Subcutaneous Rapid Dissolution Microneedle Patch Integrated with CuO2 and Disulfiram for Augmented Antimelanoma Efficacy through Multimodal Synergy of Photothermal Therapy, Chemodynamic Therapy, and Chemotherapy.

Melanoma is a malignancy of the skin that is resistant to conventional treatment, necessitating the development of effective and safe new therapies. The percutaneous microneedle (MN) system has garnered increasing interest as a viable treatment option due to its high efficacy, minimal invasiveness, painlessness, and secure benefits. In this investigation, a sensitive MN system with multiple functions was created to combat melanoma effectively. This MN system utilized polyvinylpyrrolidone (PVP) as microneedle substrates and biocompatibility panax notoginseng polysaccharide (PNPS) as microneedle tips, which encapsulated PVP-stabilized CuO2 nanoparticles as a therapeutic agent and disulfiram-containing F127 micelles to enhance the tumor treatment effect. The MN system had sufficient mechanical properties to pierce the skin, and the excellent water solubility of PNPS brought high-speed dissolution properties under the bio conditions, allowing the MNs to effectively penetrate the skin and deliver the CuO2 nanoparticles as well as the drug-loaded micelles to the melanoma site. CuO2 nanoparticles released by the MN system generated Cu2+ and H2O2 in the tumor acidic environment to achieve self-supply of hydrogen peroxide to chemodynamic therapy (CDT). In addition, Cu2+ was chelated with disulfiram to produce CuET, which killed tumor cells. And the MN system had excellent near-infrared (NIR) photothermal properties due to the loading of CuO2 nanoparticles and induced localized thermotherapy in the melanoma region to further inhibit tumor growth. Thus, the designed MN system accomplished effective tumor suppression and minimal side effects in vivo via combined therapy, offering patients a safe and effective option for melanoma treatment.

Disulfiram/Copper Induce Ferroptosis in Triple-Negative Breast Cancer Cell Line MDA-MB-231.

BACKGROUND: The complex formed by disulfiram (DSF) and copper (Cu) is safe and effective for the prevention and treatment of triple-negative breast cancer (TNBC). Although previous studies have shown that DSF/Cu induces ferroptosis, the mechanism remains unclear. METHODS: The mitochondrial morphology of TNBC treated with DSF/Cu was observed by transmission microscopy, and intracellular levels of iron, lipid reactive oxygen species (ROS), malondialdehyde, and glutathione were evaluated to detect the presence of ferroptosis. Target genes for the DSF/Cu-activated ferroptosis signaling pathway were examined by transcriptome sequencing analysis. Expression of the target gene, HOMX1, was detected by qRT-PCR, immunofluorescence and western blot. RESULTS: The mitochondria of TNBC cells were significantly atrophied following treatment with DSF/Cu for 24 h. Addition of DSF/Cu supplement resulted in significant up-regulation of intracellular iron, lipid ROS and malondialdehyde levels, and significant down-regulation of glutathione levels, all of which are important markers of ferroptosis. Transcriptome analysis confirmed that DSF/Cu activated the ferroptosis signaling pathway and up-regulated several ferroptosis target genes associated with redox regulation, especially heme oxygenase-1 (HMOX-1). Inhibition of ferroptosis by addition of the ROS scavenger N-acetyl-L-cysteine (NAC) significantly increased the viability of DSF/Cu-treated TNBC cells. CONCLUSIONS: These results show that DSF/Cu increases lipid peroxidation and causes a sharp increase in HMOX1 activity, thereby inducing TNBC cell death through ferroptosis. DSF/Cu is a promising therapeutic drug for TNBC and could lead to ferroptosis-mediated therapeutic strategies for human cancer.

Combination Therapy of Curcumin and Disulfiram Synergistically Inhibits the Growth of B16-F10 Melanoma Cells by Inducing Oxidative Stress.

Oxidative stress plays a central role in the pathophysiology of melanoma. Curcumin (CUR) is a polyphenolic phytochemical that stimulates reactive oxygen species (ROS) production, while disulfiram (DSS) is a US FDA-approved drug for the treatment of alcoholism that can act by inhibiting the intracellular antioxidant system. Therefore, we hypothesized that they act synergistically against melanoma cells. Herein, we aimed to study the antitumor potential of the combination of CUR with DSS in B16-F10 melanoma cells using in vitro and in vivo models. The cytotoxic effects of different combination ratios of CUR and DSS were evaluated using the Alamar Blue method, allowing the production of isobolograms. Apoptosis detection, DNA fragmentation, cell cycle distribution, and mitochondrial superoxide levels were quantified by flow cytometry. Tumor development in vivo was evaluated using C57BL/6 mice bearing B16-F10 cells. The combinations ratios of 1:2, 1:3, and 2:3 showed synergic effects. B16-F10 cells treated with these combinations showed improved apoptotic cell death and DNA fragmentation. Enhanced mitochondrial superoxide levels were observed at combination ratios of 1:2 and 1:3, indicating increased oxidative stress. In vivo tumor growth inhibition for CUR (20 mg/kg), DSS (60 mg/kg), and their combination were 17.0%, 19.8%, and 28.8%, respectively. This study provided data on the potential cytotoxic activity of the combination of CUR with DSS and may provide a useful tool for the development of a therapeutic combination against melanoma.

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment.

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET-a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.

Copper (α)/cis-platinum-loaded nanogels as an adjuvant potentiate disulfiram's antitumor efficacy.

Disulfiram (DSF) is nontoxic and exerts anticancer activity by forming highly toxic chelates via its metabolite diethyldithiocarbamate with transition metal ions. However, there are not enough such ions in the human body to maintain the therapeutic effect. Herein, we report nanogels that complex copper ions or cis-platinum (CDDP) for tumor metal delivery to potentiate DSF's antitumor efficacy. We synthesized zwitterionic poly[N-(3-(methacryloyloxy-2-hydroxy)propyl)]-N-methyl glycine (PGMA-SAR) capable of chelating copper ions or CDDP and formed nanogels with suitable size and zeta potential. The intravenously injected nanogels circulated long in the blood compartment and delivered a high concentration of metal ions to the tumor. Separately administered DSF could sequester the metal ions from the nanogels and form highly cytotoxic complexes with potent in vitro and in vivo anticancer activity. This study provides a new strategy to potentiate DSF in anticancer treatment.

Leveraging disulfiram to treat cancer: Mechanisms of action, delivery strategies, and treatment regimens.

Disulfiram (DSF) has been used as an alcoholism drug for 70 years. Recently, it has attracted increasing attention owing to the distinguished anticancer activity, which can be further potentiated by the supplementation of Cu2+. Although encouraging anticancer results are obtained in lab, the clinical outcomes of oral DSF are not satisfactory, which urges an in-depth understanding of the underlying mechanisms, bottlenecks, and proposal of potential methods to address the dilemma. In this review, a critical summarization of various molecular biological anticancer mechanisms of DSF/Cu2+ is provided and the predicament of orally delivering DSF in clinical oncotherapy is explained by the metabolic barriers. We highlight the recent advances in the DSF/Cu2+ delivery strategies and the emerging treatment regimens for cancer treatment. Last but not the least, we summarize the clinical trials regarding DSF and make a prospect of DSF/Cu-based cancer therapy.

Co-delivery of nanoparticle and molecular drug by hollow mesoporous organosilica for tumor-activated and photothermal-augmented chemotherapy of breast cancer.

BACKGROUND: In comparison with traditional therapeutics, it is highly preferable to develop a combinatorial therapeutic modality for nanomedicine and photothermal hyperthermia to achieve safe, efficient, and localized delivery of chemotherapeutic drugs into tumor tissues and exert tumor-activated nanotherapy. Biocompatible organic-inorganic hybrid hollow mesoporous organosilica nanoparticles (HMONs) have shown high performance in molecular imaging and drug delivery as compared to other inorganic nanosystems. Disulfiram (DSF), an alcohol-abuse drug, can act as a chemotherapeutic agent according to its recently reported effectiveness for cancer chemotherapy, whose activity strongly depends on copper ions. RESULTS: In this work, a therapeutic construction with high biosafety and efficiency was proposed and developed for synergistic tumor-activated and photothermal-augmented chemotherapy in breast tumor eradication both in vitro and in vivo. The proposed strategy is based on the employment of HMONs to integrate ultrasmall photothermal CuS particles onto the surface of the organosilica and the molecular drug DSF inside the mesopores and hollow interior. The ultrasmall CuS acted as both photothermal agent under near-infrared (NIR) irradiation for photonic tumor hyperthermia and Cu2+ self-supplier in an acidic tumor microenvironment to activate the nontoxic DSF drug into a highly toxic diethyldithiocarbamate (DTC)-copper complex for enhanced DSF chemotherapy, which effectively achieved a remarkable synergistic in-situ anticancer outcome with minimal side effects. CONCLUSION: This work provides a representative paradigm on the engineering of combinatorial therapeutic nanomedicine with both exogenous response for photonic tumor ablation and endogenous tumor microenvironment-responsive in-situ toxicity activation of a molecular drug (DSF) for augmented tumor chemotherapy.

The interaction of disulfiram and H2S metabolism in inhibition of aldehyde dehydrogenase activity and liver cancer cell growth.

Disulfiram (DSF), a sulfur-containing compound, has been used to treat chronic alcoholism and cancer for decades by inactivating aldehyde dehydrogenase (ALDH). Hydrogen sulfide (H2S) is a new gasotransmitter and regulates various cellular functions by S-sulfhydrating cysteine in the target proteins. H2S exhibits similar properties to DSF in the sensitization of cancer cells. The interaction of DSF and H2S on ALDH activity and liver cancer cell survival are not clear. Here it was demonstrated that DSF facilitated H2S release from thiol-containing compounds, and DSF and H2S were both capable of regulating ALDH through inhibition of gene expression and enzymatic activity. The supplement of H2S sensitized human liver cancer cells (HepG2) to DSF-inhibited cell viability. The expression of cystathionine gamma-lyase (a major H2S-generating enzyme) was lower but ALDH was higher in mouse liver cancer stem cells (Dt81Hepa1-6) in comparison with their parental cells (Hepa1-6), and H2S was able to inhibit liver cancer stem cell adhesion. In conclusion, these data point to the potential of combining DSF and H2S for inhibition of cancer cell growth and tumor development by targeting ALDH.

Co-treatment with disulfiram and glycyrrhizic acid suppresses the inflammatory response of chondrocytes.

BACKGROUND: Osteoarthritis (OA) is a kind of systemic musculoskeletal disorder and a most important factor for causing disability and physical painfulness. Nevertheless, due to the fact that OA can be triggered by multiple etiological factors, this disease is hard to be cured. Therefore, it is of great necessity for us to find novel targets or drugs for OA treatment. MATERIALS AND METHODS: The chondrocytes were treated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to induce pyroptosis in OA. The cell proliferation was detected by Cell Counting Kit-8 assay (CCK-8 assay). Enzyme-linked immunosorbent assay (ELISA) was used for the detection of pyroptosis-related inflammatory factors. Then, the antagonists for gasdermin D (GSDMD) (disulfiram) and high mobility group box 1 (HMGB1) (glycyrrhizic acid) were used to treat the cell model to observe the effects of disulfiram and glycyrrhizic acid on the proliferation of chondrocytes in OA. The protein levels of pyroptosis-related inflammatory factors were measured by western blot, and the levels of aldehyde dehydrogenase (ALDH) and reactive oxygen species (ROS) were measured by corresponding commercial kits. RESULTS: After chondrocytes were induced by LPS and ATP, the cell proliferation was decreased and the expressions of pyroptosis-related inflammatory factors were increased. Disulfiram and glycyrrhizic acid treatment led to enhanced cell proliferation and increased expressions of pyroptosis-related inflammatory factors, while disulfiram showed better alleviative effects on the inflammation in chondrocytes in OA. However, co-treatment with disulfiram at a high concentration and glycyrrhizic acid did not result in higher proliferation of chondrocytes and alleviated inflammation, but led to oxidative stress. CONCLUSION: In conclusion, co-treatment with disulfiram and glycyrrhizic acid at a standard concentration suppresses the inflammatory response of chondrocytes, which may provide guidance for the use of the drugs in the treatment of OA.

Copper(II)-disulfiram loaded melanin-dots for cancer theranostics.

Copper(II) diethyldithiocarbamate complex (CuET), the metabolite of disulfiram complexed with copper, is the component responsible for cancer treatment efficacy of disulfiram. But the hydrophobic property of CuET limits its use in vivo, and an appropriate drug delivery system needs to be developed. Ultrasmall melanin nanoparticle (M-Dot) with excellent biosafety and biocompatibility properties has been synthesized in our previous studies. Herein we prepared CuET loaded with M-Dots through hydrophobic interaction, which could enhance the water solubility significantly. After the administration of M-Dots-CuET in mice tumor models, the nanoparticles showed good tumor accumulation as evidenced by the enhanced photoacoustic signal in tumor regions. M-Dots-CuET also displayed excellent tumor inhibition capability, and the tumor growth inhibition value (TGI) was 45.1%. When combined with photothermal therapy, the TGI reached up to 78.6%. In summary, M-Dots-CuET provide a new potential strategy for cancer theranostics.

Diethyldithiocarbamate-copper nanocomplex reinforces disulfiram chemotherapeutic efficacy through light-triggered nuclear targeting.

To circumvent the huge cost, long R&D time and the difficulty to identify the targets of new drugs, repurposing the ones that have been clinically approved has been considered as a viable strategy to treat different diseases. In the current study, we outlined the rationale for repurposing disulfiram (DSF, an old alcohol-aversion drug) to treat primary breast cancer and its metastases. Methods: To overcome a few shortcomings of the individual administration of DSF, such as the dependence on copper ions (Cu2+) and limited capability in selective targeting, we here artificially synthesized the active form of DSF, diethyldithiocarbamate (DTC)-Cu complex (CuET) for cancer therapeutics. To achieve a greater efficacy in vivo, smart nanomedicines were devised through a one-step self-assembly of three functional components including a chemically stable and biocompatible phase-change material (PCM), the robust anticancer drug (CuET) and a near-infrared (NIR) dye (DIR), namely CuET/DIR NPs. A number of in vitro assays were performed including the photothermal efficacy, light-triggered drug release behavior, nuclear localization, DNA damage and induction of apoptosis of CuET/DIR NPs and molecular mechanisms underlying CuET-induced repression on cancer metastatic behaviors. Meanwhile, the mice bearing 4T1-LG12-drived orthotopic tumors were employed to evaluate in vivo biodistribution and anti-tumor effect of CuET/DIR NPs. The intravenous injection model was employed to reflect the changes of the intrinsic metastatic propensity of 4T1-LG12 cells responding to CuET/DIR NPs. Results: The rationally designed nanomedicines have self-traceability for bioimaging, long blood circulation time for enhanced drug accumulation in the tumor site and photo-responsive release of the anticancer drugs. Moreover, our data unearthed that CuET/DIR nanomedicines behave like "Trojan horse" to transport CuET into the cytoplasm, realizing substantial intracellular accumulation. Upon NIR laser irradiation, massive CuET would be triggered to release from the nanomedicines and reach a high local concentration towards the nucleus, where the pro-apoptotic effects were conducted. Importantly, our CuET/DIR nanomedicines revealed a pronounced capability to leash breast cancer metastases through inhibition on EMT. Additionally, these nanomedicines showed great biocompatibility in animals. Conclusion: These combined data unearthed a remarkably enhanced tumor-killing efficacy of our CuET nanomedicines through nuclear targeting. This work may open a new research area of repurposing DSF as innovative therapeutic agents to treat breast cancer and its metastases.

FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

Targeting the NPL4 Adaptor of p97/VCP Segregase by Disulfiram as an Emerging Cancer Vulnerability Evokes Replication Stress and DNA Damage while Silencing the ATR Pathway.

Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.

Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper.

Therapies for lung cancer patients initially elicit desirable responses, but the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer vs. normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50-150 nM) combined with physiological levels of Cu (15 µM CuSO4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O2, relative to 4 or 21% O2. This selective toxicity of DSF/Cu was associated with differential Cu ionophore capabilities. DSF/Cu treatment caused a >20-fold increase in cellular Cu in NSCLCs, with nearly two-fold higher Cu present in NSCLCs vs. HBECs and in cancer cells at 1% O2vs. 21% O2. DSF toxicity was shown to be dependent on the retention of Cu as well as oxidative stress mechanisms, including the production of superoxide, peroxide, lipid peroxidation, and mitochondrial damage. DSF was also shown to selectively (relative to HBECs) enhance radiation and chemotherapy-induced NSCLC killing and reduce radiation and chemotherapy resistance in hypoxia. Finally, DSF decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF could be a promising adjuvant to enhance cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.

Soybean lecithin stabilizes disulfiram nanosuspensions with a high drug-loading content: remarkably improved antitumor efficacy.

Disulfiram (DSF) has been considered as "Repurposing drug" in cancer therapy in recent years based on its good antitumor efficacy. DSF is traditionally used as an oral drug in the treatment of alcoholism. To overcome its rapid degradation and instability, DSF nanosuspensions (DSF/SPC-NSps) were prepared using soybean lecithin (SPC) as a stabilizer of high drug-loaded content (44.36 ± 1.09%). Comprehensive characterization of the nanosuspensions was performed, and cell cytotoxicity, in vivo antitumor efficacy and biodistribution were studied. DSF/SPC-NSps, having a spherical appearance with particle size of 155 nm, could remain very stable in different physiological media, and sustained release. The in vitro MTT assay indicated that the cytotoxicity of DSF/SPC-NSps was enhanced remarkably compared to free DSF against the 4T1 cell line. The IC50 value decreased by 11-fold (1.23 vs. 13.93 µg/mL, p < 0.01). DSF/SPC-NSps groups administered via intravenous injections exhibited better antitumor efficacy compared to the commercial paclitaxel injection (PTX injection) and had a dose-dependent effect in vivo. Notably, DSF/SPC-NSps exhibited similar antitumor activity following oral administration as PTX administration via injection into a vein. These results suggest that the prepared nanosuspensions can be used as a stable delivery vehicle for disulfiram, which has potential application in breast cancer chemotherapy.

Inhibitory effect on ovarian cancer ALDH+ stem-like cells by Disulfiram and Copper treatment through ALDH and ROS modulation.

BACKGROUND: Disulfiram (DSF) is a drug used for treatment of alcoholism that has also displayed promising anti-cancer activity. It unfolds its effects by inhibiting the enzyme activity of aldehyde dehydrogenase (ALDH) isoforms. METHODS: MTT assay, spheroid formation, clonogenicity assay, qRT-PCR, and ALDH enzyme activity analysis were performed using ovarian cancer cell lines IGROV1, SKOV3 and SKOV3IP1. Cell cycle analyses and measurement of intracellular reactive oxygen species (ROS) were carried out by flow cytometry. ALDH+ and ALDH- cells were isolated by FACS sorting. RESULTS: 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). DSF with or without the cofactor copper (Cu2+) exhibited cytotoxicity dose- and time-dependent and enhanced cisplatin-induced apoptosis. DSF + 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 DSF (0.08 µM). CONCLUSION: Our findings provide evidence that DSF might be employed as a novel adjuvant chemotherapeutic agent in combination with cisplatin for treatment of ovarian cancer.

Repurposing disulfiram for treatment of Staphylococcus aureus infections.

BACKGROUND: Antimicrobial resistance is an urgent threat affecting healthcare systems worldwide. Identification of novel molecules capable of escaping current resistance mechanisms and exhibiting potent activity against highly drug-resistant strains is the unmet need of the hour. METHODS: Whole cell growth inhibition assays were used to screen and identify novel inhibitors. The hit compounds were tested against Vero cells to determine the selectivity index, followed by time-kill kinetics against Staphylococcus aureus. The ability of disulfiram to synergize with several approved drugs utilized for the treatment of S. aureus was determined using fractional inhibitory concentration indexes, followed by its ability to decimate staphyloccocal infections ex vivo. Finally, the in-vivo potential of disulfiram was determined in a neutropenic murine model of S. aureus infection. RESULTS: The screening showed that disulfiram has equipotent antibacterial activity against S. aureus, including clinical drug-resistant strains (minimum inhibitory concentration 8-16 mg/L). Disulfiram exhibited concentration-dependent bactericidal activity (∼7 log10 colony-forming units/mL reduction), synergized with linezolid and gentamycin against S. aureus, eradicated staphylococcal biofilms (64-fold better than vancomycin), decimated intracellular S. aureus better than vancomycin, exhibited longer post antibiotic effect than vancomycin, and reduced bacterial counts in murine thigh as well as vancomycin at 50 mg/kg. CONCLUSION: Taken together, disulfiram exhibits all the characteristics required for repurposing as an antibacterial targeting staphylococcal infections.

Disulfiram, an alcohol dependence therapy, can inhibit the in vitro growth of Francisella tularensis.

Disulfiram (DSF) can help treat alcohol dependency by inhibiting aldehyde dehydrogenase (ALDH). Genomic analysis revealed that Francisella tularensis, the causative agent of tularemia, has lost all but one ALDH-like domain and that this domain retains the target of DSF. In this study, minimum inhibitory concentration (MIC) assays demonstrated that both DSF and its primary metabolite diethyldithiocarbamate (DDC) have strong antimicrobial activity against F. tularensis strain SCHU S4, with the MIC of DSF determined as 2 µg/mL in comparison with 8 µg/mL for DDC. The activity of DSF was further confirmed using an in vitro human macrophage infection assay. Francisella tularensis bacteria in DSF-treated cells were reduced in comparison with untreated and DDC-treated cells, comparable with that observed in doxycycline-treated cells. This suggests that DSF may be suitable for further investigation as an in vivo therapy for tularemia.

Antibacterial activity of disulfiram and its metabolites.

AIMS: Disulfiram (Antabuse™) and its metabolites formed in vivo were evaluated as antibacterial agents against thirty species of Gram-positive and Gram-negative bacteria. The synergistic potential of disulfiram (DSF) and metabolite diethyldithiocarbamate (DDTC) with approved antibiotics were also compared by isobologram (checkerboard) analysis. METHODS AND RESULTS: Standard microdilution susceptibility testing showed that most DSF metabolites did not possess appreciable antibacterial activity except for DDTC in Bacillus anthracis. Checkerboard studies revealed similarities between the combination drug effects of DSF and DDTC with standard antibiotics. CONCLUSIONS: It was concluded from the susceptibility data that the metabolites would not extend the antibacterial spectrum of DSF in vivo. The data also suggest that the DDTC by-product of DSF metabolism potentiates the antibacterial activity of DSF as both a standalone and combination agent. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a greater understanding of the antibacterial effects of Antabuse and its metabolites. This research also demonstrates the potential application of DSF as an antibiotic adjuvant for the treatment of resistant staph infections.

Anti-cancer effects of disulfiram in head and neck squamous cell carcinoma via autophagic cell death.

BACKGROUND: Disulfiram (DSF), which is used to treat alcohol dependence, has been reported to have anti-cancer effects in various malignant tumors. In this study, we investigated the anti-cancer effects and mechanism of DSF in HNSCC. METHODS: Head and neck squamous carcinoma cell lines (FaDu and Hep2) were used to analyze the anti-cancer effects of DSF. The anti-cancer effects of DSF were confirmed in vivo using a xenograft tumor model. RESULTS: The anti-cancer effects of DSF in HNSCC were found to be copper (Cu) dependent. Specifically, DSF/Cu markedly inhibited HNSCC at a concentration of 1 µM. After DSF/Cu administration, production of reactive oxygen species (ROS) was remarkable starting at 0.5 µM, suggesting that the inhibitory effects of DSF/Cu on HNSCC are mediated through the formation of ROS. The levels of phospho-JNK, phospho-cJun and phospho-p38 were increased after DSF/Cu treatment while levels of phospho-Akt were decreased. These results suggested that the inhibitory effects of DSF/Cu on HNSCC cells involve ROS formation and down-regulation of Akt-signaling. Through these molecular mechanisms, DSF ultimately induce the inhibitory effects on HNSCC cell lines mainly through autophagic cell death, not apoptotic cell death. Lastly, we investigated the clinical relevance of DSF/Cu using a HNSCC xenograft animal model, which showed that tumor growth was remarkably decreased by DSF (50 mg/kg injection). CONCLUSION: In treating patients with HNSCC, DSF may contribute to improved HNSCC patient's survival. The characteristic anti-cancer effects of DSF on HNSCC may suggest new therapeutic potential for this medication in HNSCC patients.