We have previously demonstrated that clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) acts as a zinc ionophore and induces apoptosis of human cancer cells; however, the mechanisms of clioquinol/zinc-induced apoptotic cell death remain to be elucidated further. Using fluorescence-labelled probes, the present study has examined intracellular zinc distribution after clioquinol treatment in human cancer cells in order to identify cellular targets for zinc ionophores. DU 145, a human prostate cancer line, was chosen as a model system for the present study, and results were confirmed in other human cancer cell lines. Although treatment of cancer cells with 50 μM ZnCl2 for 3 days had no effect on cell viability, addition of clioquinol dramatically enhanced the cytotoxicity, confirming our previous observations. The ionophore activity of clioquinol was confirmed using fluorescence microscopy. Intracellular free zinc was found to be concentrated in lysosomes, indicating that lysosomes are the primary target of zinc ionophores. Furthermore, lysosomal integrity was disrupted after addition of clioquinol and zinc to the cells, as shown by redistribution of both Acridine Orange and cathepsin D. Clioquinol plus zinc resulted in a cleavage of Bid (BH3-interacting domain death agonist), a hallmark of lysosome-mediated apoptotic cell death. Thus the present study demonstrates for the first time that clioquinol generates free zinc in lysosomes, leading to their disruption and apoptotic cell death.
The involvement of metals in carcinogenesis has been well recognized [1–3]. More recently, metal-binding compounds have been considered to be potential anticancer agents and have demonstrated anticancer activity . Although some compounds appear to act via metal chelation [5–7], others appear to increase intracellular metal concentrations, suggesting a different mechanism of action [8–10].
One such compound is clioquinol (5-chloro-7-iodo-8-hydroxyquinoline), an 8-hydroxyquinoline derivative, which has anticancer activity in vitro and in vivo . Clioquinol was previously used as an antibiotic in animals and humans and has been studied in clinical trials of Alzheimer's disease, without causing toxicity [11,12]. Importantly, the plasma concentration of clioquinol in patients taking 700 mg daily was close to the dose required to kill human cancer cells in vitro .
We have found previously that clioquinol induces apoptosis, and its actions can be potentiated by both copper and zinc . We have directly demonstrated that clioquinol can transport zinc into cells , and we suspect that it may also serve as a copper ionophore. Others have found that the copper–clioquinol complex can inhibit proteasome activity [13,14]. It remains uncertain whether clioquinol–zinc complexes have similar activity, or if they function via a different mechanism to induce apoptosis.
Since the clioquinol–zinc complex can cross the plasma membrane, we postulated that it might also cross intracellular membranes, thereby increasing the zinc concentration in one or more cellular organelles. Because others have shown that apoptosis can follow injury to intracellular organelles such as mitochondria  and lysosomes , we explored the possibility that clioquinol targets zinc to either of these sites. As described below, we found that clioquinol targets zinc to lysosomes, and is associated with an increase in lysosomal permeability, release of lysosomal enzymes into the cytoplasm, and cleavage of the pro-apoptotic protein Bid (BH3-interacting domain death agonist).
The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reagent was purchased from Promega. Fluorescent probes for the detection of lysosomes (LysoTracker), intracellular zinc , mitochondria (MitoTracker) and cathepsin D (pepstatin A–BODIPY FL conjugate) were purchased from Invitrogen. Antibodies against Bid and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were purchased from Cell Signaling Technology. All other reagents, including clioquinol and ZnCl2, were of analytic grade and obtained from Sigma.
Cell culture and cell viability assay
DU 145 cells were obtained from the American Type Culture Collection. Cells were cultivated in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FCS (fetal calf serum), 100 units/ml penicillin and 100 μg/ml streptomycin, and routinely grown in a 75-cm3 flask under a humid environment at 37 °C, 5% CO2. Cell viability was analysed with a modified tetrazolium assay using MTS reagent, following the manufacturer's protocol. In brief, DU 145 cells were plated in a 96-well tissue culture plate (5000 cells per well) with 100 μl of medium, which ensured a 40–60% confluence after 24 h of growth. The medium was then replaced with 100 μl of fresh medium containing clioquinol and ZnCl2 at various concentrations, and the cells were grown for designated periods. To each well, 20 μl of the MTS solution was added, and cells were incubated at 37 °C for 1 h to allow colour development. The plate was read at 490 nm and data were expressed as percentages of the values obtained from untreated control cells.
Fluorescence microscopic detection of lysosomes, mitochondria and intracellular zinc ions
Intracellular distribution of lysosomes, mitochondria and free zinc ions was analysed with the LysoTracker, MitoTracker and FluoZin-3 probes using fluorescence microscopy following the manufacturer's instructions. DU 145 cells were plated in a 12-well plate with 3×105 cells per well. At 24 h after plating, the cells were treated with 5–10 μM clioquinol and 50 μM ZnCl2 for 30 min. The medium was replaced with fresh medium (DMEM) containing 50 nM LysoTracker or 100 nM MitoTracker, and 1 μM FluoZin-3. After incubating for 30 min, the medium was removed and the cells were washed three times with HBSS (Hanks balanced salt solution) and viewed on a Nikon Eclipse TE2000-U microscope. Zinc was detected as a green colour (FluoZin-3®; excitation 490/20 nm and emission 528/38 nm), and lysosomes and mitochondria as a red colour (LysoTracker and MitoTracker; excitation 555/28 nm and emission 617/73 nm). Co-localization of zinc ions and lysosomes was determined by overlapping the green and red images. Fluorescent intensities were quantified using the NIS-Elements AR software (Nikon Instruments).
Lysosome membrane permeability analysis
AO (Acridine Orange) uptake and intracellular distribution were used to determine the changes in lysosomal membrane permeability [18–21]. DU 145 cells were plated in a 12-well plate at 3×105 cells per well. After incubation for 24 h, the cells were treated with 10 μM clioquinol and 50 μM ZnCl2 for 2 h. AO was then added to each well (at a final concentration of 2.5 μg/ml). After 30 min of incubation with AO, cells were washed twice with HBSS and examined with a fluorescence microscope (Nikon Eclipse TE2000-U). The excitation and emission wavelength of red fluorescence was 555/28 nm and 617/73 nm, and that of green fluorescence was 490/20 nm and 528/38 nm. AO concentrated in lysosomes emits a granular red fluorescence, whereas AO in the cytosol emits a diffuse green fluorescence . A reduction in granular red fluorescence combined with an increased diffuse cytosolic green fluorescence indicates a relocation of AO from the lysosomes to the cytosol, following a change in lysosome permeability.
Additional evidence of a change in lysosomal permeability was obtained by visualizing the intracellular distribution of the lysosomal aspartic endopeptidase, cathepsin D. Cathepsin D, which can mediate apoptotic cell death , was visualized using a fluorescent probe consisting of a conjugate of pepstatin A and BODIPY FL (Invitrogen).
Western blot analysis
Western blotting was performed as previously described [8,22,23]. Briefly, after treatment with clioquinol and ZnCl2, DU 145 cells were lysed in a lysis buffer containing 50 mM Tris/HCl (pH 7.4), 100 mM NaCl, 5 mM Na/EDTA, 1 mM PMSF, 0.1% SDS, 1% (v/v) Triton X-100 and 2% (v/v) glycerol. The lysates were separated on SDS/PAGE (15% gels), transferred to a PVDF membrane, and blotted with antibodies against Bid and GAPDH.
Clioquinol acts as a zinc ionophore
Treatment of DU 145 cells with clioquinol and zinc resulted in a time-dependent cytotoxicity (Figure 1), as measured with the MTS assay. Although 10 μM clioquinol or 50 μM ZnCl2 alone did not alter cell viability, the combination of the two led to a significant reduction of the viability in a time-dependent manner, with 30% reduction after 6 h of treatment, and 80% after 48 h. These data confirm our previous findings in other types of human cancer cells . We then determined whether clioquinol could transport zinc ions into DU 145 cells. Using the fluorescent probe FluoZin-3 , we found that clioquinol dramatically enhanced the intracellular free zinc level after 30 min of treatment with 50 μM ZnCl2 (Figure 2). This finding is consistent with our previous studies in which the zinc ionophore activity of clioquinol was analysed using different experimental approaches [8,22].
Effects of clioquinol and zinc on the viability of DU 145 cells
Effects of clioquinol on free intracellular zinc levels in DU 145 cells
Clioquinol targets zinc to lysosomes
To determine whether the diffuse fluorescence shown in Figure 2 truly reflected cytoplasmic distribution, the concentration of clioquinol used was reduced to 5 μM. Upon examination of cells under high power (Figure 3), a punctate distribution of FluoZin-3 was observed, suggesting that the free zinc was in membrane-bound organelles, and not in the cytoplasm. We therefore used organelle-specific fluorescent probes for lysosomes (LysoTracker) and mitochondria (MitoTracker) to identify the zinc-containing vesicles. As shown in Figure 3, intracellular free zinc was found to co-localize with lysosomes (Figure 3A), but not mitochondria (Figure 3B). Because lysosomes have been shown to concentrate zinc , our findings could be explained by dissociation of clioquinol–zinc complexes in the cytoplasm (followed by free zinc uptake into lysosomes), or within the acidic milieu of the lysosome. To investigate these possibilities, we pre-treated cells with ammonium chloride, a compound known to raise lysosomal pH , and then exposed them to clioquinol and zinc. As shown in Figure 4, pre-treatment with ammonium chloride decreased the amount of free zinc labelling in DU 145 cells. These findings suggest that clioquinol–zinc complexes dissociate after reaching the more acidic environment of the lysosome.
Effects of clioquinol on intracellular zinc distribution in DU 145 cells
Effects of ammonium chloride on the zinc ionophore activity of clioquinol in DU 145 cells
Clioquinol plus zinc enhances lysosomal permeability
Since disruption of lysosomal integrity has been linked with the induction of apoptosis , we postulated that the rapid increase in lysosomal zinc led to lysosomal instability. We utilized the observation of others that the fluorescence characteristics and distribution of AO is altered when lysosomal membrane permeability is altered . AO-loaded control DU 145 cells exhibited a pattern of granular red fluorescence (Figure 5), consistent with reports in other cell types . Treatment of the cells with 10 μM clioquinol and 50 μM ZnCl2 resulted in a loss of this pattern and a significant increase in diffuse green fluorescence within 1 h, consistent with an increase in lysosomal permeability. After 2 h of treatment, DU 145 cells showed nuclear condensations and membrane blebbings, features of apoptotic cell death. The alteration of lysosomal permeability by clioquinol and zinc was also observed in the MCF7 (human breast cancer) and HT-29 (human colon cancer) cell lines (results not shown).
Effects of clioquinol and zinc on AO staining of DU 145 cells
To confirm these findings, we examined the intracellular distribution of a lysosomal enzyme, cathepsin D , using a fluorescent probe (pepstatin A–BODIPY FL). As shown in Figure 6, treatment with clioquinol and zinc for 2 h altered the intracellular distribution of cathepsin D, confirming and extending the results obtained with AO.
Effects of clioquinol and zinc on cathepsin D distribution in DU 145 cells
Induction of apoptosis by lysosomal disruption has been demonstrated to be accompanied by the cleavage of the pro-apoptotic Bcl-2-family member, Bid . We used Western blotting to probe the extracts of DU 145 cells that had been treated with clioquinol plus ZnCl2 for 4 h. As shown in Figure 7, the combination caused a reduction of native Bid content (after normalization for GAPDH content). This finding establishes a link between the increase in free intracellular zinc content and the induction of apoptosis.
Effects of clioquinol and zinc on Bid expression in DU 145 cells
Studies from cultured tumour cells and animal models have demonstrated that clioquinol kills cancer cells and has the potential to be a clinically used anticancer agent [8,13,14]. The anticancer action of clioquinol has been demonstrated to be amplified by co-administration of the transition metals, copper and zinc . Although the complex of copper and clioquinol inhibits proteasome activity, the same has not been demonstrated for the zinc–clioquinol combination, leaving open the possibility that a different mechanism of action is responsible for its cytotoxic effects. The results from the present study demonstrate for the first time that clioquinol targets zinc to lysosomes, leading to alterations of lysosome integrity and lysosome-mediated apoptotic cell death.
After confirming that clioquinol increased intracellular zinc levels in the human prostate cancer line, DU-145, we found morphological evidence that the resulting free zinc was not diffusely distributed in a cytoplasmic pattern. Using fluorescent organelle-specific probes, we found that the free intracellular zinc co-localized with lysosomes, but not mitochondria. The significance of this finding was shown by demonstrating that clioquinol–zinc treatment caused a redistribution of AO, a small molecule normally staining lysosomes, as well as the lysosomal protein, cathepsin D. Finally, to demonstrate that these morphological findings were accompanied by appropriate biochemical events, Western blot analysis was used to show a cleavage of the pro-apoptotic protein, Bid, a marker of lysosomal-mediated apoptosis induction . Thus we have identified lysosomes as cellular targets of the clioquinol–zinc combination. These findings not only contribute to our understanding of the anticancer action of clioquinol, but they may also provide a novel mechanism for considering the toxicity of zinc in eukaryotic cells, with possible implications for other transition metals.
Our studies were stimulated by the observation that clioquinol transports zinc across the plasma membrane ([8,22] and observations in the present study). Given that the pKa of clioquinol for zinc is 7 , and that cytosol is slightly more acidic than the extracellular space, we thought it likely that the clioquinol–zinc complex dissociated in the cytosol, in accordance with the initial images obtained using FluoZn-3 (Figure 2). Using a lower concentration of clioquinol (Figure 3), we found a morphological pattern that suggested that the free zinc was sequestered within organelles. We subsequently found that the free zinc was generated within lysosomes and not mitochondria. This finding is consistent with the observation that the lysosomal pH is <5 , whereas the pH of the mitochondrial matrix is >7.5 . These results suggest that the chemical properties of cell-permeant metal-containing complexes determine whether the complex is toxic to cells. The moderate affinity of clioquinol for zinc  and a pKa lying between that of the extracellular space and the lysosomal compartment provides conditions leading to lysosome-mediated apoptotic cell death.
Previous studies have demonstrated that intralysosomal irons (redox active) contribute to oxidant-induced alterations of lysosomal stability and cell death . A recent study reported that an increase in intracellular zinc level is involved in the apoptotic pathway induced by acute hydrogen peroxide exposure in epithelial cells . These studies may suggest that zinc overloading in the lysosomes could trigger apoptotic cell death through enhancing cellular oxidative stress; however, whether an accumulation of zinc in the lysosomal compartment affects the homoeostasis of lysosomal redox-active irons, thereby leading to lysosomal disability and apoptotic cell death, requires further investigation.
Zinc deficiency is associated with several human malignances [32,33] and is believed to be associated with DNA damage and tumour growth . Restoration or an increase in intracellular zinc levels have been reported to induce apoptosis of tumour cells [8–10,35], suggesting that zinc ionophores may serve as anticancer agents. Other studies have reported that an increase in intracellular levels of zinc down-regulates NF-κB (nuclear factor κB) signalling [8,36], a signal transduction pathway that is a well-established molecular target for cancer therapy . Although a connection between changes in lysosome permeability and NF-κB activity has previously been described [37,38], it is not yet known whether clioquinol–zinc complexes cause a down-regulation of NF-κB signalling due to alterations in lysosomal permeability.
We acknowledge the technical assistance of Jessica R. Lou.
This work was supported by the Oklahoma Center for the Advancement of Science and Technology [grant number HR04-021]; the American Cancer Society (Institutional Seed Grant) [grant number IRG-05-066-01]; the China Scholarship Council; and the College of Medicine, University of Oklahoma Health Sciences Center [grant number COM22134].