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¹ Cannon Research Center, Carolinas Medical Center, Charlotte, North Carolina; ² U.S. Environmental Protection Agency, Research Triangle Park, North Carolina; ³ Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina; and ⁴ AAI Research, Wilmington, North Carolina

Requests for reprints: Thomas P. Kennedy, Department of Medicine, University of Utah Medical Center, Wintrobe 743A, 50 North Medical Drive, Salt Lake City, UT 84132. Phone: 801-581-7806; Fax: 801-585-3355. E-mail: Thomas.Kennedy{at}hsc.utah.edu

	Abstract

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The thiocarbamate alcoholism drug disulfiram blocks the P-glycoprotein extrusion pump, inhibits the transcription factor nuclear factor-B, sensitizes tumors to chemotherapy, reduces angiogenesis, and inhibits tumor growth in mice. Thiocarbamates react with critical thiols and also complex metal ions. Using melanoma as the paradigm, we tested whether disulfiram might inhibit growth by forming mixed disulfides with critical thiols in a mechanism facilitated by metal ions. Disulfiram given to melanoma cells in combination with Cu²⁺ or Zn²⁺ decreased expression of cyclin A and reduced proliferation in vitro at lower concentrations than disulfiram alone. In electrophoretic mobility shift assays, disulfiram decreased transcription factor binding to the cyclic AMP-responsive element in a manner potentiated by Cu²⁺ ions and by the presence of glutathione, suggesting that thiocarbamates might disrupt transcription factor binding by inducing S-glutathionylation of the transcription factor DNA binding region. Disulfiram inhibited growth and angiogenesis in melanomas transplanted in severe combined immunodeficient mice, and these effects were potentiated by Zn²⁺ supplementation. The combination of oral zinc gluconate and disulfiram at currently approved doses for alcoholism also induced >50% reduction in hepatic metastases and produced clinical remission in a patient with stage IV metastatic ocular melanoma, who has continued on oral zinc gluconate and disulfiram therapy for 53 continuous months with negligible side effects. These findings present a novel strategy for treating metastatic melanoma by employing an old drug toward a new therapeutic use.

	Introduction

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In the quest for effective therapies for human cancer, it is occasionally possible to apply an already approved drug toward a new use. This strategy has been most commonly used to apply cancer chemotherapeutic agents approved for one type of malignancy to the treatment of others but may also lend itself to antineoplastic application of older drugs approved for nononcologic diseases. Recently, several laboratories have investigated the aldehyde dehydrogenase inhibitor tetraethylthiuram disulfide, or disulfiram, a relatively nontoxic [oral LD₅₀ of 8.6 g/kg (1)] dithiocarbamate disulfide long used for alcohol aversion therapy (2). Disulfiram reverses in vitro resistance of human tumors to chemotherapy drugs by blocking maturation of the P-glycoprotein membrane pump that extrudes chemotherapeutic agents from the cell (3). Disulfiram also inhibits activation of nuclear factor-B (NF-B) induced in human colorectal cancer cell lines by the chemotherapeutic agent 5-fluorouracil and enhances the apoptotic effect in vitro when the two are used in combination (4). Additionally, disulfiram inhibits DNA topoisomerases (5), induces apoptosis in cultured melanoma cells (6), reduces angiogenesis (7, 8), inhibits matrix metalloproteinases and cancer cell invasiveness (7), and retards growth of C6 glioma and Lewis lung carcinoma in mice (9). However, the mechanism for effects of disulfiram is still not clear, and the use of disulfiram is yet to be reported in the treatment of human malignancies.

The antineoplastic activity of disulfiram has been attributed to proapoptotic redox-related mitochondrial membrane permeabilization (6), zinc complexation with subsequent inhibition of Zn²⁺-dependent matrix metalloproteinases (7), or Cu²⁺ complexation with inactivation of Cu/Zn superoxide dismutase (8, 9) and consequently diminished cellular generation of H₂O₂ from dismutation of superoxide anion (O₂^–; refs. 8, 9). Dithiocarbamates possess a RR'NC(S)SR'' functional group, giving them the ability to complex metals (10) and react with sulfhydryl groups (10) and glutathione (11). After oxidation to their corresponding disulfides, dithiocarbamates can inhibit critical sulfhydryls by forming mixed disulfides with critical cellular thiols (12), leading to such diverse effects as inhibition of caspases (12) but stimulation of mitochondrial permeability transition (13) and subsequent Bcl-independent apoptosis (14). In normal cells, the effects of other dithiocarbamates are potentiated by metals such as Cu²⁺ or Zn²⁺ (15). We therefore postulated that disulfiram might inhibit cellular proliferation of malignant tumor tumors by forming mixed disulfides, which disrupt vital protein functions, and that this process might be dependent on the presence of certain metal ions.

One potential use for this approach is treatment of malignant melanoma, a tumor notoriously resistant to radiation and traditional chemotherapeutic agents but independently sensitive in vitro to disulfiram (6) or metals (16). In this report, we show that disulfiram reduces activating transcription factor/cyclic AMP-responsive element binding protein (ATF/CREB) transcription factor DNA binding, cyclin A expression, cell cycle progression, and melanoma proliferation in vitro and in severe combined immunodeficient (SCID) mice in a manner dependent on and facilitated by copper and other heavy metal ions. In addition, we present the use of this strategy in a patient with stage IV ocular melanoma and hepatic metastases, who has experienced considerable tumor regression and remains clinically well after 53 continuous months of therapy with oral disulfiram and zinc gluconate.

	Materials and Methods

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Cells
Human malignant cell lines were obtained from American Type Culture Collection (Rockville, MD). Melanoma cells lines CRL1585 and CRL1619 were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (FBS) and passed with nonenzymatic cell dissociation solution (Sigma Chemical Co., St. Louis, MO). The prostate adenocarcinoma cell line CRL1435 (PC-3) and the ovarian cancer cell lines HTB75 and HTB77 were also cultured in RPMI 1640 with 10% FBS but passed with 0.05% trypsin and 0.53 mmol/L EDTA. The squamous lung carcinoma NCI-H520 and the adenosquamous lung carcinoma NCI-H596 cell lines were grown in RPMI 1640 supplemented with 10% FBS, 10 mmol/L HEPES, and 1.0 mmol/L sodium pyruvate and passed with trypsin/EDTA. All of the above were grown in a 37°C humidified environment containing 5% CO₂/air. The breast carcinoma cell line MDA-MB-453 was grown in a 37°C humidified environment with free atmospheric gas exchange, Leibovitz's L-15 medium with 2 mmol/L L-glutamine and 10% FBS, and was passed with trypsin/EDTA.

Cell Treatments
Because others have suggested that the disulfide form of dithiocarbamates is the active proximate chemical form that mediates mixed disulfide formation with protein thiols (11–13), we did most of our experiments with the tetraethylthiuram disulfide disulfiram (Sigma Chemical), which does not have a free thiol to act as an antioxidant. Malignant melanoma cells grown to confluence on 100 x 15 mm plastic Petri dishes were treated with 0 to 5 µmol/L disulfiram. These doses were chosen to approximate the steady-state plasma and tissue concentrations reported previously in humans treated with disulfiram (17). Disulfiram is converted to its bis(diethyldithiocarbamato)copper(II) complex after passage through the acid environment of the stomach (2). Therefore, Cu²⁺ was added along with disulfiram in some experiments to stimulate formation of the disulfiram-copper chelate form in which the drug is systemically absorbed. Disulfiram was dissolved in DMSO to a final concentration of <0.3% to 0.5%. Equal volumes of DMSO were added to control experiments.

The effect of disulfiram (0.15–5.0 µmol/L) or sodium diethyldithiocarbamate (1.0 µmol/L) on proliferation of malignant cell lines was studied in cultures stimulated with 10% FBS. Cell numbers were quantitated 24 to 72 hours later, as outlined below. In some experiments, disulfiram was added immediately after cells were plated. In other experiments, cells were plated and allowed to grow for 24 to 72 hours before fresh medium with disulfiram was added and cell numbers were assayed 24 to 72 hours later. Synergy was studied between disulfiram and N,N'-bis(2-chloroethyl-N-nitrosourea (carmustine, 1.0–1,000 µmol/L) or cisplatin (0.1–100 µg/mL) added to medium. The effect of metal ions on disulfiram was studied with 0.2 to 10 µmol/L Cu²⁺ (provided as CuSO₄), Zn²⁺ (as ZnCl₂), Ag⁺ (as silver lactate), or Au³⁺ (as HAuCl₄·3H₂O) ions added to growth medium, buffered to physiologic pH. To provide a biologically relevant source of copper, medium was supplemented with human ceruloplasmin at doses replicating low and high normal adult serum concentrations (250 and 500 mg/mL).

To determine whether disulfiram and metal ions might directly influence transcription factor binding, 5 µmol/L disulfiram and/or 1.6 µmol/L CuSO₄ (final concentrations) were added to the binding reaction of nuclear protein obtained from control cells stimulated with 10% FBS alone in the absence of drugs or metal ions. The binding reaction was done using either 2.5 mmol/L DTT or 3.0 mmol/L glutathione as the buffer reducing agent.

In additional experiments, the effect of disulfiram was studied on expression of CRE-regulated cell cycle proteins and proteins influencing apoptosis. Confluent cells were treated with 5 µmol/L disulfiram or 5 µmol/L disulfiram + 1.6 µmol/L CuSO₄ for 2 to 48 hours. Cells were lysed and levels of the proapoptotic protein p53, the antiapoptotic protein Bcl-2, the cyclin inhibitor p21^WAF1/Cip1, and the cyclin A and cyclin B1 were measured by immunoblots, as described below.

Potential redox effects of disulfiram were studied in three sets of experiments. The importance of cellular glutathione in thiocarbamate toxicity was studied by measuring levels of intracellular glutathione after treatment with disulfiram. Confluent monolayers were treated with disulfiram (5 µmol/L), with or without 1.6 µmol/L CuSO₄, and cells were harvested 24 hours later for measurement of glutathione. To assess whether a pro-oxidant effect of disulfiram accounts for growth inhibition, we studied the effect of the potent lipophilic antioxidant probucol (1.0–1,000 µmol/L) on the antiproliferative effect of disulfiram. Finally, generation of intracellular oxidants in response to disulfiram (0.625–5 µmol/L), Cu²⁺ (0.2–1.6 µmol/L CuSO₄), or 1.25 µmol/L disulfiram + various concentrations of Cu²⁺ was measured directly, as outlined below.

Dithiocarbamates have been reported to inhibit proliferation of malignant cells by reducing cyclooxygenase-2 production of mitogenic prostaglandins (18). To explore the role of cyclooxygenase inhibition on tumor growth, cells were cultured with or without disulfiram in the presence or absence of the cyclooxygenase-1 and cyclooxygenase-2 inhibitors indomethacin (5 µg/mL) or sodium salicylate (1 mmol/L). Dithiocarbamates have also been shown to increase cytoplasmic levels of nitric oxide (NO^.) by decomposing S-nitrosoglutathione (19). NO^. could in turn induce mitochondrial permeability transition and apoptosis. To probe whether disulfiram might be inducing growth retardation by altering NO^. production, proliferation was studied with and without disulfiram in the presence and absence of the NO^. synthase inhibitor N-nitro-L-arginine added to growth medium (100 µmol/L).

Finally, several dithiocarbamate effects have been attributed to increasing the intracellular levels of cupric ions (15, 20). To further probe the role of cupric ions in mediating cytotoxicity from disulfiram, cells were cultured with or without addition of the impermeate Cu²⁺ chelator bathocuproinedisulfonic acid (BCPS, 50 or 100 µmol/L) added to medium to sequester Cu²⁺ in the extracellular compartment. Cells were also treated for 12 hours with various concentrations of disulfiram (0.625–5.0 µmol/L) and intracellular copper levels were measured as outlined below.

Electrophoretic Mobility Shift Assays
Nuclear protein was isolated and DNA binding reactions were done and quantitated as detailed previously (21) using consensus oligonucleotides 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and 3'-TCTCTAACGGACTGCAGTCTCTCGATC-5' for the CRE and 5'-AGTTGAGGGGACTTTCCCAGGC-3' and 3'-TCAACTCCCCTGAAAGGGTCCG-5' for NF-B (p50; Promega, Madison, WI). Competition experiments were done with 10x unlabeled wild-type oligonucleotide sequences for CRE or NF-B. Supershift experiments were done by incubating the binding reaction with 1 µg supershifting antibody (Santa Cruz Biotechnology, Santa Cruz, CA) prior to electrophoresis.

Measurement of Proliferation in Cell Cultures
Proliferation of cultured cells seeded into 24-well uncoated plastic plates (Costar, Corning, NY) at 50,000 cells per well was quantitated as detailed previously (22) using a colorimetric method based on metabolic reduction of the soluble yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble purple formazan by the action of mitochondrial succinyl dehydrogenase. This assay was confirmed by experiments in which cells were stained with Wright's modified Giemsa, counterstained with eosin, and counted directly at a magnification of x100 using a 1 mm² ocular grid.

Measurement of Apoptosis
Apoptosis was studied by terminal deoxynucleotidyl transferase–dependent 3'-OH fluorescein end labeling of DNA fragments using a Fluorescein-FragEL DNA fragmentation detection kit (Oncogene Research Products, Cambridge, MA), by fluorescent-labeled Annexin V staining of phosphatidylserine translocated to the membrane surface using the Annexin V-FLUOS staining kit (Roche Molecular Biochemical, Indianapolis, IN), and by visually assessing endonuclease-dependent DNA fragmentation on ethidium bromide–stained agarose gels.

DNA Cell Cycle Measurements
To study the effect of disulfiram on the DNA cell cycle, confluent cells were treated with 10% FBS + DMSO vehicle, 10% FBS and DMSO vehicle + 250 mg/mL ceruloplasmin as a source of Cu²⁺, 10% FBS + 5 µmol/L disulfiram, or 10% FBS + 5 µmol/L disulfiram and 250 mg/mL ceruloplasmin. After 24 hours, cells were trypsinized, washed twice in cold Dulbecco's PBS with 1 mmol/L EDTA and 1% bovine serum albumin, fixed for 30 minutes in ice-cold 70% ethanol, and stained by incubation for 30 minutes at 37°C in a 10 mg/mL solution of propidium iodide in Dulbecco's PBS and 1 mg/mL RNase A. DNA cell cycle measurements were made using a FACStarPlus flow cytometer (Becton Dickinson, San Jose, CA).

Immunoblots for Proteins
Immunoblots were done and quantitated as described previously (22) using primary rabbit polyclonal antibodies against human Bcl-2, p53, p21^WAF1/Cip1, cyclin A and cyclin B1, and peroxidase-labeled donkey polyclonal anti-rabbit IgG (Santa Cruz Biotechnology).

Measurement of Intracellular Copper
Cells were cultured in 12-well plastic tissue culture plates at an initial plating density of 50,000 cells per well, grown to confluence, and treated with disulfiram or DMSO vehicle, as outlined above. Medium was removed and cells were washed twice with Dulbecco's PBS. Cells were scraped into 1.0 mL of 3 N HCl/10.0% trichloroacetic acid and hydrolyzed at 70°C for 16 hours. The hydrolysate was centrifuged at 600 x g for 10 minutes to remove debris and copper was measured in the supernatant using inductively coupled plasma emission spectroscopy (model P30, Perkin-Elmer, Norwalk, CT) at wavelengths of 325.754 and 224.700 nm. To minimize metal contamination, plastic ware rather than glassware was used in these experiments, and double-distilled deionized water was used for all aqueous medium. Results are reported as nanograms of copper per culture well.

Measurement of Intracellular Generation of Reactive Oxygen Species
Generation of reactive oxygen species in response to disulfiram with or without CuSO₄ was studied using 2'7'-dichlorofluorescin diacetate (Molecular Probes, Eugene, OR) and a modification of methods reported previously (23). Cells were plated in 24-well plastic plates at 50,000 cells per well and grown to confluence. Medium was aspirated from wells and replaced with 100 µL medium containing 10 µmol/L 2'7'-dichlorofluorescin diacetate, and plates were incubated at 37°C for 30 minutes. The 2'7'-dichlorofluorescin diacetate containing medium was aspirated, cells were washed twice with medium alone, and fresh medium (100 µL) was added to wells. With the plate on the fluorescence microplate reader (HTS 7000), cells were stimulated with 25 µL medium containing 5x concentrations of disulfiram and/or CuSO₄ to provide final concentrations of 0 to 5.0 µmol/L disulfiram and/or 0 to 1.6 µmol/L CuSO₄, respectively. The relative concentration of dichlorofluorescin was measured immediately by monitoring fluorescence at 37°C using an excitation wavelength of 485 nm and emission wavelength of 535 nm.

Measurement of Intracellular Glutathione
Disulfiram (5 µmol/L), with or without 1.6 µmol/L CuSO₄, was added to cells grown to confluence on 100 x 15 mm plastic dishes, and cells were harvested 24 hours later for measurement of glutathione using the 5,5'-dithiobis(2-nitrobenzoic acid)-glutathione reductase recycling assay (24).

Synthesis of Thiocarbamate-Metal Chelates
Synthesis of diethyldithiocarbamato-metal complexes is known in the literature. Typically, aqueous solutions of a metal ion (e.g., CuCl₂) and sodium or ammonium diethyldithiocarbamate are mixed and the desired complex was separated by extraction into an organic phase such as dichloromethane. The stoichiometric ratio between metal ion and diethyldithiocarbamate salt can influence the final stoichiometry of the product. Identical complexes were synthesized starting with disulfiram rather than diethyldithiocarbamate. All diethyldithiocarbamato-metal complexes were characterized by a single crystal X-ray diffraction and structures were reported in the Cambridge Crystallographic Database. The Ag⁺ and Zn²⁺ diethyldithiocarbamate complexes were found to be polymeric. As an example, the results for the Au³⁺ complex are detailed.

Study of Antitumor Activity of Disulfiram and Zinc Supplementation In vivo
Adult female CB17-SCID mice (Harlan, Indianapolis, IN) were housed in a protected laminar flow facility with access to water and either a standard diet containing 87 ppm zinc or a zinc-supplemented diet (Harlan) containing 1,000 ppm Zn²⁺ as zinc acetate. Mice were injected s.c. in the right groin with 5 x 10⁶ cells from a highly aggressive malignant melanoma obtained from a Carolinas Medical Center patient. The frozen tumor was passaged twice in SCID mice to adapt it to in vivo growth before use in these experiments. On the day of tumor injection, all mice began daily administration of drug. Drug was given in a total volume of 0.2 mL by gastric gavage via smooth Teflon-tipped needles inserted transorally into the stomach. Four groups were studied: tumor control (n = 10; 0.2 mL olive oil daily; zinc diet of 87 ppm); zinc-supplemented control (n = 10; 0.2 mL olive oil daily; zinc diet of 1,000 ppm); disulfiram (n = 10; 200 mg/kg/d disulfiram in 0.2 mL olive oil; zinc diet of 87 ppm); and zinc-supplemented diet + disulfiram (n = 10; 200 mg/kg/d disulfiram in 0.2 mL olive oil; zinc diet of 1,000 ppm). Mice were examined daily, the tumor was measured in two dimensions, and the tumor volume was estimated using the formula for an elipse. When estimated tumor volume approached 500 mm³ within any animal, all mice were euthanized. This protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Carolinas Medical Center. Tumors were excised, weighed, fixed in formalin, sectioned, and stained with H&E or immunostained for factor VIII. Slides were coded and examined by a blinded observer who identified vessels as deposits of red cells. For each slide, the number of vessels was counted in four different fields representative of the tumor. The average number of vessels per field was averaged per biopsy specimen and used to evaluate tumor vascularity.

	Results

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Disulfiram Inhibits Melanoma Proliferation in a Metal-Dependent Fashion
In concentrations reported in humans (17), disulfiram inhibited melanoma proliferation in vitro in a dose-dependent fashion, with near complete growth inhibition at 5 µmol/L (P < 0.001; Fig. 1), and increased the number of apoptotic cells in culture (Fig. 2). Within the same concentration ranges, disulfiram likewise inhibited growth of other malignant cells (IC₅₀: 2.5 µmol/L for CRL1585 melanoma, 2.5 µmol/L for PC-3 prostate adenocarcinoma; 0.625 µmol/L for H520 squamous cell lung cancer, 1.25 µmol/L for H596 adenosquamous cell lung cancer, and 0.625 µmol/L for MDA-MB-453 breast carcinoma). Disulfiram also augmented the antiproliferative effect of cisplatin or carmustine on melanoma cells (4 ± 1% inhibition of growth at 24 hours with 100 ng/mL cisplatin alone versus 17 ± 3% inhibition with cisplatin and 2.5 µmol/L disulfiram; P < 0.05; 46 ± 7% stimulation of growth at 24 hours with 10 µmol/L carmustine alone versus 75 ± 6% inhibition of growth with carmustine and 0.6 µmol/L disulfiram; P < 0.001), suggesting that it might reduce resistance to chemotherapy, as reported recently (3, 4).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Disulfiram inhibits proliferation of CRL1619 human melanoma cells. Cells stimulated with 10% FBS were plated at a density of 50,000 cells per well, and DMSO vehicle (5 µL/mL) or disulfiram was added to wells at the indicated concentrations. After 24, 48, 72, or 96 hours, proliferation was quantitated by assessing the cell number–dependent reduction of the soluble yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, measured as the absorbance at 540 nm (A540). Two-way ANOVA shows P < 0.001 for group, time, and group-time interaction. *, P < 0.01, at similar culture time versus DMSO vehicle. +, P < 0.001, at similar culture time point versus DMSO vehicle.

View larger version (71K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Disulfiram induces apoptosis in melanoma measured by 3'-OH fluorescein end labeling of DNA fragments. A, CRL1619 melanoma cells treated with DMSO vehicle and 3'-OH fluorescein end labeled. B, CRL1619 melanoma cells treated with disulfiram (5 µmol/L) and 3'-OH fluorescein end labeled. Cells were grown to confluence on 35 mm Petri dishes or on glass slides and treated for 12 hours with disulfiram or DMSO as vehicle. Apoptosis was studied by terminal deoxynucleotidyl transferase–dependent 3'-OH fluorescein end labeling of DNA fragments using a Fluorescein-FragEL DNA fragmentation detection kit.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Complexation of Cu2+ reduces the antiproliferative activity of disulfiram. CRL1619 human melanoma cells stimulated with 10% FBS were plated at a density of 50,000 cells per well and treated with the indicated concentrations of DMSO vehicle (5 µL/mL) or disulfiram (DS, 1.25 µmol/L) with or without the cell impermeate Cu2+ chelator BCPS to complex Cu2+ and trap it in the extracellular medium. BCPS reversed growth the antiproliferative activity of disulfiram in a dose-dependent manner. % Growth inhibition at 48 hours: 48 ± 2% with disulfiram (1.25 µmol/L); 11 ± 2% with disulfiram + BCPS (100 µmol/L); 3 ± 3% with BCPS alone (100 µmol/L). *, P < 0.001 versus untreated. +, P < 0.001 versus disulfiram.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Supplementation of growth medium with Cu2+ or Zn2+ enhances the antiproliferative activity of disulfiram. CRL1619 human melanoma cells stimulated with 10% FBS were plated at a density of 50,000 cells per well and treated with the indicated concentrations of DMSO vehicle (5 µL/mL) or disulfiram (DS, 0.625 µmol/L) and concentrations of CuSO4, ZnCl2, or metal ions + DMSO or disulfiram. After another 24 hours, proliferation was quantitated as in Fig. 1. Addition of CuSO4 (0.2 µmol/L) to medium converts disulfiram (0.625 µmol/L) from IC50 to IC100 of drug. *, P < 0.01 and +, P < 0.001 compared with no CuSO4 or ZnCl2.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Disulfiram combined with Cu2+ induces S-phase cell cycle arrest in CRL1619 melanoma cells and apoptosis. Unsynchronized CRL1619 melanoma cells were grown in the presence of DMSO vehicle, disulfiram (5 µmol/L), or disulfiram (5 µmol/L) + ceruloplasmin (250 mg/mL) as a source of Cu2+. After 24 hours, cells were harvested and flow cytometric cell cycle analysis was done. The proportion of nuclei in each phase of the cell cycle was determined with ModFit DNA analysis software. Cells in G0-G1 and G2-M are in red, cells in S phase are hatched, and apoptotic cells are in blue. Disulfiram increases the portion of cells in S phase. The combination of disulfiram and ceruloplasmin further increases the number of cells in S phase, prevents progression into the G2-M cell cycle, and induces apoptosis. Six percent of cells are apoptotic, over two thirds of cells are in S phase, and none are in G2-M.

NF-B inhibition by thiocarbamates has been associated recently with facilitation of intracellular zinc transport (27), and zinc supplementation increases the toxicity of thiocarbamates for vascular smooth muscle cells (15). Zinc substantially enhanced the antiproliferative potential of disulfiram against melanoma cells (Fig. 4). Dithiocarbamates can also chelate other metals (28), and gold and silver salts also enhanced the antiproliferative activity of disulfiram (growth inhibition: 45 ± 5% with 0.15 µmol/L disulfiram; 0 ± 0% with 5 µmol/L silver lactate alone; 71 ± 7% with disulfiram + silver lactate, P < 0.001; 0 ± 0% with 5 µmol/L gold tetrachloride alone; 99 ± 1% with disulfiram + gold tetrachloride, P < 0.001). In light of these findings, we synthesized chelates of disulfiram with Au³⁺, Cu²⁺, Zn²⁺, Ag⁺, Ga³⁺, or Fe³⁺. X-ray crystallography confirmed the structures as diethylthiocarbamato complexes of respective metal ions (complexes with Au³⁺ shown in Fig. 6; others are in online supplemental data). To confirm that the proximate reactive dithiocarbamate structure important for promoting cellular mixed disulfide formation is the thiolate anion generated from fully reduced dithiocarbamates by metals, we compared the antiproliferative activity of the thiolate sodium diethyldithiocarbamate alone or in the presence of a low concentration of DTT to promote formation of the fully reduced thioacid. Sodium diethyldithiocarbamate alone (1 µmol/L) decreased melanoma proliferation by 92 ± 2% after 48 hours (P < 0.001), but growth was inhibited by only 24 ± 3% (P < 0.001) with simultaneous addition of a concentration of DTT (100 µmol/L), which does not affect proliferation of melanoma cells by itself (0 ± 0 %). Thus, the function of metals may be to facilitate formation of the dithiocarbamate anion, which might condense into mixed disulfides with critical protein sulfhydryls (11–13).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 6. X-ray crystallographic structure of Au3+ diethyldithiocarbamate. Complexes were generated as outlined in Materials and Methods. A Nonius Kappa-CCD diffractometer was used to collect X-ray diffraction data. The crystal diffracted well and a data set was collected to 27.5° in using Mo K radiation ( = 0.71073 Å). Least squares refinement on the cell variables revealed an orthorhombic unit cell with a = 11.5167(5), b = 7.2472(2), c = 12.9350(7) Å, and a volume of 1,079.6(1) Å3. Examination of the systematic absences showed the space group to be Pnma (#62). The structure was solved by direct methods using SIR92 and revealed the crystal to be dichloro(diethylthiocarbamato)gold(III). The structure was confirmed by the successful solution and refinement of the 83 independent variables for the 893 reflections [I > 3(I)] to R factors of 3.3% and 3.2%, with an ESD of 1.499. The gold complex is a square planar coordination complex in which the Au and the four coordinated atoms sit on a mirror at x, 0.25, z. The organic ligand was found to be disordered with the diethylamine ligand occupying two sites related to each other through the mirror plane. This compound inhibited CRL1619 melanoma growth by 81 ± 1% after exposure for 48 hours to a concentration as low as 0.25 µmol/L.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Disulfiram and metals inhibit transcription factor binding to the CRE. A, CRL1619 melanoma cells exhibit constitutive DNA binding activity to the CRE (lane 1). CRL1619 melanoma cells were grown to 60% confluence on 100 x 15 mm plastic Petri dishes, nuclear protein was harvested, and electrophoretic mobility shift assays were done using the consensus oligonucleotides 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and 3'-TCTCTAACGGACTGCAGTCTCTCGATC-5' for the CRE, end labeled by phosphorylation with [-32P]ATP and T4 polynucleotide kinase. CRE complexes (I and II) are labeled. Supershift experiments done by incubating the binding reaction with antibody (1 µg) before addition of labeled probe show that complex II contains ATF2 (lane 5), whereas complex I is composed primarily of CREB1 (lane 2), with some ATF1 (lane 4). Competition experiments in lanes 6–8 show specificity of the DNA binding reaction: untreated (lane 6); with 10x unlabeled CRE probe added to binding reaction (lane 7); with 10x unlabeled NF-B probe added to binding reaction (lane 8). B, treatment of melanoma cells with disulfiram and Cu2+ inhibits transcription factor binding to CRE. CRL1619 melanoma cells were grown to 80% confluence, nuclear protein was harvested, and electrophoretic mobility shift assays were done for the CRE. Top, treatment of cultures for 6, 12, or 24 hours with the combination of disulfiram (5 µmol/L) and CuSO4 (Cu, 1.6 µmol/L) substantially interrupted transcription factor binding to CRE. The ATF2 containing complex II has proven to be the more sensitive to inhibition. Bottom, electrophoretic mobility shift assays were done using nuclear protein from replicate experiments (n = 4) in which near confluent cells were treated for 8 hours and densitometry was done on the ATF2 containing complex II. The combination of disulfiram + Cu2+ reduced DNA binding by half. *, P < 0.05 compared with other treatments. C, the inhibitory effects of disulfiram or disulfiram + Cu2+ on transcription factor binding are potentiated in the presence of glutathione (GSH). Electrophoretic mobility shift assays were done with addition of disulfiram or disulfiram + CuSO4 (Cu, 1.6 µmol/L) directly to the binding reaction of nuclear protein and oligonucleotides. Disulfiram alone reduced DNA binding to CRE in the upper ATF2 containing complex II (lane 3). This was magnified when disulfiram was combined with Cu2+ ions (lane 5). Results are consistent with modest disruption of ATF2 binding to CRE from formation of mixed disulfides between disulfiram and cysteines in the DNA binding region and greater disruption when Cu2+ is present to enhance mixed disulfide formation. However, reduction in CRE binding was much more pronounced when the binding reaction was done with GSH instead of DTT as the reducing agent (lane 7 for disulfiram, lane 9 for disulfiram + Cu2+). Inhibition of ATF2 containing complex II binding to CRE by disulfiram and Cu2+ in the presence of GSH was reversed by simultaneous addition of the potent uncharged reducing agent DTT (lane 10).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Disulfiram and Cu2+ reduce expression of the cell cycle protein cyclin A. Although disulfiram or Cu2+ alone had little effect, treatment with the combination of disulfiram + Cu2+ reduced expression of cyclin A by 24 hours, which would be expected to produce a site of cell cycle arrest consistent with that seen in Fig. 3. CRL1619 melanoma cells were plated at equal densities, grown to 80% confluence, and, in replicate experiments (n = 4 each), treated with DMSO vehicle (lanes 1–4), disulfiram (5 µmol/L, lanes 5–8), CuSO4 (Cu, 1.6 µmol/L, lanes 9–12), or the combination of disulfiram and CuSO4 (lanes 13–16). After 24 hours, immunoblots were done to assay for cyclin A. Bottom, quantitation of experiments by densitometry. *, P < 0.05 compared with all other treatments.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 9. Disulfiram + zinc supplementation decreases malignant melanoma growth in mice. Adult female CB17-SCID mice were injected s.c. in the right groin with 5 x 106 cells from a highly aggressive malignant human melanoma. Mice were fed either a standard diet containing zinc (87 ppm) or a zinc-supplemented diet containing Zn2+ (1,000 ppm) as zinc acetate. On the day of tumor injection, all mice began daily oral gavage of olive oil (0.2 mL) as a control or olive oil (0.2 mL) containing the indicated drug. Four groups were studied: tumor control (n = 10; 0.2 mL olive oil daily; standard zinc diet of 87 ppm); zinc-supplemented control (Zn; n = 10; 0.2 mL olive oil daily; zinc diet of 1,000 ppm); disulfiram (n = 10; 200 mg/kg/d disulfiram in 0.2 mL olive oil; zinc diet of 87 ppm); and zinc-supplemented diet + disulfiram (n = 10; 200 mg/kg/d disulfiram in 0.2 mL olive oil; zinc diet of 1,000 ppm). When estimated tumor volume in controls approached 500 mm3, all mice were euthanized and tumors were excised and weighed. Zn2+ supplementation alone had no affect on tumor growth, but disulfiram alone and disulfiram + Zn2+ supplementation all significantly inhibited tumor growth. *, P < 0.05 versus tumors in controls or Zn. +, P < 0.001 versus tumors in controls or Zn.

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   Figure 10. Disulfiram and zinc gluconate reduce hepatic tumor volume in a patient with metastatic ocular melanoma. Computed axial tomograms (top) and positron emission spectrographs (bottom) of a 64-year-old woman with stage IV ocular melanoma metastatic to the liver. Before treatment, she had a 5.5 cm central liver metastasis (white arrows). After 3 months of treatment with disulfiram (500 mg/d) and zinc gluconate (50 mg thrice daily), the hepatic metastasis had decreased in volume by >50% in both scans (white arrows). After continuing treatment with disulfiram (250 mg/d) and the same dose of zinc gluconate, the lesion remained stable in size at 10 and 14 months (white arrows). She continues to be clinically well and free of drug side effects on disulfiram and zinc gluconate. After 53 continuous months of treatment with this regimen, she has experienced no quantifiable malignant progression. A follow-up abdominal computed tomography scan after 42 months of therapy shows that the hepatic tumor burden has remained small.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

One potential mechanism explaining the antiproliferative activity of disulfiram is inhibition of transcription factor DNA binding, which we have shown to be sensitive to disruption by disulfiram in a manner potentiated by heavy metal ions. Melanomas are dependent for growth and metastasis on activation of distinct transcription factors, such as ATF/CREB (36–38) and NF-B (41). ATF/CREB transcription factors, in particular, play prominent roles in cell proliferation and survival (39, 42), and others have suggested molecular disruption of ATF/CREB-mediated transcription for controlling melanoma growth (37, 38). In addition to having an important cell cycle regulatory function, NF-B induces expression of several antiapoptotic genes such as TRAF, c-IAP, IXAP, A1/Bft-1, and IEX-1L (43). Malignancies with constitutive activation of NF-B such as melanoma (41) or those in which NF-B is induced by radiation or chemotherapy (43) are resistant to the proapoptotic effects of most current cancer therapies, and strategies that inhibit NF-B sensitize tumors to chemotherapeutic drugs (43, 44). Similar to our findings that disulfiram increased susceptibility of melanomas to cisplatin or carmustine, others have shown recently that disulfiram increases the susceptibility of human coloretal cancer cell lines to 5-fluorouracil by inhibiting NF-B (4). In this study, Wang et al. (4) found that disulfiram did not prevent degradation of its inhibitor IB but did inhibit DNA binding of NF-B in vitro. These results agree with our studies showing direct in vitro inhibition of ATF/CREB DNA binding in nuclear extracts from melanoma cells in a manner enhanced by cupric ions and glutathione and support our hypothesis that disulfiram disrupts DNA transcription factor binding by forming mixed disulfides that induce S-glutathionylation of critical cysteines in the transcription factor DNA binding region. This mechanism has been also suggested for how NO^. inhibits the activity of c-Jun, Fos, ATF/CREB, Myb, and Rel/NF-B family transcription factors (45).

Other important cellular targets for disulfiram also exist, including Cys⁵⁶ of the adenine nucleotide translocator that enforces permeability transition pore complex opening in mitochondria and release of cytochrome c and apoptosis inducing factor (14). Mitochondrial permeability transition induced by disulfiram (13) would also be expected to sensitize resistant malignancies to chemotherapeutic treatment (46). However, all of these critical thiols would be expected to behave similarly, with formation of mixed disulfides and perhaps S-glutathionylation catalyzed by disulfiram in a fashion enhanced by presence of copper or other similar metal ions. Disulfiram has long been known to form mixed disulfides with cysteine thiols (47). Furthermore, the drug is absorbed as its bis(diethyldithiocarbamato)copper(II) complex (2), further suggesting that a heavy metal-thiolate chelate may be the active drug facilitating mixed disulfide formation. Additional support for a thiolate-metal complex as the proximate effector is provided by results showing that the copper chelator BCPS reduces and medium supplementation with Cu²⁺ or other heavy metal ions facilitates melanoma growth inhibition by disulfiram, that the reducing agent DTT reverses growth inhibition from the thiolate diethyldithiocarbamate, and that X-ray crystallography confirms the structure of dithiocarbamate-metal ion complexes. A growing literature points to cysteine mixed disulfide formation followed by glutathione conjugation as a unique mechanism for regulating activity of cellular proteins (11, 14, 45, 46, 48), including the process by which pyrollidine dithiocarbamate inhibits NF-B (49). Thus, dithiocarbamate-metal ion complexes might inhibit cellular growth and sensitize tumors to chemotherapy by cysteine modification at multiple sites.

Employed at the currently approved dose of 250 mg/d, disulfiram seems safe and is readily available for application to several novel treatment strategies for malignancies. First, as originally reported by Schreck et al. (50), disulfiram inhibits activation of NF-B and can could be used to block activation of this transcription factor by conventional cancer chemotherapy, thereby sensitizing tumors to the effects of currently available drugs (4). In this role, disulfiram would be aided by its ability to block the P-glycoprotein extrusion pump used to extrude chemotherapeutic agents from malignant cells (3), another mechanism for tumor drug resistance. However, the systemic levels of disulfiram (10 µmol/L) that might be required to replicate in vivo the NF-B inhibition and chemotherapy sensitization reported by Wang et al. (4) in vitro are achievable in vivo only with doses of disulfiram (500 mg) that produce considerable nausea and other systemic side effects (2). Similar to the experience with our melanoma patient, concurrent supplementation with metal ions might provide NF-B inhibition using much lower doses of disulfiram. Second, as suggested by Marikovsky et al. (9) and confirmed by Shiah et al. (7), disulfiram might be potentially investigated as an angiogenesis inhibitor. Although its antiangiogenic effect has been postulated recently as complexation by disulfiram of zinc ions from matrix metalloproteinases (7), our results from melanomas transplanted into SCID mice showing fewer vessels in disulfiram + zinc–treated animals (2.0 ± 0.7 vessels per field) compared with those treated with disulfiram alone (2.5 ± 0.7 vessels per field) are not consistent with this mechanism. Furthermore, the disulfiram concentration required to effectively inhibit angiogenesis (5–10 µmol/L) in reported studies (7) can likely be achieved only with relatively high doses in man (2). Concurrent supplementation with metal ions might also lower the effective antiangiogenic dose of disulfiram to one better tolerated. Finally, as shown by our experience with malignant melanoma in SCID mice and in the reported patient, disulfiram might offer potential in some tumors as complexing agents for delivery of antiproliferative metal ions to tumor cells. Currently, cisplatin is the only approved metal-based chemotherapy. However, zinc and arsenic are antiproliferative for tumors (16, 51, 52) and zinc and other metals inhibit NF-B and activator protein-1 (53–56). Therefore, as a potential solution for several current therapeutic difficulties facing clinical oncology, disulfiram may present a novel and readily applicable drug to effect metal-thiolate induced mixed disulfide modification of protein thiols critical for tumor cell drug resistance and growth. In light of our findings and those of others (3–9), we suggest that randomized clinical trials of this strategy are warranted.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: This work is the subject of U.S. patents 6,548,540; 6,589,987; and 6,706,759, which are owned by the Charlotte-Mecklenberg Hospital Authority.

Received 3/29/04; revised 5/14/04; accepted 6/18/04.

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