[Medline]
  43. Barak LS, et al. Constitutive desensitization: a new paradigm for g protein-coupled receptor regulation. Assay Drug Dev. Technol (2003) 1:339–346.[CrossRef][Web of Science][Medline]
  44. Maudsley S, et al. The origins of diversity and specificity in g protein-coupled receptor signaling. J. Pharmacol. Exp. Ther (2005) 314:485–494.[Abstract/Free Full Text]
  45. Uzgare AR, et al. Enhanced redundancy in Akt and mitogen-activated protein kinase-induced survival of malignant versus normal prostate epithelial cells. Cancer Res (2004) 64:6190–6199.[Abstract/Free Full Text]
  46. Bertram J, et al. Inhibition of the phosphatidylinositol 3'-kinase pathway promotes autocrine Fas-induced death of phosphatase and tensin homologue-deficient prostate cancer cells. Cancer Res (2006) 66:4781–4788.[Abstract/Free Full Text]
  47. Liao Z, et al. Increased phospho-AKT is associated with loss of the androgen receptor during the progression of N-methyl-N-nitrosourea-induced prostate carcinogenesis in rats. Prostate (2005) 64:186–199.[CrossRef][Web of Science][Medline]
  48. Lee JT Jr, et al. Phosphatidylinositol 3'-kinase activation leads to multidrug resistance protein-1 expression and subsequent chemoresistance in advanced prostate cancer cells. Cancer Res (2004) 64:8397–8404.[Abstract/Free Full Text]
  49. Li X, et al. Activation of extracellular signal-regulated kinase by 12-hydroxyheptadecatrienoic acid in prostate cancer PC3 cells. Arch. Biochem. Biophys (2007) 467:20–30.[CrossRef][Web of Science]
  50. Chen J, et al. Niaspan increases angiogenesis and improves functional recovery after stroke. Ann. Neurol (2007) 7:7.
  51. Park KS, et al. Extracellular zinc stimulates ERK-dependent activation of p21(Cip/WAF1) and inhibits proliferation of colorectal cancer cells. Br. J. Pharmacol (2002) 137:597–607.[CrossRef][Web of Science][Medline]
  52. Tanaka Y, et al. Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway. J. Biol. Chem (2003) 278:33753–33762.[Abstract/Free Full Text]
  53. Stewart JR, et al. Protein kinase C-{alpha} mediates epidermal growth factor receptor transactivation in human prostate cancer cells. Mol. Cancer Ther (2005) 4:726–732.[Abstract/Free Full Text]
  54. Oh SY, et al. Differential modulation of zinc-stimulated p21(Cip/WAF1) and cyclin D1 induction by inhibition of PI3 kinase in HT-29 colorectal cancer cells. Exp. Mol. Med (2002) 34:27–31.[Web of Science][Medline]
Received October 10, 2007; revised December 21, 2007; accepted January 19, 2008.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Neurosci.Home page
L. Besser, E. Chorin, I. Sekler, W. F. Silverman, S. Atkin, J. T. Russell, and M. Hershfinkel
Synaptically Released Zinc Triggers Metabotropic Signaling via a Zinc-Sensing Receptor in the Hippocampus
J. Neurosci., March 4, 2009; 29(9): 2890 - 2901.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Figure 1
Right arrow Supplementary Data
Right arrow All Versions of this Article:
29/9/1692    most recent
bgn027v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dubi, N.
Right arrow Articles by Hershfinkel, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dubi, N.
Right arrow Articles by Hershfinkel, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?