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CELLULAR AND MOLECULAR

Clioquinol Promotes Cancer Cell Toxicity through Tumor Necrosis Factor Release from Macrophages

Centre for Neuroscience and Department of Pathology, University of Melbourne, Victoria, Australia (T.D., G.F., A.C., P.J.C., A.R.W.); and Mental Health Research Institute, Parkville, Victoria, Australia. (T.D., G.F., A.C., P.J.C., A.R.W.)

Received August 16, 2007; accepted October 15, 2007.

	Abstract

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Copper has an important role in cancer growth, angiogenesis, and metastasis. Previous studies have shown that cell-permeable metal ligands, including clioquinol (CQ) and pyrrolidine dithiocarbamate, inhibit cancer cell growth in cell culture and in vivo. The mechanism of action has not been fully determined but may involve metal-mediated inhibition of cancer cell proteasome activity. However, these studies do not fully account for the ability of cell-permeable metal ligands to inhibit cancer cell growth without affecting normal cells. In this study, we examined the effect of CQ on macrophage-mediated inhibition of HeLa cancer cell growth in vitro. When CQ was added to RAW 264.7 macrophage-HeLa cell cocultures, a substantial increase in HeLa cell toxicity was observed compared with CQ treatment of HeLa cells cultured alone. Transfer of conditioned medium from CQ-treated macrophages to HeLa cells also induced HeLa cell toxicity, demonstrating the role of secreted factors in the macrophage-mediated effect. Further investigation revealed that CQ induced copper-dependent activation of macrophages and release of tumor necrosis factor (TNF) .In studies with recombinant TNF, we showed that the level of TNF released by CQ-treated macrophages was sufficient to induce HeLa cell toxicity. Moreover, the toxic effect of conditioned medium from CQ-treated macrophages could be prevented by addition of neutralizing antibodies to TNF. These studies demonstrate that CQ can induce cancer cell toxicity through metal-dependent release of TNF from macrophages. Our results may help to explain the targeted inhibition of tumor growth in vivo by CQ.

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQ) is a lipophilic compound capable of complexing with Cu and zinc (Di vaira et al., 2004). Initially, CQ was used as an antibiotic for treatment of diarrhea and skin infections (Yassin et al., 2000); however, its use was stopped in some countries after a possible association with subacute myelo-optic neuropathy in Japan (Richards, 1971; Yassin et al., 2000; Di Vaira et al., 2004). Subsequent epidemiological evidence does not support this link, and it appears that vitamin B₁₂ deficiency may have had a role in the syndrome (Chen et al., 2007; Ding et al., 2005). Several recent studies have generated interest in CQ as a modulator of metal homeostasis in neurodegenerative disorders. CQ has been shown to reduce or prevent the formation of amyloid plaques in transgenic Alzheimer's disease mice (Cherny et al., 2001) and conferred benefit to Alzheimer's disease patients in a small clinical trial (Ritchie et al., 2003). The compound has also shown positive effects in a Parkinson's disease mouse model and inhibited aggregation of huntingtin in a cell model of Huntington's disease (Nguyen et al., 2005).

Because of the association between Cu and cancer growth and metastasis, a number of groups have investigated the potential of CQ as an anticancer drug. Ding et al. (2005) demonstrated caspase-dependent apoptosis in a number of cancer cell lines treated with CQ. This toxic effect was related to the ionophoric activity of CQ because CQ-mediated cellular Cu uptake resulted in increased cytotoxicity. Subsequent CQ treatment of mice with xenograft tumors revealed a substantial reduction in tumor size but without evidence of broad cytotoxicity. A potential role for the nuclear factor B (NFB) was investigated, but only small changes in activity were observed after CQ treatment of cancer cells (Ding et al., 2005). Daniel et al. (2005) also reported specific anticancer effects of CQ. CQ and another Cu ionophore, pyrrolidine dithiocarbamate (PDTC), were found to induce metal-dependent inhibition of the proteasome in human breast cancer cells. Subsequently, Chen et al. (2007) reported that CQ-Cu complexes targeted chymotrypsin-like activity of the proteasome.

Although these studies have highlighted the potential for CQ as a therapeutic agent for treatment of cancer, little information is available on how CQ specifically targets cancer cells, particularly in vivo. The metal ionophore effect of CQ has been demonstrated in many different cell types (Treiber et al., 2004; Benvenisti-Zarom et al., 2005; White et al., 2006; Masuda et al., 2007). Although CQ may specifically target the proteasome in some cancer cell lines (Chen et al., 2007), it is uncertain whether this extends to all cancer cells and whether this is the primary anticancer mechanism in vivo (Ding et al., 2005). An alternative mode of action for CQ-mediated anticancer activity is through the immune system. To further investigate the potential role of immune cells in anticancer effects by CQ, we examined whether macrophage-mediated cancer inhibition could play a role in CQ activity. RAW 264.7 macrophages were cocultured with HeLa cancer cells in the presence of CQ. We found that the presence of macrophages greatly potentiated the toxic effect of CQ toward HeLa cells. Further examination showed that tumor necrosis factor (TNF) release from CQ-treated macrophages induced toxic effects on cocultured HeLa cells. Continued investigation of how CQ induces macrophage activation may help to identify novel metal-mediated signal transduction pathways involved in anticancer responses.

	Materials and Methods

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Cell Culture. Raw 264.7 macrophages were obtained from Dr. David Thomas (Ian Potter Cancer Genomic Centre, Peter MacCallum Institute, Melbourne, Australia) and Dr. Dmitri Sviridov (Baker Heart Research Institute, Melbourne, Australia). HeLa cells were a gift from Dr. Patrick Sexton (Howard Florey Institute, Melbourne, Australia). Raw 264.7 macrophages were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), 16 mM HEPES, and 50 µg/ml penicillin/streptomycin. For experiments, cells were plated into 24- or 6-well plates or 100-mm dishes (Nalge Nunc International, Naperville, IL) at a dilution of 1:4 in RPMI medium supplemented with 10% FCS. Cells were grown for 1 to 2 days until 80 to 90% confluent and used for experiments. HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% FCS, 2 mM glutamine, 10 mM HEPES, and 50 µg/ml penicillin/streptomycin. Cells were passaged at 1:20 dilution and seeded at 1:10 into 24-well plates or onto glass coverslips for experiments. Cells were maintained 1 to 2 days until 80 to 90% confluent before experimental treatments.

CQ Treatment. The medium used for the coculture and transfer experiments was Opti-MEM (Invitrogen, Carlsbad, CA) without serum. For coculture experiments, HeLa cells on coverslips were added to wells containing macrophages. CQ (10 mM stock in dimethyl sulfoxide) and/or Cu(II) (10 mM in dH2O) was added to macrophage-HeLa cell cocultures at indicated concentrations for 24 h. Coverslips of HeLa cells were then removed, and cell viability of macrophages and HeLa cells was determined separately using the MTT assay as described previously (White et al., 1999) or Trypan blue staining with 0.4% Trypan blue (Sigma-Aldrich, St. Louis, MO). For experiments involving transfer of conditioned medium, macrophages were treated with CQ or metals at indicated concentrations for 2 or 24 h, and medium was transferred to separate cultures of HeLa cells for a further 24 h. This was followed by MTT or Trypan blue assay of cell viability. Where indicated, CQ was also added alone to HeLa cells for 24 h. In some experiments, the Cu(I)-selective metal ligand, bathocuproine sulfonate (BCS), was added at a concentration of 200 µM. Experiments were also performed in media pretreated with Chelex 100 resin to deplete residual Cu levels. We have reported previously that Chelex 100 treatment reduces Cu levels in the medium by approximately 10-fold (White et al., 2004).

NFB Assay. Nuclear extracts of CQ-treated and control macrophages were obtained using a nuclear extraction kit (Active Motif, Carlsbad, CA). NFB activity was determined in extracts using the NFB p50 transcription factor ELISA (Active Motif) as per the kit's instructions.

Multiplex Analysis of Cytokine Release. Macrophages were treated with CQ (10 µM) for 6 h, and levels of cytokines in the conditioned medium were determined using the ChemiArray Mouse Inflammation Antibody Array I (Chemicon International, Temecula, CA) as per kit instructions. Measurement of TNF levels in conditioned medium was also measured using a murine TNF ELISA (Assay Designs) as per kit insert. As a positive control, macrophages were treated with 100 ng/ml lipopolysaccharide (LPS) (Sigma-Aldrich), and TNF levels were determined in conditioned medium.

Treatment with TNF. To determine the effect of TNF on HeLa cell viability, cocultures were treated with recombinant murine TNF (eBioscience, San Diego, CA) at a concentration of 10.0 and 100 ng/ml for 24 h. Cell viability was determined using the MTT assay.

TNF Neutralization. Neutralizing antibody to TNF was purchased from eBioscience. Antibody was added to culture medium at 10 µg/ml together with CQ (10 µM). HeLa cells were cocultured with macrophages in medium containing TNF antibody, and the effect on HeLa cell viability was determined by MTT assay. As a control, cells were also treated with neutralizing antibodies to interleukin (IL)-12p40/p70 (eBioscience) at a concentration of 10 µg/ml.

Statistical Analysis. All data described in graphical representations are mean ± S.E.M. from three to six separate tests unless stated. Results were analyzed on raw data using a two-tailed Student's t test.

	Results

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CQ Stimulates Macrophages to Release Soluble Toxic Factors That Reduce HeLa Cell Viability. Initially, we examined the effect of coculturing macrophages with HeLa cells in the presence of CQ. Cocultures of macrophages and HeLa cells were exposed to CQ for 24 h, and the viability of each cell type was determined separately. In addition, HeLa cells were also grown separately in CQ-supplemented medium to determine the basal effect of CQ on this cell type. A concentration range of 1 to 10 µMCQwas used because higher concentrations (25–100 µM CQ) were found to be highly toxic to HeLa cells cultured in the absence of macrophages (data not shown). As shown in Fig. 1A, treatment of macrophages had little effect on the viability of this cell type. A small (17%) but significant increase in macrophage viability was observed after treatment with 10 µMCQ (Fig. 1A). In contrast, HeLa cells grown alone in CQ-supplemented medium revealed decreased cell viability (Fig. 1B), and this effect was significantly exacerbated when HeLa cells were cocultured with macrophages (Fig. 1B). CQ had no significant effect on macrophage viability in cocultures (data not shown). We then confirmed that the loss of HeLa MTT reduction induced by CQ in macrophage cocultures was due to a decrease in viability and not simply altered cell metabolism. Measurement of Trypan blue and MTT staining of HeLa cells from macrophage cocultures revealed a close correlation in cell toxicity between both methods (Fig. 1C). This confirmed the loss of HeLa cell viability induced by CQ in the cocultures. Our data established that macrophages can increase the toxic effects of CQ toward HeLa cancer cells. We then examined whether the toxic effect of CQ-treated macrophages was mediated by secreted molecules. This was achieved by transferring conditioned medium from CQ-treated macrophages to HeLa cells. Macrophages were cultured for 2 or 24 h in medium supplemented with 1 to 10 µM CQ, and then the conditioned medium was applied to HeLa cell cultures for a further 24 h. Conditioned medium from macrophages treated with CQ for 2 h had little effect on HeLa cell viability, but when conditioned medium was transferred from macrophages treated for 24 h, it induced a significant toxic effect on the HeLa cells (Fig. 1D). Conditioned media from macrophages treated for 24 h with 10 µM CQ reduced HeLa cell viability to less than 50%, which was analogous to the coculture effect in Fig. 1, B and C. This result indicates that treatment of macrophages with CQ results in increased secretion of factors that are inhibitory to HeLa cancer cell survival.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Macrophages promote CQ-mediated inhibition of HeLa cell growth. A, Raw 264.7 macrophages were treated with CQ (1–10 M) for 24 h, and cell viability was determined using the MTT assay. CQ slightly but significantly increased macrophage cell viability at 10 µM(*, p < 0.05). B, HeLa cells were cocultured with macrophages for 24 h in the presence of 1 to 10 µM CQ. HeLa cells were also treated with CQ without macrophages for 24 h. Viability of HeLa cells was determined by MTT assay, and this revealed a significantly greater loss of viability when HeLa cells were cultured with CQ and macrophages compared with CQ alone (*, p < 0.05; **, p < 0.01). C, HeLa cells were cocultured with macrophages for 24 h in the presence of 1 to 10 µMCQ for 24 h. The viability of the HeLa cells was determined by Trypan blue exclusion assay and compared with viability obtained with MTT assay. The Trypan blue and MTT assays both produced very similar measures of cell viability (*, p < 0.05; **, p < 0.01 compared with untreated control). D, HeLa cells were cultured for 24 h in media that had been preincubated on macrophage cultures for 2 or 24 h with CQ (1–10 µM). A significant decrease in HeLa cell viability was observed when cells were treated with macrophage-conditioned medium that had been exposed to CQ for 24 h (*, p < 0.05; **, p < 0.01).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Macrophage-mediated CQ effects on HeLa cells are Cu-dependent. A, HeLa cells were cocultured with RAW 264.7 macrophages for 24 h in the presence or absence of CQ (1–10 µM) or CQ and BCS (200 µM). Treatment with BCS significantly decreased HeLa cell toxicity (*, p < 0.05; **, p < 0.01). B, HeLa cells were cultured for 24 h in media that had been preincubated on macrophage cultures for 24 h with CQ (1–10 µM) or CQ and BCS (200 µM). BCS treatment significantly inhibited HeLa cell toxicity (*, p < 0.05; **; p < 0.01).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Cu promotes HeLa cell toxicity by macrophages. A, HeLa cells were treated with 1 to 10 µM Cu(II) or cocultured with macrophages and 1 to 10 µM Cu(II) for 24 h. HeLa cells cocultured with macrophages and 10 µM Cu(II) revealed a significant increase in cell toxicity compared with HeLa cells treated with 10 µM Cu(II) alone (**, p < 0.01). B, HeLa cells were cocultured with macrophages and CQ alone (1–10 µM) or CQ and Cu(II) (1–10 µM each) for 24 h. Treatment with CQ-Cu induced the same level of HeLa cell toxicity as CQ alone. C, HeLa cells were cultured with macrophages in Chelex 100 treated-medium (Chelex medium) for 24 h with CQ and increasing concentrations of Cu(II). Depletion of Cu using Chelex 100 prevented HeLa cell toxicity from CQ and macrophages. Addition of Cu(II) to the medium restored the toxic effects CQ in the cocultures (**, p < 0.01).

CQ Induces up-Regulation of Cytokine Release from Macrophages. Next, we examined how CQ induced HeLa cell toxicity by macrophages. The metal ionophore activity of CQ is similar to PDTC (Chen et al., 2007), and PDTC is a potent inhibitor of NFB signaling (Sherman et al., 1993). NFB activity was measured in nuclear extracts of macrophages treated with 10 µM CQ for 24 h. However, we found no evidence of altered NFB activity (Fig. 4). Because CQ promoted release of a soluble factor(s) from macrophages that induced HeLa cell toxicity, we next examined the levels of cytokines in conditioned macrophage medium. Macrophages were maintained in CQ-treated (10 µM) medium for 24 h, and the cytokine levels in the media were determined using a Mouse Inflammation Antibody Array I (Chemicon). This analysis revealed up-regulation of a number of cytokines in conditioned medium after CQ treatment (Table 1). This included a range of interleukins as well as tissue inhibitors of matrix metalloproteases, interferon, and TNF.

View larger version (21K): [in this window] [in a new window]   Fig. 4. NFB activity in macrophages treated with CQ. RAW 264.7 macrophages were treated with CQ, Cu(II), or CQ plus Cu(II) (10 µM each) for 24 h, and NFB activity was measured in nuclear extracts. No significant changes to NFB levels were observed after any treatment.

CQ Induces TNF Release from Macrophages and Inhibition of HeLa Cell Viability. One of the most highly elevated cytokines in CQ-treated macrophage medium was TNF (Table 1), and this cytokine has well known anticancer properties (Mocellin et al., 2007). Therefore, we examined the role of TNF further in CQ-treated macrophages and macrophage-HeLa cell cocultures. Treatment of macrophages with CQ or Cu(II) (10 µM each) for 24 h induced an approximate 900% increase in TNF levels, whereas Cu(II) induced over 1200% increase (Fig. 5A). This was in the same order of magnitude as the level of TNF induced by treating macrophages with 100 ng/ml LPS (Fig. 5A). A dose-response effect was also observed, with increasing concentrations of CQ (1–10 µM) inducing greater release of TNF from macrophages (Fig. 5B). Interestingly, the level of TNF release induced by 10 µM CQ was substantially higher when measured by ELISA (900%) than by antibody array (135%) (Table 1). The difference in the observed magnitude of TNF release between the two techniques may indicate suboptimal binding conditions used in the array. The binding environment is a compromise for all the cytokines and cannot be optimized for an individual antibody-antigen interaction. However, these data nonetheless confirm that TNF was significantly up-regulated in macrophage cultures treated with CQ. Interestingly, when macrophages were cotreated with CQ and Cu (10 µM each), no further elevation in TNF levels were observed compared with CQ alone (Fig. 5A). This was consistent with our finding that addition of CQ and Cu had no greater toxic effect than CQ alone on HeLa cells in cocultures (Fig. 3B).

View larger version (30K): [in this window] [in a new window]   Fig. 5. CQ induces TNF release from macrophages, which induces HeLa cell toxicity. A, RAW 264.7 macrophages were treated with CQ, Cu(II), or CQ plus Cu(II) (10 µM each) for 24 h, and TNF levels were determined in conditioned media by ELISA. LPS (100 ng/ml) was added as a positive control. Treatment of macrophages with CQ or Cu induced a significant increase in TNF levels in the conditioned medium. Treatment with CQ and Cu(II) together did not induce greater levels of TNF than CQ alone. Cotreatment with BCS (200 µM) or treatment in Chelex medium significantly inhibited TNF release compared with CQ treatment alone in normal medium. Addition of Cu(II) (10 µM) to Chelex medium restored TNF release after treatment with CQ (**, p < 0.01 compared with untreated cultures; ***, p < 0.01 compared with CQ-treated macrophages in normal medium). B, RAW 264.7 macrophages were treated with 1 to 10 µM CQ for 24 h, and levels of TNF were measured in conditioned medium. All concentrations of CQ increased TNF levels relative to control (*, p < 0.05; **, p < 0.01 compared with untreated control). C, HeLa cells were treated with conditioned medium from macrophages cultured with 10 µM CQ for 24 h or cultured in fresh medium containing 10.0 or 100 ng/ml recombinant TNF. After 24 h, HeLa cell viability was determined and revealed that TNF induced a similar level of cytotoxicity compared with the conditioned medium (**, p < 0.01 compared with untreated controls). D, HeLa cells were cocultured with macrophages and 10 µM CQ for 24 h with or without neutralizing antibody to TNF or IL-12p40/p70 (10 µg/ml). Antibody to TNF significantly increased HeLa cell viability compared with treatment with CQ alone in cocultures (**, p < 0.01).

We then further examined the role of Cu in CQ-mediated TNF release by macrophages. Cotreatment of macrophages with 10 µM CQ and 200 µM BCS for 24 h revealed that chelation of extracellular Cu by BCS prevented CQ-mediated TNF release (Fig. 5A). Likewise, Chelex treatment (Cu depletion) of medium also inhibited TNF release by CQ (Fig. 4A). Returning Cu(II) to the medium of Chelex-treated cultures restored the ability of CQ to induce TNF release (Fig. 5A), confirming that CQ requires extracellular Cu to mediate TNF release from macrophages.

Although these studies demonstrated that CQ induced TNF release from macrophages, it was still uncertain whether this cytokine was responsible for the loss of HeLa cell viability in the cocultures. Therefore, HeLa cell cultures were treated with recombinant human TNF at a concentration analogous to the level of TNF measured by ELISA in culture medium from CQ-treated macrophages (approximately 10.0 ng/ml). HeLa cultures maintained in conditioned medium from CQ-treated macrophages induced a high level of toxicity as before (approximately 45% viability, Fig. 5B). Treatment of separate HeLa cell cultures with TNF at 10.0 or 100 ng/ml also induced toxicity (Fig. 5B). This toxicity was less than induced by 10 µM CQ, suggesting that one or more of the additional cytokines up-regulated by CQ could also contribute to the toxicity in cocultures. Therefore, to further confirm that TNF was inducing toxicity in HeLa cells cocultured with macrophages, we treated cocultures with CQ and neutralizing antibody to TNF. Addition of the neutralizing antibody significantly inhibited the toxic effects of CQ against HeLa cells when cultured with macrophages. Again, the inhibition by the neutralizing antibody to TNF was not complete and is consistent with a role for additional macrophage-released cytokines in HeLa cell toxicity. Antibodies to the unrelated cytokine, IL-12p40/p70, but which also showed increased levels in CQ-treated macrophages, had no effect on HeLa cell viability. This demonstrated that addition of antibody per se does not inhibit CQ toxicity. These findings demonstrate that CQ is able to induce TNF release from macrophages, which in turn is toxic to HeLa cells. The data may have important implications for understanding how CQ induces specific antitumor activity in mouse models of cancer and potentially in human cancer patients.

	Discussion

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This report describes a novel mechanism for the anticancer activity of CQ. Although previous reports have described the toxicity of several cancer cell lines by CQ, this is the first to demonstrate that CQ can induce cancer toxicity through macrophage activation. Our studies revealed that CQ promotes the release of TNF from macrophages in vitro with the TNF subsequently inducing toxicity in HeLa cancer cells. Although we did not investigate the cell toxicity pathway in this study, it is known that TNF can induce apoptosis in cancer cells. However, activation of the cell death pathway in HeLa cells normally requires inhibition of protein synthesis in addition to TNF treatment (White et al., 1999). Protein synthesis inhibition was not required to induce the toxic effects observed in our cultures by CQ macrophage-conditioned media or recombinant TNF. Therefore, it is likely that the inhibition of HeLa cell viability in our cultures (as assessed by the MTT and Trypan blue assays) did not reflect apoptosis but instead was TNF-mediated inhibition of cell growth.

Previous in vitro studies have demonstrated a specific targeting of some cancer cell lines directly by CQ. In these studies, CQ induced metal-dependent inhibition of proteasome activity resulting in activation of cell death pathways and cancer cell apoptosis (Chen et al., 2007). CQ has also been demonstrated to induce antitumor effects in vivo (Ding et al., 2005; Chen et al., 2007), but the mechanism of action remains unclear. Our report suggests CQ-mediated cancer cell toxicity may involve Cu-dependent activation of macrophages, resulting in specific and localized attack on cancer cells. This indirect CQ effect may occur in addition to reported direct effects of CQ on cancer cell proteasome activity. Whether this effect occurs with alternative cancer cell types is not known. Alternatively, TNF could exacerbate proteasome inhibition induced by CQ as reported with other proteasomal inhibitors (Rice et al., 2001).

We found that the ability of CQ to induce macrophage activation and TNF release was dependent on extracellular Cu. CQ is a Cu ionophore (White et al., 2006; Chen et al., 2007) and is therefore able to facilitate the uptake of Cu into cells including macrophages. Depletion of Cu from the culture medium prevented CQ-mediated TNF release and anti-HeLa cell activity while restoring even 100 nM Cu to the medium induced toxic effects to HeLa cells in the presence of macrophages and CQ. Because Cu levels can reach micromolar concentrations in vivo (Wataha et al., 1996), our findings suggest that CQ could induce similar uptake of extracellular Cu in vivo. Interestingly, little is known about the role of Cu in direct stimulation of inflammatory responses by macrophages (Sidoti-de Fraisse et al., 1998). This is the first report that ligand-mediated metal uptake can induce release of TNF and a number of interleukins from macrophages. Previously, Wataha et al. (1996) reported that Cu and other metals could potentiate LPS-mediated release of IL-1 in human THP1 macrophages, but Cu had no effect on TNF in these cells. In contrast, Cuderi (1990) observed increased TNF mRNA in peripheral blood monocytes treated with Cu. It is possible that metals may have various effects on different immune cell populations or could act in concert with other, unidentified molecules. Whatever the mechanism, our findings together with recent reports by Ding et al. (2005) and Chen et al. (2007) further support the use of CQ as an anticancer metal ionophore rather than a metal chelator. This mode of action contrasts with cell-impermeable metal chelators such as tetrathiomolybdate and desferrioxamine that chelate extracellular Cu, thus preventing angiogenesis (Chen et al., 2007).

The mechanism of CQ-mediated Cu uptake and TNF release is uncertain. We have previously reported that epithelial cells and neuronal cell lines treated with CQ-Cu complexes revealed robust activation of cell signaling pathways involving phosphoinositol-3-kinase and downstream modulation of mitogen-activated protein kinases such as c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase. CQ-Cu complexes also modified phosphorylation of glycogen synthase kinase 3 (White et al., 2006). However, similar kinase activation by CQ or Cu was not observed in our macrophage cultures (data not shown). An alternative mechanism could involve metal-mediated modulation of TNF release from cell membranes through increased metalloprotease activity because metalloproteases are responsible for cleavage of pro-TNF at the cell surface (Bala and Failla, 1992). This is supported by our studies in other cell types, which have shown that CQ can induce increased production of matrix metalloproteases (White et al., 2006). Alternatively, previous studies have shown that Cu can induce release of IL-2, IL-6, and IL-8 from cells in culture (Schmalz et al., 1998; Bar-Or et al., 2003; Ju et al., 2006). Our protein array study revealed that levels of IL-2 and IL-6 were increased in culture medium of macrophages treated with CQ (Table 1). Although we did not substantiate this finding by ELISA, it is possible that elevated levels of interleukins may induce TNF release from the macrophages. If this does occur, it may be triggered through Cu-dependent modulation of transcription factors that result in elevated cytokine production (Qiao et al., 2007). The specific pathway(s) activated by Cu may be dependent on which cellular compartment the metal complexes are localized to. This may also vary between different metal-ligand complexes such as CQ-Cu and PDTC-Cu. Such differences may explain why CQ did not significantly modulate NFB activity in this study or a previous report (Ding et al., 2005), despite similarities in cellular metal uptake to the NFB inhibitor, PDTC (Daniel et al., 2005).

It is noteworthy that our findings have significant implications for the development of anticancer agents based on metal ligands. Our studies have demonstrated that CQ is able to enhance proinflammatory responses via a metal ionophore effect. Although this may potentially allow more localized targeting of tumors through macrophage-dependent tumor cell toxicity, our data also suggest that metals could have an important and as yet uncharacterized role in inflammation. The level of metal found to stimulate release of TNF from macrophages in this study was well within levels found in biological fluids (Wataha et al., 1996). Whether there are protective mechanisms in vivo that modulate metal-mediated inflammation is not known. Further studies are necessary to characterize the role of metals such as Cu in proinflammatory responses and whether metal ionophores can be developed into specific anticancer agents that target tumors through immune cell stimulation.

	Footnotes

 
This work was supported by the National Health and Medical Research Council of Australia (Grant 400202). A.R.W. was supported by an RD Wright Fellowship from the National Health and Medical Research Council of Australia.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.130377.

ABBREVIATIONS: CQ, clioquinol, 5-chloro-7-iodo-8-hydroxyquinoline; PDTC, pyrrolidine dithiocarbamate; TNF, tumor necrosis factor; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; BCS, bathocuproine sulfonate; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; IL, interleukin; NFB, nuclear factor B.

Address correspondence to: Dr. Anthony R. White, Centre for Neuroscience and Department of Pathology, University of Melbourne, Victoria, Australia 3010. E-mail: arwhite{at}unimelb.edu.au

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