Zinc is a potential therapeutic for chemoresistant ovarian cancer.

Ovarian cancer is the leading cause of death from gynecological cancer. The high mortality rate reflets the lack of early diagnosis and limited treatment alternatives. We have observed a number of properties of zinc cytotoxicity that make it attractive from a therapeutic standpoint. Using SKOV3 and ES2 cells, ovarian cancer cell lines that demonstrate varied degrees of resistance to known therapeutics, we show that zinc killing is time and concentration dependent. Death is preceded by distinct changes in cell shape and size. The effects of zinc are additive with cisplatin or doxorubicin, whose morphological effects are distinct from those of zinc. Cytotoxicity of paclitaxel is minimal, making it difficult to determine additivity with zinc. Paclitaxel results in changes in cell shape and size similar to those of zinc but has different effects on cell cycle progression and cyclin expression. The data indicate that the means by which zinc kills ovarian cancer cells is distinct from currently used chemotherapeutics. Based on the properties reported here, zinc has the potential to be developed as either a primary treatment or as a second line of defense against cancers that have developed resistance to currently used chemotherapeutics.

Zinc functions as a cytotoxic agent for prostate cancer cells independent of culture and growth conditions.

The effects of zinc on the viability of PC3, LNCaP and DU145 prostate cancer cell lines in vitro were examined. The data indicate that, despite their distinctly different gene expression profiles, morphology and tissue origin, all cell lines responded to zinc in a similar time and dose dependent manner. Experiments using pyrithione indicated that cell death is mediated by internalized zinc. Zinc effects on cells plated as monolayers were compared to its effects on cells plated in a collagen matrix. Although the rate of cell growth in the matrix was delayed compared to cells in 2-dimensional cultures, the cytotoxic effects of zinc were unaltered. Using both 2-dimensional and 3-dimensional cultures, we observed that zinc cytotoxicity was independent of both the culture conditions and the rate of cell growth, results that contrast the activity of the current chemotherapeutics used to treat prostate cancer. The attractive properties of zinc cytotoxicity demonstrated in this paper suggest that is can be developed as a novel and effective chemotherapeutic agent for prostate cancer treatment.

Direct intra-tumoral injection of zinc-acetate halts tumor growth in a xenograft model of prostate cancer.

Intracellular levels of zinc have shown a strong inverse correlation to growth and malignancy of prostate cancer. To date, studies of zinc supplementation in prostate cancer have been equivocal and have not accounted for bioavailability of zinc. Therefore, we hypothesized that direct intra-tumoral injection of zinc could impact prostate cancer growth. In this study, we evaluated the cytotoxic properties of the pH neutral salt zinc acetate on the prostate cancer cell lines PC3, DU145 and LNCaP. Zinc acetate killed prostate cancer cell lines in vitro, independent of androgen sensitivity, in a dose-dependent manner in a range between 200 and 600 microM. Cell death occurred rapidly with 50% cell death by six hours and maximal cell death by 18 hours. We next established a xenograft model of prostate cancer and tested an experimental treatment protocol of direct intra-tumoral injection of zinc acetate. We found that zinc treatments halted the growth of the prostate cancer tumors and substantially extended the survival of the animals, whilst causing no detectable cytoxicity to other tissues. Thus, our studies form a solid proof-of-concept that direct intra-tumoral injection of zinc acetate could be a safe and effective treatment strategy for prostate cancer.

Zinc induces ERK-dependent cell death through a specific Ras isoform.

The effect of Zn on p53-independent cell death was examined in IIC9 embryonic fibroblasts. Despite the fact that these cells are p53-minus, Zn-mediated death occurs via an apoptotic mechanism. Death is facilitated by the presence of the Zn ionophore, pyrithione, indicating that intracellular Zn initiates the death response. Our investigations of the mechanism of Zn action demonstrate that Zn induces the death of IIC9 cells in a manner that is ERK-dependent. Expression of dn-(dominant negative)Ras attenuates ERK1/2 activation by Zn, and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments confirm that Zn treatment activates Ras and identified H-Ras as the specific isoform activated. This contrasts the activation of N-Ras that occurs when IIC9 cells are stimulated with thrombin. Thus, although the prolonged activation of the Ras/ERK pathway by Zn is similar to that seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation.

Zinc inhibition of adenylyl cyclase correlates with conformational changes in the enzyme.

We have previously demonstrated that Zn(2+) inhibits hormone and forskolin stimulation of cAMP synthesis in intact N18TG2 cells, corresponding plasma membranes, and of recombinant adenylyl cyclase isoforms. If, however, the enzyme is pre-activated by hormone or forskolin, Zn(2+) inhibition is attenuated [J. Biol. Chem. 277 (2002) 11859]. We have extended our analyses of this inhibition to investigations of soluble adenylyl cyclase, composed of the CI and CII domains of the full-length protein. The properties of Zn(2+) inhibition of the soluble enzyme parallel that of the full-length protein, including the fact that inhibition is not competitive with Mg(2+). By monitoring intrinsic and extrinsic fluorescence, we demonstrate changes in enzyme conformers in response to the addition of varied effectors. The data suggest a possible mechanism by which Zn(2+) inhibits adenylyl cyclase activity.

Nitric oxide and the other cyclic nucleotide.

Nitric oxide (NO) participates in the regulation of the daily activities of cells as well as in cytotoxic events. Elucidating the mechanism(s) by which NO carries out its diverse functions has been the goal of numerous laboratories. In the cardiovascular system, evidence indicates that NO mediates its effects via an activation of soluble guanylyl cyclase (sGC). In other tissues, it is not clear if sGC is an exclusive target for NO or what the functions of cGMP might be. It is also unlikely that the diversity of NO actions is explained solely by changes in cGMP. This review focuses on the evidence that NO modulates cAMP signalling, with specific attention to the effects of NO on adenylyl cyclase (AC) as the target of NO regulation.

Zinc inhibition of cAMP signaling.

Zn(2+) is required as either a catalytic or structural component for a large number of enzymes and thus contributes to a variety of important biological processes. We report here that low micromolar concentrations of Zn(2+) inhibited hormone- or forskolin-stimulated cAMP production in N18TG2 neuroblastoma cells. Similarly, low concentrations inhibited hormone- and forskolin-stimulated adenylyl cyclase (AC) activity in membrane preparations and did so primarily by altering the V(max) of the enzyme. Zn(2+) also inhibited recombinant isoforms, indicating that this reflects a direct interaction with the enzyme. The IC(50) for Zn(2+) inhibition was approximately 1-2 microm with a Hill coefficient of 1.33. The dose-response curve for Zn(2+) inhibition was identical for AC1, AC5, and AC6 as well as for the C441R mutant of AC5 whose defect appears to be in one of the catalytic metal binding sites. However, AC2 displayed a distinct dose-response curve. These data in combination with the findings that Zn(2+) inhibition was not competitive with Mg(2+) or Mg(2+)/ATP suggest that the inhibitory Zn(2+) binding site is distinct from the metal binding sites involved in catalysis. The prestimulated enzyme was found to be less susceptible to Zn(2+) inhibition, suggesting that the ability of Zn(2+) to inhibit AC could be significantly influenced by the coincidence timing of the input signals to the enzyme.