Exposure to zinc induces apoptosis in PC-3 and BPH cells, which accumulate high intracellular levels of zinc, but not in HPR-1 cells, which do not accumulate high levels of zinc. Once initiated, the induction of apoptosis is not reversed by the removal of zinc, i.e., it is an irreversible process. The apoptogenic effect is due to a direct effect of zinc on mitochondria that results in the release of cytochrome c. The cell specificity of zinc induction of apoptogenesis is dependent on the ability of the cells to accumulate high levels of intracellular zinc and on the ability of the mitochondria to respond to the direct effect of zinc.

The prostate gland of humans and other animals accumulates a level of zinc that is 3-10 times greater than that found in other tissues. Associated with this ability to accumulate zinc is a rapid zinc uptake process in human prostate cells, which we previously identified as the hZIP1 zinc transporter. We now provide additional evidence that hZIP1 is an important operational transporter that allows for the transport and accumulation of zinc. The studies reveal that hZIP1 (SLC39A1) but not hZIP2 (SLC39A2) is expressed in the zinc-accumulating human prostate cell lines, LNCaP and PC-3. Transfected PC-3 cells that overexpress hZIP1 exhibit increased uptake and accumulation of zinc. The V(max) for zinc uptake was increased with no change in K(m). Along with the increased intracellular accumulation of zinc, the overexpression of hZIP1 also results in the inhibition of growth of PC-3 cells. Down-regulation of hZIP1 by treatment of PC-3 cells with hZIP1 antisense oligonucleotide resulted in a decreased zinc uptake. Uptake of zinc from zinc chelated with citrate was as rapid as from free zinc ions; however, the cells did not take up zinc chelated with EDTA. The cellular uptake of zinc is not dependent upon an available pool of free Zn(2+) ions. Instead, the mechanism of transport appears to involve the transport of zinc from low molecular weight ligands that exist in circulation as relatively loosely bound complexes with zinc.

BackgroundThe 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.ResultsThe 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.ConclusionThis 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.

Although the total zinc content of cells generally approximates 0.2 mM, the cytosolic free zinc ion concentration is negligible (subnanomolar concentrtations). However, all reported studies of effects of zinc on cellular respiration and terminal oxidation involved μM-mM levels of free zinc ions. Prostate cells and their mitochondria accumulate 3-10 fold more zinc than other mammalian cells. We considered that a cytosolic pool of mobile reactive low molecular weight zinc ligands could inhibit respiration and terminal oxidation. The effects of ZnLigands, especially ZnCitrate, versus free Zn + + ions on respiration and terminal oxidation were studied with prostate and liver mitochondria. ZnLigands were equally as effective as free Zn + + ions in the inhibition of respiration and terminal oxidation of both prostate and liver mitochondria, which supports our concept that zinc can be transferred from cytosolic donor ZnLigands directly to zinc-binding sites of terminal oxidation components. Also, the respiration and specific activities of terminal oxidation components of prostate mitochondria are 20-50% of liver mitochondria. Zinc inhibition and inherently low levels of electron transport components are likely major factors responsible for the low respiration that characterizes prostate cells.

Prostate cells accumulate high cellular and mitochondrial concentrations of zinc, generally 3-10-fold higher than other mammalian cells. However, the mechanism of mitochondrial import and accumulation of zinc from cytosolic sources of zinc has not been established for these cells or for any mammalian cells. Since the cytosolic concentration of free Zn(2+) ions is negligible (estimates vary from 10(-9) to 10(-15) M), we postulated that loosely bound zinc-ligand complexes (Zn-Ligands) serve as zinc donor sources for mitochondrial import. Zinc chelated with citrate (Zn-Cit) is a major form of zinc in prostate and represents an important potential cytosolic source of transportable zinc into mitochondria. The mitochondrial uptake transport of zinc was studied with isolated mitochondrial preparations obtained from rat ventral prostate. The uptake rates of zinc from Zn-Ligands (citrate, aspartate, histidine, cysteine) and from ZnCl(2) (free Zn(2+)) were essentially the same. No zinc uptake occurred from either Zn-EDTA, or Zn-EGTA. Zinc uptake exhibited Michaelis-Menten kinetics and characteristics of a functional energy-independent facilitative transporter associated with the mitochondrial inner membrane. The uptake and accumulation of zinc from various Zn-Ligand preparations with logK(f) (formation constant) values less than 11 was the same as for ZnCl(2;) and was dependent upon the total zinc concentration independent of the free Zn(2+) ion concentration. Zn-Ligands with logK(f) values greater than 11 were not zinc donors. Therefore the putative zinc transporter exhibits an effective logK(f) of approximately 11 and involves a direct exchange of zinc from Zn-Ligand to transporter. The uptake of zinc by liver mitochondria exhibited transport kinetics similar to prostate mitochondria. The results demonstrate the existence of a mitochondrial zinc uptake transporter that exists for the import of zinc from cytosolic Zn-Ligands. This provides the mechanism for mitochondrial zinc accumulation from the cytosol which contains a negligible concentration of free Zn(2+). The uniquely high accumulation of mitochondrial zinc in prostate cells appears to be due to their high cytosolic level of zinc-transportable ligands, particularly Zn-Cit.