The findings described above allow us to define the relationship between the down-regulated MT expression and the decreased endogenous level of zinc in human prostate malignant cells and tissues. The disturbance of zinc homeostasis in prostate would significantly impair the cellular metabolism and growth as discussed previously [5, 6]. Now, a critical issue could be raised: Does the low level of cellular zinc lead responsively to a suppressed endogenous MT level or does the down-regulated MT expression result in a significant depletion of cellular zinc underlying the tumorigenesis of prostate tissues. Most recently, an important study on the mechanism of MT gene regulation by metal response element-binding transcription factor-1 (MTF-1) was reported by Dr. R. Tjian's group . In this study, the robust response of the MT genes to metal was used as a model; MTF-1, a core facilitating factor, and two central regulatory factors, transcription factor II D (TFIID) and mediator (MED), were identified as major players in modulating the transcriptional response of MT to metal. The MTF-1 recruits TFIID, which then binds MT promoter DNA and subsequently activates transcription upon recruitment of the MED . The appropriate expression of the endogenous MT genes is achieved only when these coactivators are balanced. Intriguingly, they found that the MTF-1 requires different coactivator subunits depending on the context of the core promoter (the stability of the multi-subunit coactivator complexes can be compromised by loss of a single subunit), leading to the potential control of fine-tune transcriptional regulation. We observed that the endogenous level of MT in the prostate cells is cell-type specific and that the oncological trend of the endogenous MT may be closely related to MTF-1-involved MT gene regulation.
The alteration of MT1/2 gene expression was not only found in human prostate, but was also discovered in primary human hepatocellular carcinomas , in which the suppression of MT1/2 gene expression is mediated through phosphatidylinositol 3-kinase (PI3K) signaling pathway by the inactivation of the CCAAT/enhancer binding protein α (C/EBPα). On the other hand, MTF-1 interacted with C/EBPα as a complex and subsequently activated MT1/2 gene expression. An early study also indicated that in prostate cancer cell lines the MT1 promoter activity was suppressed by promoter methylation of cytosine within the CpG dinucleotide region . Besides the MT promoter methylation, the possibility of dysfunctional interaction of MTF-1 with nuclear factor I (NFI) was also seriously considered . These findings highlight insights for future investigations for the regulation of MT gene expression in association with the prostate zinc homeostasis and tumorigenesis.
MTs, as zinc donors/receptors, play a key role for zinc-related proteins and enzymes, which are involved in many crucial cellular functions including cell metabolism, signaling transduction pathways, and nuclear gene transcriptional regulation. Concordantly with the functions, the extensive cellular distribution of MT1/2 was observed in normal prostate HPR-1 cells with the intensity of immunofluorescence staining in the cytosol and even stronger signals in the nuclei (Fig. 3). However, a substantive decrease of MT1/2 intensity was displayed in both BPH and PC-3 cells, which confirmed the result of the Western blot analysis. In extension of previous observations of high IHC staining in the nuclei of BPH cells , we found that normal HPR-1 cells exhibit much more aggregation of MT1/2 in nuclei, than BPH and PC-3 cells do. This suggests that the suppression of MT expression and the alteration of MT distribution in BPH and PC-3 cells may negatively influence the cellular function.
The results of the suppressed MT1/2 expression in prostate malignant cells led us to further investigate MT1/2 expression in human prostatic tissues including the normal, BPH, and adenocarcinomas. At the present time, only few studies provided limited information of MT1/2 expression in human prostate tissues. Among these data, the correlation of the positive IHC staining of MT1/2 and the adenocarcinoma status was variable ranging from 33% (15/45) , 67% (20/30)  to 100% (5/5) ; while some reports indicated that the Gleason grades of the tumor tissue scoring from low to high reversely corresponded to the intensity of MT1/2 IHC staining [32, 33].
In this study, normal and BPH tissues give images of intensive and/or patchy MT1/2 IHC staining, which are consistent with some previous observations [30, 31, 33]. Moreover, we determined the negative and/or weak MT1/2 IHC staining in the majority of adenocarcinomatous cells (72%), which is in agreement with one of most recent reports (67%) , while other studies showed higher staining signals of MT1/2 in prostate malignant tissues, 33% and 0%, respectively [32, 33]. The discrepancy of the IHC staining data may result from the differential MT1/2 antibodies and the variable staining methods. Apparently, examining more prostate malignant tissues with a standard IHC method will provide significant information of MT1/2 expression, which could be considered as the candidate gene used for early diagnosis and gene therapy for prevention and targeting of prostate caner.
In addition to MT1/2, we have also determined, for the first time, the zinc regulation of MT 3 expression at both transcriptional and translational levels in three prostate cell models. Although the MT 3 was initially thought to be a neuron-specific MT isoform possessing a neuronal cell growth inhibitory activity [18, 34], in recent years the demonstration of a wider, non-neural distribution of human MT 3 [35, 36] motivated a study of MT 3 in prostate, in which a variable nature of MT 3 expression was detected in prostate tissues and cells . Contrary to expectations based on the previous study , we found that among three cell models, only BPH cells profoundly express the highest endogenous level of MT 3, which was also significantly up-regulated by zinc at both mRNA and protein levels (Figs. 5 and 7). Also, neither normal HPR-1 nor malignant PC-3 cells seem to express essential amounts of MT 3 or to respond to zinc induction of the MT 3, thereby suggesting a cell-type specific nature of the MT 3 gene expression and regulation. The characteristic inhibitory effect of MT 3 on cell growth was discriminated by promoting cell necrosis from cell apoptosis in human proximal tubule cells [38, 39]. We have previously demonstrated that zinc inhibits prostate BPH and PC-3 cells growth mainly by cell apoptosis through the mitochondrial pathway . Now with the recognition of the zinc effect on MT 3 in BPH cells, it is possible that the role of MT 3 as a growth inhibitory factor may also involve in zinc-induced cell death in BPH cells.
Using human prostate cell models, we demonstrated that zinc treatment, at least in in vitro cell culture system, can restore the cellular zinc especially in the malignant cells, in which the endogenous zinc was depleted possibly due to the down-regulated MT expression through the MTF-1 involved mechanism. In turn, the restored zinc subsequently enhances MT gene expression significantly to secure the physiological and/or pharmacological level of zinc which will be recruited quickly by the proteins and enzymes to resume their bioactivities and eventually to control cell proliferation and/or death. Hence, zinc supplement may serve as a potential approach to modulate the molecular environment in prostate BPH and malignant cells aiming to enhance their responses to other chemotherapeutical treatment.