J Nucl Med. 2013 Jun;54(6):953-60. doi: 10.2967/jnumed.112.113324.

The role of copper in disulfiram-induced toxicity and radiosensitization of cancer cells.

Rae C, Tesson M, Babich JW, Boyd M, Sorensen A, Mairs RJ.

Radiation Oncology, Institute of Cancer Sciences, Cancer Research United Kingdom Beatson Laboratories, Glasgow, University of Glasgow, Glasgow, United Kingdom.

 

Abstract

Disulfiram has been used for several decades in the treatment of alcoholism.  It now shows promise as an anticancer drug and radiosensitizer.  Proposed mechanisms of action include the induction of oxidative stress and inhibition of proteasome activity.  Our purpose was to determine the potential of disulfiram to enhance the antitumor efficacy of external-beam g-irradiation and 131I-metaiodobenzylguanidine (131I-MIBG), a radiopharmaceutical used for the therapy of neuroendocrine tumors.  Methods: The role of copper in disulfiram-induced toxicity was investigated by clonogenic assay after treatment of human SK-N-BE(2c) neuroblastoma and UVW/noradrenaline transporter (NAT) glioma cells.  The synergistic interaction between disulfiram and radiotherapy was evaluated by combination-index analysis.  Tumor growth delay was determined in vitro using multicellular tumor spheroids and in vivo using human tumor xenografts in athymic mice.  Results: Escalating disulfiram dosage caused a biphasic reduction in the surviving fraction of clonogens.  Clonogenic cell kill after treatment with disulfiram concentrations less than 4 mM was copper-dependent, whereas cytotoxicity at concentrations greater than 10 mM was caused by oxidative stress.  The cytotoxic effect of disulfiram was maximal when administered with equimolar copper.  Likewise, disulfiram radiosensitization of tumor cells was copper-dependent.  Furthermore, disulfiram treatment enhanced the toxicity of 131I-MIBG to spheroids and xenografts expressing the noradrenaline transporter.  Conclusion: The results demonstrate that the cytotoxicity of disulfiram was copper-dependent; molar excess of disulfiram relative to copper resulted in attenuation of disulfiram-mediated cytotoxicity, copper was required for the radiosensitizing activity of Disulfiram, and copper-complexed disulfiram enhanced the efficacy not only of external beam radiation but also of targeted radionuclide therapy in the form of 131I-MIBG.  Therefore, disulfiram may have anticancer potential in combination with radiotherapy.

KEYWORDS: 131I-metaiodobenzylguanidine, copper, disulfiram, neuroblastoma radiosensitizer

PMID 23616582

 

SUPPLEMENT

Disulfiram is an agent which has been used clinically for several decades.  Therefore it has a well-characterised pharmacokinetic profile and is considered to have negligible, reversible side-effects.  Disulfiram has a range of activities which suggest its potential as an anti-cancer agent.  It inhibits proteasome activity and NF-kB activation which are anti-proliferative and pro-apoptotic, respectively.  It also chelates metal ions, including Zn2+ and Cu2+, preventing their use as cofactors in enzymes involved in angiogenesis and redox regulation.  Many of these effects are pertinent to the treatment of cancer cells which have high proteasome and NF-kB activities as well as high levels of oxidative stress and intracellular copper due to elevated proliferative rate and metabolic activity.  Disulfiram can also enhance the potency of chemotherapeutic agents and radiation and decrease resistance to these therapies.

The childhood cancer, neuroblastoma, is devastating.  It accounts for approximately 15% of all childhood cancer deaths.  Long-term survival of high-risk neuroblastoma is less than 40%.  Increasing the copper content of neuroblastoma cells results in DNA damage, cell cycle arrest and apoptosis.  Intracellular transport of copper is enhanced by disulfiram and disulfiram analogs.  It is noteworthy that disulfiram administered to children induced no side-effect.

Neuroblastoma cells express high levels of the noradrenaline transporter (NAT), so are susceptible to targeted radiotherapy with the noradrenaline analog meta-iodobenzylguanidine (131I-MIBG).  Previously we demonstrated increased efficacy of 131I-MIBG therapy using radiosensitizing drugs.  Having hypothesized that disulfiram would also sensitize neuroblastoma cells to radiotherapy, this was subsequently demonstrated in vitro and in vivo and the importance of copper concentration was established.

We tested the ability of disulfiram, as a single agent or in combination with radiation, to kill NAT-expressing tumor cells.  At clinically achievable concentrations (< 2 mM), disulfiram induced cell kill, decreased clonogenic survival and synergistically enhanced the anti-tumor effect of external beam radiation or 131I-MIBG.  Both the cytotoxic and radiosensitizing effects of disulfiram were only achieved in the presence of copper, and were prevented by addition of a competing copper chelator.

The importance of the study is twofold.  It demonstrates the potential to combine targeted radiotherapy of neuroblastoma with disulfiram – an inexpensive and safe drug, suitable for use in paediatric patients.  It also shows the importance of copper concentration to the efficacy of disulfiram. One of the principal mechanisms underlying disulfiram’s toxicity is the formation of an active disulfiram-copper complex, allowing some degree of selectivity for cancer cells which contain elevated levels of copper, as shown in the figure below.

Colin Rae fig1

The administration of disulfiram with copper supplementation may elevate the copper content of malignant cells to cytocidal levels, or maintain pre-existing high copper concentration in malignant cells.  This therapeutic strategy could be applicable to various tumor types.

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