Aldehyde-dehydrogenase (ALDH1); gasdermin D (GSDMD)

Disulfiram-copper complex efficiently reduces proteasome activity in cultured breast cancer MDA-MB-231 and MCF10DCIS.com cells prior to causing apoptotic cancer cell death, but not in normal immortalized MCF-10A cells [1]. A commercially utilized anti-alcoholic medication called disulfiram (DS) significantly and dose-dependently suppresses both constitutive and 5-FU-induced NF-activation. DisuLfiram has little effect on 5-FU-induced IkappaBalpha degradation, although it does reduce NF-kappaB nuclear translocation and DNA binding activity. Disulfiram synergistically increased 5-FU's cytotoxicity on both DLD-1 and RKO (WT) cell lines while also markedly enhancing 5-FU's apoptotic effect on those lines. Additionally, 5-FU chemoresistance in the 5-FU-resistant cell line H630 (5-FU) is successfully eliminated in vitro by DisuLfiram [2]. CuCl2 greatly increased DSF-induced cell death to less than 10% of the control, while oseltamivir decreased the number of viable cells [3]. At lower concentrations than disulfiram alone, disulfiram in combination with Cu2+ or Zn2+ in melanoma cells decreases cyclin A expression and inhibits in vitro proliferation [4]. The combination of DisuLfiram (0.1 nM-10 μM; 72 h) and Cu2+ increases its cytotoxicity against ovarian cancer cell lines [1].

Disulfiram dramatically reduced tumor development (74%) in mice containing MDA-MB-231 tumor xenografts; this was linked to proteasome inhibition and apoptosis induction [1]. In tumor tissues, ubiquitinated proteins and the natural proteasome substrates p27 and Bax accumulate, and these indicators of proteasome inhibition are used to gauge the extent of proteasome suppression. Caspases being activated and apoptotic cell nuclei forming are signs of apoptosis [1]. Disulfiram inhibits the nuclear factor-kappaB transcription factor, limits the P-glycoprotein extrusion pump, decreases angiogenesis, makes tumors more susceptible to chemotherapy, and stops tumor growth in mice [4]. When melanoma tumors are transplanted into severe combined immunodeficient mice, disulfiram reduces their development and angiogenesis; Zn2+ supplementation amplifies these effects [4].

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In addition, when administered to mice bearing MDA-MB-231 tumor xenografts, DSF significantly inhibited the tumor growth (by 74%), associated with in vivo proteasome inhibition (as measured by decreased levels of tumor tissue proteasome activity and accumulation of ubiquitinated proteins and natural proteasome substrates p27 and Bax) and apoptosis induction (as shown by caspase activation and apoptotic nuclei formation). Our study shows that inhibition of the proteasomal activity can be achieved by targeting tumor cellular copper with the nontoxic compound DSF, resulting in selective apoptosis induction within tumor cells.[1] 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[4].

Electrophoretic mobility-shift assay [2]
The extraction of nuclear and cytoplasmic protein and electrophoretic mobility-shift assay (EMSA) analysis were carried out as described previously.32 To test the effect of Disulfiram (DS) and 5-FU on the DNA binding activity of NF-κB, the cells were incubated with the compounds at indicated concentrations and time length. The nuclear protein concentration was determined using DC Protein Assay. Equal amount of the nuclear extract (5 μg) was incubated with 1 μg poly(dIdC) in binding buffer [50 mM Tris (pH 7.6), 250 mM KCl, 25 mM DTT, 5mM EDTA and 25% glycerol] for 10 min at room temperature. Approximately 20,000 cpm of 32P-labeled NF-κB DNA probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was added and the reaction was incubated at room temperature for 20 min. The double-stranded oligonucleotides containing AP-1, AP-2, SP-1, Oct-1, CREB or TFIID consensus sequences were purchased from xxx. The EMSA for these transcription factors were carried out using the same conditions as those for NF-κB. For supershift assay and binding specificity determination, 5 μg of nuclear extract from RKOWT cells treated with 5-FU was incubated with 0.4 μg of antibody (p65, p50, p52, C-Rel, Rel-B or VEGF or 20 × wild-type or mutant (5′-AGTTGATATTACTTTTATAGGC-3′) unlabeled NF-κB probe for 30 min before EMSA analysis. The complexes were separated on a 6% polyacrylamide gel and exposed for autoradiography. The band intensity was analysed using Molecular Analyst software.

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ALDH activity was inhibited in ovarian cancer stem cells (the proportion of ALDH+ cells was reduced from 21.7% to 0.391%, 8.4% to 0%, 6.88% to 0.05% in cell lines IGROV1, SKOV3, and SKOV3IP1, respectively). Disulfiram (DS) with or without the cofactor copper (Cu2+) exhibited cytotoxicity dose- and time-dependent and enhanced cisplatin-induced apoptosis. Disulfiram (DS) + Cu2+ increased intracellular ROS levels triggering apoptosis of ovarian cancer stem cells (CSC). Significantly more colony and spheroid formation was observed in ALDH+ compared with ALDH- cells (P < 0.01). Moreover, ALDH+ cells were more resistant to cisplatin treatment compared with ALDH-cells (P < 0.05) and also exhibited a lower basal level of ROS. However, no significant difference in ROS accumulation nor in cellular viability was observed in ALDH + cells in comparison to ALDH- cells after pre-treatment with Disulfiram (DS) (0.08 μM) [6].

The effect of disulfiram (0.15-5.0 μM) or sodium diethyldithiocarbamate (1.0 μM) on proliferation of malignant cell lines is studied in cultures stimulated with 10% FBS. Cell numbers are quantitated 24 to 72 hours later, as outlined below. In some experiments, disulfiram is added immediately after cells are plated. In other experiments, cells are plated and allowed to grow for 24 to 72 hours before fresh medium with disulfiram is added and cell numbers are assayed 24 to 72 hours later. Synergy is studied between disulfiram and N,N′-bis(2-chloroethyl-N-nitrosourea (carmustine, 1.0-1,000 μM) or cisplatin (0.1-100 μg/mL) added to medium. The effect of metal ions on disulfiram is studied with 0.2 to 10 μM 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 is supplemented with human ceruloplasmin at doses replicating low and high normal adult serum concentrations (250 and 500 mg/mL) [4].

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Western blot analysis [2]
The nuclear or cytoplasmic protein (60 μg/lane) was electrophoresed through a 10% SDS-PAGE and transferred to a PVDF membrane. The blots were stained with primary (NF-κB p50, 1:500; NF-κB p65 and IκBα, 1:100) and secondary antibodies respectively for 1 hr at room temperature. The loading quantity of protein was verified by staining the same membranes with anti-α-tubulin antibody for cytoplasmic protein or SimpleBlue SafeStain for nuclear protein. The signal was detected using an ECL Western blotting detection kit. The intensity of the bands was analysed using Molecular Analyst software.

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Deoxynucleotidyl transferase-mediated dUTP nick end labeling assay [2]
The cells were grown on SuperCell culture slides until 70% confluent and exposed to variable concentrations of 5-FU, Disulfiram (DS) or 5-FU plus Disulfiram (DS) for 24 hr. After 1 hr fixation in 4% paraformaldehyde, the cells were stained according to the manufacturer's instructions. Briefly, the cells were fixed with a freshly prepared paraformaldehyde solution (4% in PBS, pH 7.4) for 1 hr at room temperature and then permeabilised with 0.1% Triton X-100/0.1% sodium citrate for 2 min on ice. After incubation with deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction mixture and Converter-AP, the apoptotic cells were detected using a chromogenic substrate (NBT/BCIP) and examined under light microscope. Each experiment was carried out in duplicate.

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Detection of internucleosomal DNA cleavage [2]
DNA fragmentation was detected by a modified method described previously.33 Seventy percent confluent cells in 25 cm2 flasks were exposed to 5-FU, Disulfiram (DS) or 5-FU + Disulfiram (DS) for 24 hr and collected by trypsinisation. Genomic DNA samples (2 μg/lane) were electrophorised through a 1.5% agarose slab gel containing 0.5 μg/ml ethidium bromide and visualised under UV illumination.

Human breast tumor xenograft experiments. Five-week-old female athymic nude mice were purchased from Taconic Research Animal Services and housed under pathogen-free conditions according to Wayne State University animal care guidelines. The protocols of animal experiments were reviewed and approved by Institutional Laboratory Animal Care and Use Committee of Wayne State University. MDA-MB-231 cells (5 × 106) were injected s.c. at one flank of the mice. Tumor size was measured every other day. Tumor volume (V) was determined by the equation: V = (L × W2) × 0.5, where L is the length and W is the width of the tumor. When xenografts reached volumes of ∼200 mm3, the mice bearing tumors were randomly assigned to control or DSF groups (n = 10), and administered daily using either solvent control (PBS/cremophor/DMSO/ethanol, 7.5:1.5:0.5:0.5) or 50 mg/kg/d DSF. When the control tumors reached ∼1,600 mm3 (on day 29), the experiment was terminated and the mice were sacrificed. The tumors were removed and photographed and the tumor tissues were then used for multiple assays to measure proteasome inhibition and apoptotic cell death.[1]

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50 mg/kg/d; p.o.; for 29 days

Mice bearing MDA-MB-231 tumor xenografts

Absorption, Distribution and Excretion
Disulfiram is absorbed slowly from the gastrointestinal tract (80 to 90% of oral dose).
Absorption /of disulfiram is/ slow. Eighty to ninety percent of an oral dose is absorbed. /Its/ biotransformation /is predominately/ hepatic /and/ a single dose will begin to affect ethanol metabolism within 1 to 2 hours.
Disulfiram is ... completely absorbed from the human GI tract. However, a period of 12 hr is required for its full action, perhaps because, being highly sol in lipid, it is initially localized in fat. Elimination is relatively slow, and about 1/5 still remains in body at end of a week. The greater part of the absorbed drug is ... excreted in the urine as the sulfate, partly free and partly esterified.
After a single oral dose of 50, 100, 200, or 400 mg/kg, disulfiram was found in dose-dependent quantities in blood, liver, kidney, spleen, brain, muscle, and peri-epididymal adipose tissue of rats. After a 2-mo treatment, accumulation was not dose-dependent, suggesting a saturation point for various organs.
The human plasma protein binding characteristics of disulfiram and its therapeutically active metabolite, diethylthiocarbamic acid methyl ester were investigated. Both compounds were bound principally to albumin over the ranges 200-800 and 345-2756 nM, respectively. The average number of binding sites was approximately one for both substances, whereas the average association constants were 7.1X10+4 and 6.1X10+3/M, respectively.
For more Absorption, Distribution and Excretion (Complete) data for DISULFIRAM (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Hepatic.
Disulfiram is slowly metabolized in the liver to diethyldithiocarbamate, diethylamine, and carbon disulfide. Six hr after oral administration of the drug, one third of plasma disulfiram is in the form of diethyldithiocarbamate.
In rats the following metabolites of disulfiram were found: diethyldithiocarbamate; diethyldithiocarbamate s-glucuronide; inorganic sulfate; diethylamine and carbon disulfide. A small amount of S was bound to proteins as mixed disulfides. ... Metabolism of disulfiram in man is similar to that in animals.
Recently, n,n-diethylthiocarbamoyl-1-thio-beta-glucopyranosiduronic acid was isolated from combined urine of 4 men given oral doses of tetraethylthiuram disulfide.
Diethylthiocarbamic acid methyl ester, in contrast to other disulfiram metabolites, is a potent inhibitor of liver aldehyde dehydrogenase in vitro. Like disulfiram, diethylthiocarbamic acid methyl ester had a pronounced hypothermic effect in rats. This hypothermic effect and the augmented blood pressure response to ethanol challenge in rats developed rapidly with diethylthiocarbamic acid methyl ester but were somewhat delayed with disulfiram. The blood pressure response outlasted the presence of diethylthiocarbamic acid methyl ester in plasma (less than 24 hr); a significant effect was found 48 hr after pretreatment but not 72 hr after a single dose. No effect was observed when ethanol was given 15 min before diethylthiocarbamic acid methyl ester or disulfiram. These latter two observations are consistent with a function of diethylthiocarbamic acid methyl ester as a suicide inhibitor of aldehyde dehydrogenase. Since diethylthiocarbamic acid methyl ester has been reported to inhibit aldehyde dehydrogenase in vitro, even under anaerobic conditions, diethylthiocarbamic acid methyl ester may be the active metabolite of disulfiram.
For more Metabolism/Metabolites (Complete) data for DISULFIRAM (7 total), please visit the HSDB record page.
Disulfiram is completely absorbed from the human GI tract. However, a period of 12 hr is required for its full action, perhaps because, being highly sol in lipid, it is initially localized in fat. It is slowly metabolized in the liver to diethyldithiocarbamate, diethylamine, and carbon disulfide. Six hr after oral administration of the drug, one third of plasma disulfiram is in the form of diethyldithiocarbamate. Elimination is relatively slow, and about 1/5 still remains in body at end of a week. The greater part of the absorbed drug is excreted in the urine as the sulfate, partly free and partly esterified (A620, A622).

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Biological Half-Life
The elimination half-life of disulfiram in plasma is 7.3 hr. /From table/
Following a 250 mg dose, the half-lives of disulfiram, diethyldithiocarbamate and carbon disulfide are 7.3 +/-1.5 hours, 15.5 +/-4.5 hours, and 8.9 +/-1.4 hours, respectively.

Toxicity Summary
Disulfiram blocks the oxidation of alcohol at the acetaldehyde stage during alcohol metabolism following disulfiram intake causing an accumulation of acetaldehyde in the blood producing highly unpleasant symptoms. Disulfiram blocks the oxidation of alcohol through its irreversible inactivation of aldehyde dehydrogenase, which acts in the second step of ethanol utilization. In addition, disulfiram competitively binds and inhibits the peripheral benzodiazepine receptor, which may indicate some value in the treatment of the symptoms of alcohol withdrawal, however this activity has not been extensively studied.

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Hepatotoxicity
Chronic therapy with disulfiram is associated with mild serum aminotransferase elevations in up to 25% of patients, but elevations above 3 times the upper limit of normal (ULN) occur in 4% of patients or less. Importantly, disulfiram is a well established cause of clinically apparent liver injury, which can be severe and even fatal. The estimated incidence of acute liver injury is 1 per 10,000 to 30,000 patient-years of disulfiram treatment. The injury usually arises within 2 to 12 weeks of starting disulfiram, but the latency can be shorter in cases of reexposure and may arise only after 3 to 6 months, particularly with intermittent therapy. The clinical presentation resembles acute viral hepatitis and the pattern of injury is typically hepatocellular (Cases 1 and 2). Rash, fever and eosinophilia are not uncommon, but are rarely severe. The injury can be severe (Case 3) and the fatality rate is at least 10% in cases with jaundice. Rechallenge or reexposure is usually associated with rapid recurrence of liver injury and should be avoided. The clinical presentation and histology differ greatly from alcoholic hepatitis, in that disulfiram liver injury is marked by viral hepatitis-like changes of focal hepatocellular necrosis, lobular disarray and chronic inflammatory cell infiltrates with eosinophils, but without significant fat, neutrophils or Mallory bodies. In the 1980s and 1990s, disulfiram was often listed among the most common causes of acute liver injury and liver failure due to medications. More recently, disulfiram use has decreased and cases of clinically apparent liver injury from disulfiram are now rare. The majority of cases of disulfiram hepatotoxicity have been reported from Scandinavian countries.
Chronic therapy with disulfiram can cause widespread homogenous eosinophilic inclusions in hepatocytes, similar to the “ground glass” changes that can occur in chronic HBsAg carriers.
Likelihood score: A (well known cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Because no information is available on the use of disulfiram during breastfeeding, an alternate drug is preferred, especially while nursing a newborn or preterm infant. Drug labeling recommends that disulfiram not be given to nursing mothers.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Toxicity Data
LD50: 8.6g/kg (oral, rat).

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Interactions
Simultaneously feeding of the animals with disulfiram caused a potentiation of the hepatotoxicity of 1,2-dichloroethane, possibly due to an inhibition of microsomal mixed-function oxidase-mediated metabolism of 1,2-dichloroethane and to a compensatory increase in metabolism to reactive metabolites generated by glutathione-S-transferase-mediated conjugation of 1,2-dichloroethane with reduced glutathione.
Use of alcohol or alcohol containing products within 14 days of disulfiram therapy will result in disulfiram-alcohol reaction.
Chronic preoperative administration or perioperative use of hepatic enzyme inhibitors, such as disulfiram, may decrease the plasma clearance and prolong the duration of action of alfentanil.
Metabolism of bacampicillin produces low plasma concentrations of alcohol and acetaldehyde; although the risk of disulfiram-alcohol interaction appears minimal, caution is recommended if concurrent use is unavoidable. A similar reaction is thought to occur with amoxicillin and clavulanate combination.
For more Interactions (Complete) data for DISULFIRAM (35 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 500 mg/kg
LD50 Rabbit oral 2050 mg/kg
LD50 Mouse intraperitoneal 75 mg/kg
LD50 Mouse oral 1980 mg/kg
LD50 Rabbit dermal >2000 mg/kg bw

[1]. Chen D, ert al. Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and?xenografts?via?inhibition?of the proteasome?activity. Cancer Res. 2006 Nov 1;66(21):10425-33.

[2]. Disulfiram-mediated inhibition of NF-kappaB activity enhances cytotoxicity of 5-fluorouracil in human colorectal cancer cell lines. Int J Cancer. 2003 Apr 20;104(4):504-11.

[3]. Disulfiram facilitates intracellular Cu uptake and induces apoptosis in human melanoma cells. J Med Chem. 2004 Dec 30;47(27):6914-20.

[4]. Disulfiram inhibits activating transcription factor/cyclic AMP-responsive element binding protein and human melanoma growth in a metal-dependent manner in vitro, in mice and in a patient with metastatic disease. Mol Cancer Ther. 2004 Sep;3(9):1049-60.

[5]. Identification of pyroptosis inhibitors that target a reactive cysteine in gasdermin D. The Preprint Server For Biology, 2018,Jul. 10. doi: 10.1038/s41590-020-0669-6

[6]. Inhibitory effect on ovarian cancer ALDH+ stem-like cells by Disulfiram and Copper treatment through ALDH and ROS modulation. Biomed Pharmacother. 2019 Oct;118:109371.

Therapeutic Uses
Alcohol Deterrents; Enzyme Inhibitors
Disulfiram is used to help maintain sobriety in the treatment of chronic alcoholism in conjunction with supportive and psychotherapeutic measures.
Case reports suggest that disulfiram may be useful for the treatment of nickel dermatitis. However, small double-blind placebo-controlled study of patients with hand eczema and nickel allergy did not find a clinically significant difference between those treated with disulfiram and those treated with placebo. Because some patients worsen with this therapy and because patients treated for nickel dermatitis have developed disulfiram induced hepatitis, this therapy is not generally indicated.
The oral efficacy of several chelating drugs, incl disulfiram, was studied in relation to their ability to prevent lethality due to acute inhalation exposure to nickel carbonyl. Disulfiram resulted in very high, but transient, plasma levels. Small, repeated oral doses of disulfiram would be just as effective in nickel carbonyl poisoning as a single large dithiocarb dose. However, disulfiram increase the nickel retained in brain tissue, possibly accounting for its limited efficacy. Caution in the use of oral disulfiram in human nickel carbonyl intoxication is recommended.

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Drug Warnings
... Alarming reactions may result from the ingestion of even small amt of alc in persons being treated with disulfiram. Marked resp depression, cardiovascular collapse, cardiac arrhythmias, myocardial infarction, acute congestive heart failure, unconsciousness, convulsions, and sudden and unexplained fatalities have occurred.
The ingestion of alcohol by individuals previously treated with disulfiram gives rise to marked signs and symptoms. Within about 5-10 min face feels hot, and soon afterwards it is flushed and scarlet in appearance. As vasodilatation spreads over whole body, intense throbbing is felt in head and neck, and a pulsating headache may develop. Respiratory difficulties, nausea, copious vomiting, sweating, thirst, chest pain, considerable hypotension, orthostatic syncope, marked uneasiness, weakness, vertigo, blurred vision, and confusion are observed. Facial flush is replaced by pallor, and blood pressure may fall to shock level.
May decrease urinary vanilmandelic acid excretion, although ... not sufficient to interfere with diagnosis of pheochromocytoma. /Disulfiram's/ inhibition of dopamine hydroxylase ... may increase urinary concn of homovanillic acid /adverse effect, oral/
Patients receiving disulfiram should be warned to avoid cough syrups, sauces, vinegars, elixirs, and other preparations that contain alcohol. External application of alcoholic liniments or lotions, including aftershave or back rub, may be sufficient to produce a disulfiram-alcohol reaction. Patients should be cautioned that disulfiram-alcohol reactions may occur for several weeks after discontinuance of disulfiram.
For more Drug Warnings (Complete) data for DISULFIRAM (17 total), please visit the HSDB record page.

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Pharmacodynamics
Disulfiram produces a sensitivity to alcohol which results in a highly unpleasant reaction when the patient under treatment ingests even small amounts of alcohol. Disulfiram blocks the oxidation of alcohol at the acetaldehyde stage during alcohol metabolism following disulfiram intake, the concentration of acetaldehyde occurring in the blood may be 5 to 10 times higher than that found during metabolism of the same amount of alcohol alone. Accumulation of acetaldehyde in the blood produces a complex of highly unpleasant symptoms referred to hereinafter as the disulfiram-alcohol reaction. This reaction, which is proportional to the dosage of both disulfiram and alcohol, will persist as long as alcohol is being metabolized. Disulfiram does not appear to influence the rate of alcohol elimination from the body. Prolonged administration of disulfiram does not produce tolerance; the longer a patient remains on therapy, the more exquisitely sensitive he becomes to alcohol.

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Disulfiram (DSF), a member of the dithiocarbamate family capable of binding copper and an inhibitor of aldehyde dehydrogenase, is currently being used clinically for the treatment of alcoholism. Recent studies have suggested that DSF may have antitumor and chemosensitizing activities, although the detailed molecular mechanisms remain unclear. Copper has been shown to be essential for tumor angiogenesis processes. Consistently, high serum and tissue levels of copper have been found in many types of human cancers, including breast, prostate, and brain, supporting the idea that copper could be used as a potential tumor-specific target. Here we report that the DSF-copper complex potently inhibits the proteasomal activity in cultured breast cancer MDA-MB-231 and MCF10DCIS.com cells, but not normal, immortalized MCF-10A cells, before induction of apoptotic cancer cell death. Furthermore, MDA-MB-231 cells that contain copper at concentrations similar to those found in patients, when treated with just DSF, undergo proteasome inhibition and apoptosis. In addition, when administered to mice bearing MDA-MB-231 tumor xenografts, DSF significantly inhibited the tumor growth (by 74%), associated with in vivo proteasome inhibition (as measured by decreased levels of tumor tissue proteasome activity and accumulation of ubiquitinated proteins and natural proteasome substrates p27 and Bax) and apoptosis induction (as shown by caspase activation and apoptotic nuclei formation). Our study shows that inhibition of the proteasomal activity can be achieved by targeting tumor cellular copper with the nontoxic compound DSF, resulting in selective apoptosis induction within tumor cells. [1]

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5-Fluorouracil (5-FU) is the major chemotherapeutic component for colorectal cancer (CRC) and other types of solid tumours. Resistance of cancer cells to 5-FU is considered the major obstacle for successful chemotherapy. NF-kappaB is a transcription factor. Cancer cells with high NF-kappaB nuclear activity demonstrate robust chemo- and radio-resistance. We demonstrated that nuclear NF-kappaB activity in CRC cell lines, DLD-1 and RKO(WT), was significantly induced by 5-FU in a concentration- and time-dependent manner. 5-FU induced IkappaBalpha degradation and promoted both NF-kappaB nuclear translocation and its DNA binding activity. 5-FU treatment did not influence the activities of AP-1, AP-2, Oct-1, SP-1, CRE-B and TFIID. Disulfiram (DS), a clinically used anti-alcoholism drug, strongly inhibited constitutive and 5-FU-induced NF-kappaB activity in a dose-dependent manner. DS inhibited both NF-kappaB nuclear translocation and DNA binding activity but had no effect on 5-FU-induced IkappaBalpha degradation. Used in combination, DS significantly enhanced the apoptotic effect of 5-FU on DLD-1 and RKO(WT) cell lines and synergistically potentiated the cytotoxicity of 5-FU to both cell lines. DS also effectively abolished 5-FU chemoresistance in a 5-FU resistant cell line H630(5-FU) in vitro. As DS has extensive preclinical and clinical experience, translating its anticancer usage from in vitro study to clinical trials is relatively straightforward.[2]

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The alcohol-abuse deterrent disulfiram (DSF) is shown to have a highly selective toxicity against melanoma in culture, inducing a largely apoptotic response, with much lower toxicity against several other cell lines. Melanoma cell lines derived from different stages (radial, vertical, and metastatic phase) were all sensitive to DSF treatment in vitro; melanocytes were only slightly affected. A required role of extracellular Cu is demonstrated for DSF toxicity. Low concentrations of DSF alone decreased the number of viable cells, and the addition of CuCl(2) significantly enhanced the DSF-induced cell death to less than 10% of control. Significantly, the intracellular Cu concentration of melanoma cells increased rapidly upon DSF treatment. Both the intracellular Cu uptake and the toxicity induced by DSF were blocked by co-incubation with bathocuproine disulfonic acid (BCPD, 100 muM), a non-membrane-permeable Cu chelator. Chemical studies demonstrated a complicated, extracellular redox reaction between Cu(II) and DSF, which forms the complex Cu(deDTC)(2) in high yield, accompanied by oxidative decomposition of small amounts of disulfiram. The Cu complex has somewhat higher activity against melanoma and is suggested to be the active agent in DSF-induced toxicity. The redox conversion of DSF was unique to Cu(II) and not engendered by the other common biological metal ions Fe(II or III), Mn(III), and Zn(II). The implications of this work are significant both in the possible treatment of melanoma as well as in limiting the known side-effects of DSF, which we propose may be diminished by cotreatment to decrease adventitious Cu.[3]

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The thiocarbamate alcoholism drug disulfiram blocks the P-glycoprotein extrusion pump, inhibits the transcription factor nuclear factor-kappaB, 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.[4]

C10H20N2S4

296.54

296.05

C, 40.50; H, 6.80; N, 9.45; S, 43.25

97-77-8

3117

White to yellow solid

1.2±0.1 g/cm3

369.0±25.0 °C at 760 mmHg

69-71 °C(lit.)

177.0±23.2 °C

0.0±0.8 mmHg at 25°C

1.620

3.88

0

4

7

16

201

0

S=C(N(CC)CC)SSC(N(CC)CC)=S

AUZONCFQVSMFAP-UHFFFAOYSA-N

InChI=1S/C10H20N2S4/c1-5-11(6-2)9(13)15-16-10(14)12(7-3)8-4/h5-8H2,1-4H3

Bis(diethylthiocarbamyl) disulfide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.01 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30 mg/mL (101.17 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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Solubility in Formulation 5: 10 mg/mL (33.72 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)