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Introduction: Efficacious chemotherapy does not exist for treatment or prevention of prostate, liver, and pancreatic carcinomas, and some other cancers that exhibit decreased zinc in malignancy. Zinc treatment offers a potential solution; but its support has been deterred by adverse bias. Areas covered: 1. The clinical and experimental evidence for the common ZIP transporter/Zn down regulation in these cancers. 2. The evidence for a zinc approach to prevent and/or treat these carcinomas. 3. The issues that introduce bias against support for the zinc approach. Expert opinion: ZIP/Zn downregulation is a clinically established common event in prostate, hepatocellular and pancreatic cancers. 2. Compelling evidence supports the plausibility that a zinc treatment regimen will prevent development of malignancy and termination of progressing malignancy in these cancers; and likely other carcinomas that exhibit decreased zinc. 3. Scientifically-unfounded issues that oppose this ZIP/Zn relationship have introduced bias against support for research and funding of a zinc treatment approach. 4. The clinically-established and supporting experimental evidence provide the scientific credibility that should dictate the support for research and funding of a zinc approach for the treatment and possible prevention of these cancers. 5. This is in the best interest of the medical community and the public-at-large., Competing Interests: Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

The human prostate gland contains extremely high zinc levels; which is due to the specialized zinc-accumulating acinar epithelial of the peripheral zone. These cells evolved for their unique capability to produce and secrete extremely levels of citrate, which is achieved by the high cellular zinc level effects on the cell metabolism. This review highlights the specific functional and metabolic alterations that result from the accumulation of the high zinc levels, especially its effects on mitochondrial citrate metabolism and terminal oxidation. The implications of zinc in the development and progression of prostate cancer are described, which is the most consistent hallmark characteristic of prostate cancer. The requirement for decreased zinc resulting from down regulation of ZIP1 to prevent zinc cytotoxicity in the malignant cells is described as an essential early event in prostate oncogenesis. This provides the basis for the concept that an agent (such as the zinc ionophore, clioquinol) that facilitates zinc uptake and accumulation in ZIP1-deficient prostate tumors cells will markedly inhibit tumor growth. In the current absence of an efficacious chemotherapy for advanced prostate cancer, and for prevention of early development of malignancy; a zinc treatment regimen is a plausible approach that should be pursued., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Prostate cancer is the second leading cause of cancer deaths among men. The availability of animal models that represent the events and factors that exist in the natural history and biology of human prostate cancer is essential in dealing with prostate cancer. In recent decades and presently, emphasis has been directed at the development and employment of prostate cancer induced in transgenic mice. However, the important consistent hallmark characteristic and event of decrease in zinc and citrate and downregulation of ZIP1 zinc transporter in prostate malignancy has not been studied or identified in any animal model. We investigated the status of these parameters in TRAMP tumors as compared with human prostate cancer. The results show that citrate levels are markedly decreased in the developing and advancing stages of malignancy in TRAMP. Zinc levels are also decreased and ZIP1 transporter is lost in TRAMP tumors. In vitro studies show that zinc treatment of TRAMP C2 cells exhibits cytotoxic effects. Collectively, these results mimic the ZIP1, zinc, and citrate status and relationship that exist in human prostate cancer. This is the first report that establishes the existence of the human prostate zinc/citrate hallmark characteristic and relationship in an animal model. It now appears that the TRAMP model will be suitable for studies relating to the implications and role of zinc- and citrate-related metabolism in the development and progression of human prostate cancer.

Background: A marked decrease in the level of zinc is a consistent characteristic of prostate cancer; which results from down-regulation of ZIP1 zinc transporter. The aim of this study was to determine if RREB-1 transcription is involved in the down-regulation of ZIP1 gene expression; and to determine the expression of RREB-1 in benign and cancerous prostate in situ., Methods: Overexpression and siRNA knock down of RREB-1 were used to determine the effect of RREB-1 on hZIP1 abundance in PC-3 cells. Immunohistochemistry with tissue microarrays (TMAs) and tissue sections was used to determine the levels of RREB-1 expression in prostate in situ., Results: Overexpression of RREB-1 resulted in a decrease in the abundance of hZIP1 in the plasma membrane of PC-3 cells; whereas siRNA knock down significantly increased hZIP1 expression. Prostate TMAs and tissue sections showed an inverse relationship between RREB-1 and hZIP1 staining., Conclusions: RREB-1 overexpression results in down-regulation of hZIP1 and contributes to the loss of hZIP1 expression and zinc in prostate cancer. This is an early event in prostate carcinogenesis., (Copyright © 2011 Wiley-Liss, Inc.)

This minireview is prompted by the recent report of Banas et al. (J Biol Inorg Chem 15:1147-1155, 2010), which purports to show and concludes that zinc levels are increased in prostate cancer. Such a conclusion conflicts with the overwhelming corroborating clinical and experimental evidence that has amassed from numerous reports over the past approximately 60 years; these consistently show that prostate zinc levels are decreased in the development and progression of prostate cancer. We submit that this is an established relationship in prostate cancer that must be considered and described in any studies that purport to identify results that are inconsistent with this established relationship. In support of this relationship, we provide a minireview of the information that has led to the establishment of this relationship. As with most established clinical relationships, exceptions and anomalies often exist. However, these must be described and explained in the context of the established relationship, and not in the context of refutation of the established relationship, at least not until sufficient corroborating evidence overwhelms the existing evidence. This provides a background to address and to critique the report of Banas et al. Of broader and more serious implications are the widespread recalcitrance and/or lack of knowledge within the clinical and biomedical research community for recognition that zinc decrease in prostate cancer is an established relationship. This leads to misinformation and misinterpretations regarding clinical, experimental, and epidemiological issues that do not serve the best interests of the scientific, medical, and public communities.

Background: Normal prostate accumulates extremely high levels of zinc compared to other soft tissues. In contrast, the level of zinc in the prostate decreases significantly in prostate cancer. We have shown that down-regulation of the expression of the zinc transporter hZIP1 in prostate cancer is an important event that is responsible for the decrease of zinc levels. However, the mechanism of hZIP1 down-regulation is not known. We have hypothesized that hZIP1 is down-regulated through transcriptional regulation., Methods: The hZIP1 promoter was studied using luciferase reporter assays, site-directed mutagenesis, gel shift, and ChIP assay., Results: We have characterized a promoter region, downstream of the transcription start site, responsible for repression of hZIP1 transcription. We demonstrate that this region contains a binding site for the Ras-Responsive Element Binding protein 1 (RREB-1) and that the binding of RREB-1 to the hZIP1 promoter is involved in the decrease of hZIP1 transcription in PC-3 cells., Conclusion: The Ras pathway and activation of RREB-1 are involved in hZIP1 down-regulation and may play a role in the decrease of the transporter expression in prostate cancer., (Copyright 2009 Wiley-Liss, Inc.)

We have demonstrated that zinc exposure induces apoptosis in human prostate cancer cells (PC-3) and benign hyperplasia cells (BPH), but not in normal prostate cells (HPR-1). However, the mechanisms underlying the effects of zinc on prostate cancer cell growth and zinc homeostasis remain unclear. To explore the zinc effect on gene expression profiles in normal (HPR-1) and malignant prostate cells (PC-3), we conducted a time course study of Zn treatment with microarray analysis. Microarray data were evaluated and profiled using computational approach for the primary and secondary data analyses. Final analyses were focused on the genes (1) highly sensitive to zinc; (2) associated with zinc homeostasis, i.e., metallothioneins (MTs), solute zinc carriers (ZIPs) and zinc exporters (ZnTs); (3) relevant to several oncogenic pathways. Zinc-mediated mRNA levels of MT isotypes were further validated by semi-quantitative RT-PCR. Results showed that zinc effect on genome-wide expression patterns was cell-type specific, and zinc appeared to have mainly down-regulatory effects on thousands of genes (1953 in HPR-1; 3534 in PC-3) with a threshold of +/-2.5-fold, while fewer genes were up-regulated (872 in HPR-1; 571 in PC-3). The patterns of zinc effect on functional MT genes' expression provided evidence for the cell type-dependent zinc accumulation and zinc-induced apoptosis in prostate cells. In PC-3 cells, zinc significantly up-regulated the expression of MT-1 isotypes MT-1J and MT-1M, denoted previously as "nonfunctional" MT genes, and now a depictive molecular structure of MT-1J was proposed. Examination of genes involved in oncogenic pathways indicated that certain genes, e.g., Fos, Akt1, Jak3 and PI3K, were highly regulated by zinc with cell-type specificity. This work provided an extensive database on zinc-related prostate cancer research. The strategy of data analysis was devoted to finding genes highly sensitive to Zn, and the genes associated with zinc accumulation and zinc-induced apoptosis. The results indicate that zinc regulation of gene expression is cell-type specific, and MT genes play important roles in prostate malignancy.

Early detection is the key to effective treatment of prostate cancer, and to the prevention of deaths due to progression to untreatable advanced stage cancer. Because of mitigating factors, especially benign prostatic hyperplasia (BPH), that result in a low accuracy (about 60%) of prostate-specific antigen (PSA) testing, there is an urgent need for a more reliable biomarker for the identification of early stage through advanced stage prostate cancer and 'at-risk' individuals. To address this issue we propose that changes in prostatic fluid composition could provide accurate and reliable biomarkers for the screening of prostate cancer. Most notable is the consistent and significant decrease in citrate and zinc that is associated with the development and progression of prostate cancer. In this review we provide the clinical and physiological basis and the evidence in support of the utility of prostatic fluid analysis as an effective approach for screening/detection of prostate cancer, especially early stage and 'at-risk' subjects. The problem of BPH interference that plagues PSA testing is eliminated in the potential prostatic fluid biomarkers. The potential development of rapid, simple, direct, accurate clinical tests provides additional advantageous conditions. Further exploration and development of citrate, zinc and other electrolytes as prostatic fluid biomarkers are urgently needed to address this critical prostate cancer issue.

Background: The development and progression of prostate cancer requires the transformation of normal zinc-accumulating epithelial cells to malignant cells that have lost the ability to accumulate zinc. This metabolic transformation is essential so that the tumor suppressive effects of zinc can be eliminated and the malignant process can proceed. One of the major effects of zinc is its prevention of prostate cell growth by its induction of apoptosis. The accumulation of cellular zinc has a direct effect on the mitochondria that results in the release of cytochrome c, which initiates the caspase cascade that leads to apoptosis. This effect is associated with the mitochondrial pore-forming process, but the mechanism by which zinc induces the release of cytochrome c and induces mitochondrial apoptogenesis has not been resolved. The present report provides for the first time information that implicates Bax in the zinc induction of mitochondrial apoptogenesis., Results: The effects of zinc treatment on the Bax levels of PC-3 cells and on the mitochondria were determined. The exposure of isolated mitochondria to zinc results in an increase in membrane bound Bax, which is due to the mitochondrial insertion of endogenous resident Bax. The mitochondrial Bax/Bcl-2 ratio is increased by zinc treatment. Zinc treatment of PC-3 cells also increases the mitochondrial level of Bax. In addition, zinc treatment increases the cellular level of Bax and the cellular Bax/Bcl2 ratio. Down regulation of Bax in PC-3 cells eliminates the zinc induction of apoptosis. The increase in cellular Bax level appears to involve zinc induction of Bax gene expression., Conclusion: This report extends and confirms that physiological levels of zinc induce apoptosis in prostate cells. The study provides evidence that zinc is directly involved in facilitating a Bax-associated pore formation process that initiates mitochondrial apoptogenesis. This is enhanced by an additional effect of zinc on increasing the cellular level of Bax. To avoid the anti-tumor apoptogenic effects of zinc, the malignant cells in prostate cancer posses genetic/metabolic adaptations that prevent the cellular accumulation of zinc.

Background: The disturbance of zinc homeostasis featured with a significant decrease of cellular zinc level was well documented to associate with the development and progression of human prostate malignancy. We have previously reported that zinc treatment induces prostate malignant cell apoptosis through mitochondrial pathway. Metallothionein (MT) is a major receptor/donor of zinc in the cells. However, the studies on the expression of MT in association with the prostate pathological and malignant status are very limited, and the zinc regulation of MT isoform expression in prostate cells remains elusive. The goals of this study were to define the expression of endogenous MTs, the isoforms of MT 1, 2, 3 at both messenger ribonucleic acid (mRNA) and protein levels; and to investigate the zinc effect on MT expression in normal prostate, benign prostatic hyperplasia (BPH) and malignant PC-3 cells, and in relevant human tissues. Cellular MT proteins were detected by immunohistochemistry, fluorescence staining and Western blot analysis; reverse transcription polymerase chain reaction (RT-PCR) was used to determine the MT isoform-specific mRNAs., Results: Our results demonstrated a significant suppression of endogenous levels of MT1/2 in malignant PC-3 cells (95% reduction compared to the normal prostate cells) and in human adenocarcinoma tissues (73% MT1/2 negative). A moderate reduction of MT1/2 expression was observed in BPH. Zinc treatment remarkably induced MT1/2 expression in PC-3 and BPH cells, which was accordant with the restored cellular zinc level. MT 3, as a growth inhibitory factor, was detected and up-regulated by zinc mainly in BPH cells., Conclusion: This study provided evidence of the association of attenuated MT1/2 with prostate tumor progression, and the zinc induction of MT1/2 expression resulting in cellular zinc restoration. The results suggest the potential of MT1/2 as a candidate biomarker for prostate cancer and the utilization of zinc in prostate cancer prevention and treatment.

Background: In normal prostate epithelial cells low m-aconitase activity decreases citrate oxidation leading to citrate accumulation. In prostate cancer cells m-aconitase activity is increased and citrate content is lower. The effect of inhibition of m-aconitase on ATP production by prostate cancer cells (PC3) is not known nor is the contribution of glycolysis versus respiration., Methods: ATP content of PC3 cells as affected by inhibition of m-aconitase (fluoroacetate (FA), zinc), inhibition of glycolysis (2DxG), or respiration (DNP, oligomycin) was determined. The ability to maintain ATP using glucose or glutamine as sole substrate was also determined. Intermediates including ATP, lactate, glucose, and glutamine were assayed in neutralized perchloric acid (PCA) cell extracts, virgin, and conditioned medium by enzymatic fluorometry., Results: Data show that inhibition of m-aconitase, glycolysis, or respiration alone did not decrease ATP content. Inhibition of both glycolysis and respiration were required to decrease ATP content. PC3 cells were able to produce ATP with either glucose or glutamine as sole substrate. Though FA clearly inhibited m-aconitase there was no evidence that zinc had a similar effect., Conclusion: PC3 cells can support ATP production when m-aconitase is inhibited by using glycolysis or oxidation of substrate (e.g., glutamine) entering the TCA cycle distal to citrate., ((c) 2007 Wiley-Liss, Inc.)

Human prostate glandular epithelial cells have the unique capability of accumulating high levels of zinc. This is essential to inhibit m-aconitase activity so that citrate can accumulate for secretion into prostatic fluid, which is a major function of the prostate gland. As a result, the Krebs cycle is truncated with the consequence of the lost ATP production that would result from citrate oxidation. The cellular accumulation of zinc also inhibits mitochondrial terminal oxidation and respiration. In addition to these metabolic effects, zinc accumulation exhibits anti-proliferative effects via its induction of mitochondrial apoptogenesis. Zinc accumulation also inhibits the invasive/migration activities in malignant prostate cells. The anti-proliferative effects and the effects on invasion and migration occur through zinc activation of specific intracellular signaling pathways. Consequently, these effects impose anti-tumor actions by zinc. The ability of prostate cells to accumulate zinc is due to the expression and activity of the zinc uptake transporter, ZIP1. To avoid the anti-tumor effects of zinc, in prostate cancer the malignant prostate cells exhibit a silencing of ZIP1 gene expression accompanied by a depletion of cellular zinc. Therefore we regard ZIP1 as a tumor suppressor gene in prostate cancer. In addition to prostate cells, similar tumor suppressor effects of zinc have been identified in several other types of tumors.

Background: The normal human prostate glandular epithelium has the unique function of accumulating high levels of zinc. In prostate cancer this capability is lost as an early event in the development of the malignant cells. The mechanism and factors responsible for the ability of the normal epithelial cells to accumulate zinc and the loss of this capability in the malignant cells need to be identified. We previously reported that Zip1 is an important zinc uptake transporter in prostate cells and is down regulated in the malignant cells in situ along with the depletion of zinc levels. In this report we investigated the expression of two other Zip family zinc transporters, Zip2 and Zip3 in malignant versus nonmalignant (normal and BPH) glands. Zip2 and Zip3 relative protein levels were determined by immunohistochemistry analysis of human prostate tissue sections., Results: Normal and BPH glandular epithelium consistently exhibited the strong presence of both Zip 2 and Zip3; whereas both transporters consistently were essentially non-detectable in the malignant glands. This represents the first report of the expression of Zip3 in human prostate tissue; and more importantly, reveals that ZiP2 and Zip3 are down regulated in malignant cells in situ as we also had demonstrated for Zip1. Zip2 and Zip3 transporter proteins were localized predominantly at the apical cell membrane, which is in contrast to the Zip1 localization at the basolateral membrane. Zip2 and Zip3 seemingly are associated with the re-uptake of zinc from prostatic fluid., Conclusion: These results coupled with previous reports implicate Zip2 and Zip3 along with Zip1 as important zinc uptake transporters involved in the unique ability of prostate cells to accumulate high cellular zinc levels. Zip1 is important for the extraction of zinc from circulation as the primary source of cellular zinc. Zip 2 and Zip3 appear to be important for retention of the zinc in the cellular compartment. The down regulation of all three transporters in the malignant cells is consistent with the loss of zinc accumulation in these cells. Since zinc imposes tumor suppressor effects, the silencing of the gene expression for these transporters is a required event for the manifestation of the malignant activities of the neoplastic cells. This now provides new insights into the genetic/molecular events associated with the development of prostate cancer; and supports our concept of Zip1, and now Zip2 and Zip3, as tumor suppressor genes and zinc as a tumor suppressor agent.

Background: The genetic and molecular mechanisms responsible for and associated specifically with the development and progression of malignant prostate cells are largely unidentified. In addition, despite its implication in virtually all malignant cells, the role of altered cellular metabolism as an essential factor in prostate malignancy has been largely ignored. Moreover, the intermediary metabolism of normal prostate as well as malignant prostate cells is among the least studied and most poorly understood of all mammalian cells. Some important factors, especially the role of zinc, have been identified and implicated in the development and progression of prostate malignancy. In this review, we provide a current and updated integrated assessment of the relationships of intermediary metabolism in normal prostate and in prostate cancer. The experimental and clinical evidence that leads to the formulation of concepts of normal and malignant prostate metabolism is presented. The evidence for a concept of zinc as a tumor suppressor agent and Zip1 zinc transporter as a tumor-suppressor gene is described., Results: The specialized function of the normal prostate glandular epithelium to produce and secrete enormously high levels of citrate involves and requires unique intermediary metabolism activities that are not generally associated with other normal mammalian cells. The accumulation of zinc by these cells is an essential factor in this unique metabolic relationship. In malignancy, the normal zinc-accumulating citrate-producing epithelial cells are metabolically transformed to citrate-oxidizing cells that lose the ability to accumulate zinc. A genetic alteration in the expression of ZIP1 zinc transporter is associated with this metabolic transformation. These genetic/metabolic relationships have important consequences on citrate-related metabolism, bioenergetics, cell proliferation and invasive capabilities of the malignant cells, which result in tumor-suppression characteristics., Conclusion: The genetic/metabolic relationships in normal prostate glandular epithelium are driven by the unique function to accumulate and secrete citrate. The genetic/metabolic transformation of the prostate malignant cells is driven by the metabolic/bioenergetic, growth/proliferative, and invasive/migration requirements of the malignant process. Zinc is critical to these relationships. An understanding of these genetic/metabolic relationships provides new directions and opportunities for development of regimens for the prevention and treatment of prostate cancer. Important insight into the genetic/metabolic requirements of the prostate malignant process is now evolving. Most importantly at this time, an appreciation and recognition of the genetic/metabolic significance and implications in the development of prostate malignancy is imperative; and much needed research in this area is essential. Hopefully, this review will help to achieve these goals.

Background: In prostate cancer, normal citrate-producing glandular secretory epithelial cells undergo a metabolic transformation to malignant citrate-oxidizing cells. m-Aconitase is the critical step involved in this altered citrate metabolism that is essential to prostate malignancy. The limiting m-aconitase activity in prostate epithelial cells could be the result of a decreased level of m-aconitase enzyme and/or the inhibition of existing m-aconitase. Earlier studies identified zinc as an inhibitor of m-aconitase activity in prostate cells; and that the depletion of zinc in malignant cells is an important factor in this metabolic transformation. However, a possibility remains that an altered expression and level of m-aconitase enzyme might also be involved in this metabolic transformation. To address this issue, the in situ level of m-aconitase enzyme was determined by immunohistochemical analysis of prostate cancer tissue sections and malignant prostate cell lines., Results: The immunocytochemical procedure successfully identified the presence of m-aconitase localized in the mitochondrial compartment in PC-3, LNCaP, and DU-145 malignant prostate cell lines. The examination of prostate tissue sections from prostate cancer subjects demonstrated that m-aconitase enzyme is present in the glandular epithelium of normal glands, hyperplastic glands, adenocrcinomatous glands, and prostatic intraepithelial neoplastic foci. Quantitative analysis of the relative level of m-aconitase in the glandular epithelium of citrate-producing adenomatous glands versus the citrate-oxidizing adenocarcinomatous glands revealed no significant difference in m-aconitase enzyme levels. This is in contrast to the down-regulation of ZIP1 zinc transporter in the malignant glands versus hyperplastic glands that exists in the same tissue samples., Conclusion: The results demonstrate the existence of m-aconitase enzyme in the citrate-producing glandular epithelial cells; so that deficient m-aconitase enzyme is not associated with the limiting m-aconitase activity that prevents citrate oxidation in these cells. The level of m-aconitase is maintained in the malignant cells; so that an altered enzyme level is not associated with the increased m-aconitase activity. Consequently, the elevated zinc level that inhibits m-aconitase enzyme is responsible for the impaired citrate oxidation in normal and hyperplastic prostate glandular epithelial cells. Moreover, the down-regulation of ZIP1 zinc transporter and corresponding depletion of zinc results in the increase in the activity of the existing m-aconitase activity in the malignant prostate cells. The studies now define the mechanism for the metabolic transformation that characterizes the essential transition of normal citrate-producing epithelial cells to malignant citrate-oxidizing cells.

Mutations in mitochondrial DNA are frequent in cancer and the accompanying mitochondrial dysfunction and altered intermediary metabolism might contribute to, or signal, tumour pathogenesis. The metabolism of human prostate peripheral zone glandular epithelial cells is unique. Compared with many other soft tissues, these glandular epithelial cells accumulate high concentrations of zinc, which inhibits the activity of m-aconitase, an enzyme involved in citrate metabolism through Krebs cycle. This causes Krebs cycle truncation and accumulation of high concentrations of citrate to be secreted in prostatic fluid. The accumulation of zinc also inhibits terminal oxidation. Therefore, these cells exhibit inefficient energy production. In contrast, malignant transformation of the prostate is associated with an early metabolic switch, leading to decreased zinc accumulation and increased citrate oxidation. The efficient energy production in these transformed cells implies increased electron transport chain activity, increased oxygen consumption, and perhaps, excess reactive oxygen species (ROS) production compared with normal prostate epithelial cells. Because ROS have deleterious effects on DNA, proteins, and lipids, the altered intermediary metabolism may be linked with ROS production and accelerated mitochondrial DNA mutations in prostate cancer.

The role of zinc in the development and progression of prostate malignancy and its potential application in the prevention and treatment of prostate cancer (PCa) are contemporary critical issues for the medical/scientific community and the public-at-large. The overwhelming clinical and experimental evidence provides a compelling rational basis for the expectation and concept that prostate zinc accumulation is an important factor in the development and progression of prostate malignancy; and that zinc could be efficacious in the prevention and treatment of PCa. In contrast, various epidemiologic studies have produced divergent and conflicting results regarding the efficacy of dietary and supplemental zinc against PCa. Before reaching any definitive conclusions regarding this complex issue, one should have a complete understanding of the clinical and experimental evidence associated with the involvement of zinc in the normal and malignant prostate. Also, an understanding of interacting effects of confounding factors on the absorption, assimilation, and bioavailability of supplemental dietary zinc is important. The purpose of this review is to present the current state of the clinical and experimental information regarding zinc relationships in the normal prostate and in the pathogenesis PCa. The evidence in support of a potential beneficial effect of zinc supplement versus potential harmful effects on PCa is assessed. A discussion of the divergent results of the epidemiologic studies is presented along with a description of important factors and conditions that impact or mask the effects of dietary zinc on PCa development and progression. We also hope to bring more attention to the medical and research community of the critical need for concerted clinical and basic research regarding zinc and PCa, for the development of appropriate human prostate models to investigate these relationships, for further appropriately designed epidemiologic studies, and for future well-controlled clinical trials.

Background: The genetic and molecular mechanisms responsible for and associated with the development and progression of prostate malignancy are largely unidentified. The peripheral zone is the major region of the human prostate gland where malignancy develops. The normal peripheral zone glandular epithelium has the unique function of accumulating high levels of zinc. In contrast, the ability to accumulate zinc is lost in the malignant cells. The lost ability of the neoplastic epithelial cells to accumulate zinc is a consistent factor in their development of malignancy. Recent studies identified ZIP1 (SLC39A1) as an important zinc transporter involved in zinc accumulation in prostate cells. Therefore, we investigated the possibility that down-regulation of hZIP1 gene expression might be involved in the inability of malignant prostate cells to accumulate zinc. To address this issue, the expression of hZIP1 and the depletion of zinc in malignant versus non-malignant prostate glands of prostate cancer tissue sections were analyzed. hZIP1 expression was also determined in malignant prostate cell lines., Results: hZIP1 gene expression, ZIP1 transporter protein, and cellular zinc were prominent in normal peripheral zone glandular epithelium and in benign hyperplastic glands (also zinc accumulating glands). In contrast, hZIP1 gene expression and transporter protein were markedly down-regulated and zinc was depleted in adenocarcinomatous glands and in prostate intra-epithelial neoplastic foci (PIN). These changes occur early in malignancy and are sustained during its progression in the peripheral zone. hZIP1 is also expressed in the malignant cell lines LNCaP, PC-3, DU-145; and in the nonmalignant cell lines HPr-1 and BPH-1., Conclusion: The studies clearly establish that hZIP1 gene expression is down regulated and zinc is depleted in adenocarcinomatous glands. The fact that all the malignant cell lines express hZIP1 indicates that the down-regulation in adenocarcinomatous glands is likely due to in situ gene silencing. These observations, coupled with the numerous and consistent reports of loss of zinc accumulation in malignant cells in prostate cancer, lead to the plausible proposal that down regulation of hZIP1 is a critical early event in the development prostate cancer.

Zinc is an essential metal for all cells. It plays a role in a wide variety of physiological and biochemical processes. In the prostate epithelial cell the accumulation of high cellular zinc is a specialized function that is necessary for these cells to carry out the major physiological functions of production and secretion of citrate. The production of citrate and its secretion into prostatic fluid is a differentiated function of the prostate epithelial cells that is apparently important for reproduction. The loss of citrate and zinc accumulation is the most consistent and persistent characteristic of prostate malignancy. This characteristic of prostate cancer indicates that the lost ability of the malignant cells to accumulate zinc and citrate is an important factor in the development and progression of malignancy. The lost ability of the epithelial cells to accumulate zinc and thus to also accumulate citrate is the result of decreased expression of specific zinc uptake transporters. The purpose of this presentation is to review the current understanding of zinc and zinc homeostasis in the prostate and the role of zinc and zinc transporters in the normal function of the prostate and the pathogenesis of prostate cancer.

Human prostate secretory epithelial cells have the uniquely specialized function of accumulating and secreting extremely high levels of citrate. This is achieved by their ability to accumulate high cellular levels of zinc that inhibit citrate oxidation. This process of net citrate production requires unique metabolic/bioenergetic mitochondrial relationships. In prostate cancer, the malignant cells undergo a metabolic transformation from zinc-accumulating citrate-producing sane cells to citrate-oxidizing malignant cells that lost the ability to accumulate zinc. This review describes the metabolic/bioenergetic, zinc and mitochondrial relationships involved in normal and malignant prostate. Hopefully, this report will generate much needed interest and research in this neglected, but critically important, area of investigation.

The most consistent and persistent biochemical characteristic of prostate cancer (PCa) is the marked decrease in zinc and citrate levels in the malignant cells. This relationship provides compelling evidence that the lost ability of the malignant cells to accumulate zinc is an important factor in the development and progression of prostate malignancy. In addition, this relationship provides a rational basis for the concept that restoration of high zinc levels in malignant cells could be efficacious in the treatment and prevention of PCa. Epidemiological studies regarding dietary zinc effects on PCa have been conflicting and confusing. The purpose of this presentation is to present a current state of information regarding zinc relationships in the pathogenesis and treatment of PCa. We also hope to bring more attention to the medical and research community of the critical need for concerted clinical and basic research regarding zinc and PCa.

Prostate epithelial cells accumulate the highest zinc levels of any cells in the body. Evidence indicates that zinc plays critical roles in the normal function and pathology of the prostate gland. We have identified two important effects of zinc in the prostate epithelial cells: the inhibition of m-aconitase and the induction of mitochondrial apoptogenesis. However, at the present time, the effects of zinc on prostatic cells in in vivo conditions have not yet been reported. The objectives of this in vivo study were to investigate the effect of zinc on: tumorogenicity in nude mice, zinc accumulation in tumor tissues, and the levels of mitochondrial membrane permeability related proteins, Bax/Bcl-2. A tumorigenicity animal model was established using male nude mice (4-6 weeks old) with inoculation of PC-3 cells (5-10x10(6)/mL) prepared in 10% Matrigel. The mice were treated with zinc by ALZET osmotic pumps (Durect Corporation), with a releasing rate of 0.25 micro l/h for 28 days. Zinc concentrations of the tumor tissues were determined by Atomic Absorption Spectrophotometer method. Frozen sections of tumor tissues were prepared for TUNEL assay. The levels of Bax and Bcl-2 in the tumor tissues were determined by Western blot analyses. Our study demonstrated that in vivo treatment of zinc increased zinc accumulation and citrate production in PC-3 cell induced tumor tissues and inhibited tumor growth. The inhibitory effect of zinc appears to result from zinc-induced apoptosis by regulation of mitochondrial membrane permeability-related Bax/Bcl-2 proteins.

This review emphasizes the importance and role of altered intermediary metabolism of prostate cells in the pathogenesis of prostate adenocarcinoma (PCa) and the progression of malignancy. The focus of the presentation is a summary of the overwhelming evidence which implicates the metabolic transformation of citrate-producing sane cells to citrate-oxidizing malignant cells in the process of malignancy. The evidence now demonstrates that altered zinc accumulation is an important factor in this transformation. These metabolic relationships are uniquely different from the metabolic alterations associated with tumorigenesis of other mammalian cells. The metabolic transformation of zinc-accumulating citrate-producing normal prostate epithelial cells to citrate-oxidizing malignant cells has important implications on cellular bioenergetics, cell growth and apoptosis, lipogenesis, angiogenesis. Based on the metabolic considerations new concepts concerning the pathogenesis, diagnosis and treatment of prostate malignancy are presented. Unfortunately the metabolism of the prostate has been a seriously neglected and largely ignored area of prostate research. The importance of expanded research into the intermediary metabolism of normal and neoplastic prostate is essential to future significant advances in understanding and dealing with PCa., (Copyright 2000 S. Karger AG, Basel.)

The glandular epithelial cells of the human prostate gland have the unique capability and function of accumulating the highest zinc levels of any soft tissue in the body. Zinc accumulation in the prostate is regulated by prolactin and testosterone; however, little information is available concerning the mechanisms associated with zinc accumulation and its regulation in prostate epithelial cells. In the present studies the uptake and accumulation of zinc were determined in the human malignant prostate cell lines LNCaP and PC-3. The results demonstrate that LNCaP cells and PC-3 cells possess the unique capability of accumulating high levels of zinc. Zinc accumulation in both cell types is stimulated by physiological concentrations of prolactin and testosterone. The studies reveal that these cells contain a rapid zinc uptake process indicative of a plasma membrane zinc transporter. Initial kinetic studies demonstrate that the rapid uptake of zinc is effective under physiological conditions that reflect the total and mobile zinc levels in circulation. Correspondingly, genetic studies demonstrate the expression of a ZIP family zinc uptake transporter in both LNCaP and PC-3 cells. The rapid zinc uptake transport process is stimulated by treatment of cells with physiological levels of prolactin and testosterone, which possibly is the result of the regulation of the ZIP-type zinc transporter gene. These zinc-accumulating characteristics are specific for prostate cells. The studies support the concept that these prostate cells express a unique hormone-responsive, plasma membrane-associated, rapid zinc uptake transporter gene associated with their unique ability to accumulate high zinc levels.

Background: One of the major current problems involved in prostate cancer (PCa) is the unavailability of sensitive, accurate, and preferably noninvasive procedures for the diagnosis of PCa. Moreover, procedures are needed which will permit the early detection, staging, location, and estimation of the volume of malignancy, and preferably a mapping of the prostate for follow-up of progression and regression of the malignancy., Methods: The unique citrate relationships of the prostate, coupled with recent developments and technological advancements in magnetic resonance spectroscopy (MRS) for the in situ determination of citrate levels, now provides an excellent diagnostic procedure which can achieve all these goals. There exist strong, compelling basic and clinical studies in support of the employment of 1H MRS measurements of citrate and other associated metabolites in the diagnosis of PCa., Results: This review provides the background leading to the current status of MRS citrate analysis, summarizes the data from clinical trials, and describes the applications of the procedure for the diagnosis of PCa and follow-up of patients. The use of MRS studies in defining the functional, as well as pathological relationships of the prostate, is also discussed., Conclusions: This review is intended to be informative to the prostate- and oncology-interested community, and, hopefully, to engender much-needed interest and support in future research regarding the prostate relationships described in this report.

The prostate gland of humans and many other animals has the major function of accumulating and secreting extraordinarily high levels of citrate. This specialized metabolic process of "net citrate production" is the result of unique metabolic capabilities of the secretory epithelial cells. Most importantly, in prostate cancer (Pca) the capability for net citrate production is lost. In addition to citrate, the normal and BPH (benign prostatic hyperplasia) prostate also accumulates the highest levels of zinc in the body. As with citrate, in Pca the ability for high zinc accumulation is diminished. These and other correlations between zinc and citrate in the prostate have been indicative of an important role of zinc in the regulation of citrate metabolism in normal and malignant prostate epithelial cells. The link between zinc and citrate metabolism has now been established. The intramitochondrial accumulation of high zinc levels inhibits mitochondrial (m-) aconitase activity, which inhibits citrate oxidation. This essentially truncates the Krebs cycle and markedly decreases the cellular energy (ATP) production normally coupled to citrate oxidation. It is also clear that zinc accumulation in citrate-producing prostate epithelial cells is regulated by testosterone and by prolactin. These relationships form the basis for a new concept of the role of zinc and citrate-related energy metabolism in prostate malignancy. The inability of malignant prostate cells to accumulate high zinc levels results in increased citrate oxidation and the coupled ATP production essential for the progression of malignancy. The concept offers new approaches to the treatment of Pca.

Citrate production is a major physiological function of the prostate that is regulated by testosterone and prolactin. Mitochondrial aspartate aminotransferase (mAAT) is a key enzyme in the metabolic pathway of prostate citrate production. In addition, prolactin stimulates expression of mAAT in the rat lateral prostate. In this report we establish the role of prolactin in the regulation of mAAT in two prostate cancer cell lines, LNCaP and PC-3. LNCaP cells respond to hormonal stimulation with increased secretion of prostate specific products. PC-3 cells, on the other hand, are testosterone independent and apparently do not respond to other growth factors either. Results showed that both LNCaP and PC-3 cells responded to prolactin with increased mAAT activity and an increased steady state level of mAAT mRNA. Prolactin also increased protein kinase C (PKC) activity in both these cell lines. Treatment of LNCaP and PC-3 cells with the phorbol ester 12-O-tetradecanoylphorbol (TPA) caused the same effect on mAAT activity and mRNA level as prolactin. The results suggest that the diacylglycerol-PKC signal transduction system mediates the prolactin effect on mAAT. In addition, these results also show that the prolactin effect on mAAT is independent of androgens since PC-3 cells reportedly lack androgen receptor expression. Thus, these results provide evidence that prolactin is a physiological regulator of prostate function in human as well as rat prostate. In addition, the results also show that though prostate cancer cells are androgen independent, they remain responsive to prolactin. This could have important implications for the treatment and management of prostate cancer.

Normal and benign prostate hyperplasia (BPH) prostate is characterized by the presence of extraordinarily high levels of citrate. Presumably, this results from the inability of the prostate epithelial cells to oxidize citrate due to a limiting mitochondrial (m-) aconitase. In contrast, prostate carcinoma (CA) is not characterized by high citrate levels. Malignant prostate epithelial cells apparently undergo a metabolic transformation from citrate-producing to citrate-oxidizing cells. A consequence of citrate production in normal and BPH cells is an inefficient and low level of ATP production. It is proposed that the process of malignancy necessitates an energy production that cannot be provided by citrate-producing cells. Consequently, the transformation of prostate epithelial cells to citrate-oxidizing cells which increases the energy production capability is essential to the process of malignancy and metastasis. The metabolic transformation likely occurs as a premalignant or early malignant stage. This bioenergetic theory of prostate malignancy, if correct, will provide new approaches to the diagnosis and treatment of CA.