Anti-alcoholism Drug Disulfiram Research Articles

The anti-alcoholism drug disulfiram effectively ameliorates ulcerative colitis through suppressing oxidative stresses-associated pyroptotic cell death and cellular inflammation in colonic cells

BackgroundOxidative stress, cell pyroptosis and inflammation are considered as important pathogenic factors for ulcerative colitis (UC) development, and the traditional anti-alcoholism drug disulfiram (DSF) has recently been reported to exert its regulating effects on all the above cellular functions, which makes DSF as ideal therapeutic agent for UC treatment, but this issue has not been fully studied. MethodsDextran sulfate sodium (DSS)-induced animal models in C57BL/6J mice and lipopolysaccharide (LPS)-induced cellular models in colonic cell lines (HT-29 and Caco-2) for UC were respectively established. Cytokine secretion was determined by ELISA. Cell viability and proliferation were evaluated by MTT assay and EdU assay. Real-Time qPCR, Western Blot, immunofluorescent staining assay and immunohistochemistry (IHC) were employed to evaluate gene expressions. The correlations of the genes in the clinical tissues were analyzed by using the Pearson Correlation analysis. ResultsDSF restrained oxidative stress, pyroptotic cell death and cellular inflammation in UC models in vitro and in vivo, and elimination of Reactive Oxygen Species (ROS) by N-acetyl-l-cysteine (NAC) rescued cell viability in LPS-treated colonic cells (HT-29 and Caco-2). Further experiments suggested that a glycogen synthase kinase-3β (GSK-3β)/Nrf2/NLRP3 signaling cascade played critical role in this process. Mechanistically, DSF downregulated GSK-3β and NLRP3, whereas upregulated Nrf2 in LPS-treated colonic cells. Also, the regulating effects of DSF on Nrf2 and NLRP3 were abrogated by upregulating GSK-3β. Moreover, upregulation of GSK-3β abolished the protective effects of DSF on LPS-treated colonic cells. ConclusionsTaken together, data of this study indicated that DSF restrained oxidative damages-related pyroptotic cell death and inflammation via regulating the GSK-3β/Nrf2/NLRP3 pathway, leading to the suppression of LPS-induced UC development.

Anti-alcoholism drug disulfiram for targeting glioma energy metabolism using BBB-penetrating delivery of fixed-dose combination

The interconnection between tumor metabolism and the immune microenvironment governs the progression of glioma. It was revealed that the metabolic enzymes ALDH1L1 and PKM2 were overexpressed in glioma. We discovered that the anti-alcoholism drug disulfiram (DSF) can intervene in the tumor energy metabolism by inhibiting ATP supply via an alternative pathway, of which ALDH1L1 is an essential enzyme in the 10-formyl-tetrahydrofolate-NADH-ATP metabolism axis. We thus proposed a dual-inhibition strategy by using a combination therapy of a PKM2 inhibitor shikonin (SHK) and an ALDH1L1 inhibitor DSF. By simultaneously inhibiting glycolysis and NADH-ATP metabolism, the ATP generation in the tumor cells can be efficiently cut down. A BBB-penetrating hybrid albumin/lactoferrin nanosystem (termed BSA/LF NP) was developed for biomimetic codelivery of DSF/SHK for anti-glioma therapy. Due to the overexpression of SPARC and LRP-1 in both the BBB and glioma cells, the BSA/LF NP could efficiently enter the glioma. More importantly, it can deliver a fixed-dose ratio of DSF and SHK to the glioma, providing a basis for synergistic effect in combination therapy. The BSA/LF NP effectively prolonged the survival of glioma mice by interactive modulation of energy metabolism (via both glycolysis and NADH-ATP pathways) and immune microenvironment. The work provided a promising glioma-targeting strategy for a fixed-dose combination and a potent ATP-depletion treatment.

Disulfiram-loaded metal organic framework for precision cancer treatment via ultrasensitive tumor microenvironment-responsive copper chelation and radical generation

Off-target toxicity remains a major limitation of current cancer therapy, necessitating an alternative precision approach to treat cancers. Herein, a tumor microenvironment (TME)-triggered anticancer strategy was developed by constructing an anti-alcoholism drug disulfiram (DSF)-loaded, Cu-doped zeolite imidazolate frameworks-8 (DSF-Cu/ZIF-8) nanoparticle followed by PEGylation (PEG-DSF-Cu/ZIF-8) to realize in situ generation of cytotoxic compounds specifically in TME. The PEG-DSF-Cu/ZIF-8 demonstrated excellent hydrolytic stability in normal physiological conditions, guaranteeing the minimized off-target release of disulfiram and Cu ions. Under the TME condition, the PEG-DSF-Cu/ZIF-8 exhibited acidity-triggered biodegradation and the associated payload release, through which low-toxic compounds (disulfiram and Cu2+ ions) were converted to highly cytotoxic Cu-chelate product to kill cells specifically in TME. Tumor-sensitive anti-cancer performance was further enhanced by hydroxyl radical generation via TME-responsive Fenton-like reactions catalyzed by Cu+ presenting in the PEG-DSF-Cu/ZIF-8 structure and Cu+ produced during formation of the chelate product. Anti-cancer therapeutic evaluation was performed in 2D 4T1 tumor cell culture and 3D 4T1 tumor spheroids, and demonstrated highly TME-responsive, low-dose induced anti-cancer effect. This proof-of-concept work provides a nanoparticle-based drug repurposing strategy by developing a tumor-sensitive anti-cancer agent for low-toxic and efficacious cancer therapy.

The anti-alcoholism drug disulfiram produces anxiolytic effects in mice.

The anti-alcoholism drug disulfiram produces anxiolytic effects in mice.

Induction of immunogenic cell death in radiation-resistant breast cancer stem cells by repurposing anti-alcoholism drug disulfiram

BackgroundThe current successful clinical use of agents promoting robust anti-tumor immunity in cancer patients warrants noting that radiation therapy (RT) induces immunogenic cell death (ICD) of tumor cells, which can generate anti-tumor immune responses. However, breast cancer stem cells (BCSCs) are resistant to RT and RT alone usually failed to mount an anti-tumor immune response.MethodsHigh aldehyde dehydrogenase activity (ALDH)bright and CD44+/CD24−/ESA+ cancer cells, previously shown to have BCSC properties, were isolated from human MDA-MB-231 and UACC-812 breast cancer cell lines by flow cytometer. Flow sorted BCSCs and non-BCSCs were further tested for their characteristic of stemness by mammosphere formation assay. Induction of ICD in BCSCs vs. non-BCSCs in response to different in vitro treatments was determined by assessing cell apoptosis and a panel of damage-associated molecular pattern molecules (DAMPs) by flow and enzyme-linked immunosorbent assay (ELISA).ResultsWe found that ionizing radiation (IR) triggered a lower level of ICD in BCSCs than non-BCSCs. We then investigated the ability of disulfiram/cooper (DSF/Cu) which is known to preferentially induce cancer stem cells (CSCs) apoptosis to enhance IR-induced ICD of BCSCs. The results indicate that DSF/Cu induced a similar extent of IDC in both BCSCs and non-BCSCs and rendered IR-resistant BCSCs as sensitive as non-BCSCs to IR-induced ICD. IR and DSF/Cu induced ICD of BCSCs could be partly reversed by pre-treatment of BCSCs with a reactive oxygen species (ROS) scavenger and XBP1s inhibitors.ConclusionDSF/Cu rendered IR-resistant BCSCs as sensitive as non-BCSCs to IR-induced ICD. Our data demonstrate the potential of IR and DSF/Cu to induce ICD in BCSCs and non-BCSCs leading to robust immune responses against not only differentiated/differentiating breast cancer cells but also BCSCs, the root cause of cancer formation, progression and metastasis.Graphical abstract

Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties

Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.

Targeting FROUNT with Disulfiram Regulates Macrophage Responses in Cancer

Tumor-associated macrophages influence tumor progression and resistance to immune checkpoint therapy. Chemokine signaling is a key regulator of macrophage accumulation and activity. Here, we report that the chemokine receptor-binding protein FROUNT modulates tumor-associated macrophage responses and is an important regulator of tumor progression. By screening 131,200 compounds, we identified the anti-alcoholism drug disulfiram (DSF) as a potent inhibitor of FROUNT that interferes with FROUNT-chemokine receptor interaction by directly binding to FROUNT. Both DSF-treatment and FROUNT-deficiency markedly reduced tumor progression and decreased the tumor-promoting activity of macrophages. Studies using clinical specimens and FROUNT-GFP reporter mice showed that FROUNT was highly expressed in macrophages, and myeloid-specific deletion of FROUNT impaired tumor growth. Low FROUNT expression in patients with lung adenocarcinoma was correlated with better clinical outcomes. Moreover, co-treatment of DSF with an immune checkpoint antibody synergistically inhibited tumor growth. Together, inhibition of FROUNT by DSF presents a strategy for macrophage-targeted cancer therapy.

Anti-hepatocellular carcinoma properties of the anti-alcoholism drug disulfiram discovered to enzymatically inhibit the AMPK-related kinase SNARK in vitro.

We recently described that the anti-apoptotic AMPK-related kinase, SNARK, promotes transforming growth factor (TGF)-β signaling in hepatocellular carcinoma (HCC) cells, as a potentially new therapeutic target. Here we explored FDA-approved drugs inhibiting the enzymatic activity of SNARK, using an in vitro luminescence kinase assay system. Interestingly, the long-used anti-alcoholism drug disulfiram (DSF), also known as Antabuse, emerged as the top hit. Enzymatic kinetics analyses revealed that DSF inhibited SNARK kinase activity in a noncompetitive manner to ATP or phosphosubstrates. Comparative in vitro analyses of DSF analogs indicated the significance of the disulfide bond-based molecular integrity for the kinase inhibition. DSF suppressed SNARK-promoted TGF-β signaling and demonstrated anti-HCC effects. The chemical and enzymatic findings herein reveal novel pharmacological effects of and use for DSF and its derivatives, and could be conducive to prevention and inhibition of liver fibrosis and HCC.

A chemical-genetic interaction map of small molecules using high-throughput imaging in cancer cells.

Small molecules often affect multiple targets, elicit off-target effects, and induce genotype-specific responses. Chemical genetics, the mapping of the genotype dependence of a small molecule's effects across a broad spectrum of phenotypes can identify novel mechanisms of action. It can also reveal unanticipated effects and could thereby reduce high attrition rates of small molecule development pipelines. Here, we used high-content screening and image analysis to measure effects of 1,280 pharmacologically active compounds on complex phenotypes in isogenic cancer cell lines which harbor activating or inactivating mutations in key oncogenic signaling pathways. Using multiparametric chemical-genetic interaction analysis, we observed phenotypic gene-drug interactions for more than 193 compounds, with many affecting phenotypes other than cell growth. We created a resource termed the Pharmacogenetic Phenome Compendium (PGPC), which enables exploration of drug mode of action, detection of potential off-target effects, and the generation of hypotheses on drug combinations and synergism. For example, we demonstrate that MEK inhibitors amplify the viability effect of the clinically used anti-alcoholism drug disulfiram and show that the EGFR inhibitor tyrphostin AG555 has off-target activity on the proteasome. Taken together, this study demonstrates how combining multiparametric phenotyping in different genetic backgrounds can be used to predict additional mechanisms of action and to reposition clinically used drugs.

Editorial Commentary: Reversing Latency in HIV-Infected Patients

The inability of combination antiretroviral treatment (cART) to eradicate human immunodeficiency virus (HIV) infection has renewed scientific interest in curative strategies [1]. The primary barrier preventing eradication of HIV by cART is a pool of long-lived latently infected cells of which memory CD4 + T cells appear to be the most important. These latently infected cells harbor integrated proviral DNA capable of resuming HIV expression and fueling viral rebound upon cessation of cART. One strategy to deplete this viral reservoir has been termed “kick and kill,” indicating a 2-step process by which expression of the latent virus is reactivated by a latency-reversing agent followed by elimination of the infected cell by either cell apoptotic/cytolytic mechanism or by immune-mediated clearance. The hunt for pharmacological interventions capable of reactivating HIV from the resting state and hence deliver the first step of this strategy is now on. In the study by Spivak et al in this issue of Clinical Infectious Diseases, the antialcoholism drug disulfiram was tested in 15 virologically suppressed patients with HIV for its ability to reactivate latent HIV. Two questions were asked in this study: First, can disulfiram activate latent HIV as determined by increases in residual viremia? Second, can HIV reactivation with disulfiram lead to a reduction in the size of the latent reservoir? As for the first point, the jury is still out. Overall, during 14 days of disulfiram dosing, there was no significant effect on the level of residual viremia measured by the single-copy assay. Despite this, there are 2 findings supporting the latencyreversing capacity of disulfiram. In 10 patients with measurements 2 hours after the first dose, an increase in residual viremia was observed. Furthermore, during the postdosing phase of the study, an overall increase in viral particle production above baseline was noted. Another class of compounds, the histone deacetylase inhibitors (HDACi), has recently been found to reactivate HIV in CD4 T cells in vivo [2, 3] although these 2 studies investigating the potential of vorinostat to reactive HIV failed to detect any increases in plasma viremia. Unfortunately, the study by

A Pilot Study Assessing the Safety and Latency-Reversing Activity of Disulfiram in HIV-1-Infected Adults on Antiretroviral Therapy

Transcriptionally silent human immunodeficiency virus type 1 (HIV-1) DNA persists in resting memory CD4(+) T cells despite antiretroviral therapy. In a primary cell model, the antialcoholism drug disulfiram has been shown to induce HIV-1 transcription in latently infected resting memory CD4(+) T cells at concentrations achieved in vivo. We conducted a single-arm pilot study to evaluate whether 500 mg of disulfiram administered daily for 14 days to HIV-1-infected individuals on stable suppressive antiretroviral therapy would result in reversal of HIV-1 latency with a concomitant transient increase in residual viremia or depletion of the latent reservoir in resting memory CD4(+) T cells. Disulfiram was safe and well tolerated. There was a high level of subject-to-subject variability in plasma disulfiram levels. The latent reservoir did not change significantly (1.16-fold change; 95% confidence interval [CI], .70- to 1.92-fold; P = .56). During disulfiram administration, residual viremia did not change significantly compared to baseline (1.53-fold; 95% CI, .88- to 2.69-fold; P = .13), although residual viremia was estimated to increase by 1.88-fold compared to baseline during the postdosing period (95% CI, 1.03- to 3.43-fold; P = .04). In a post hoc analysis, a rapid and transient increase in viremia was noted in a subset of individuals (n = 6) with immediate postdose sampling (HIV-1 RNA increase, 2.96-fold; 95% CI, 1.29- to 6.81-fold; P = .01). Administration of disulfiram to patients on antiretroviral therapy does not reduce the size of the latent reservoir. A possible dose-related effect on residual viremia supports future studies assessing the impact of higher doses on HIV-1 production. Disulfiram affects relevant signaling pathways and can be safely administered, supporting future studies of this drug.

Mechanisms of disulfiram-induced cocaine abstinence: antabuse and cocaine relapse.

The anti-alcoholism drug disulfiram (Antabuse), which is an inhibitor of aldehyde dehydrogenase, induces an aversive reaction to alcohol consumption and thereby helps patients reduce alcohol intake. Recent clinical trials, initiated to investigate whether disulfiram could be used to treat individuals who abuse both alcohol and cocaine, have indicated that disulfiram effectively decreases cocaine consumption. Yet the ability of disulfiram to curb cocaine intake cannot be explained by the disruption of ethanol metabolism. Here, we synthesize clinical and animal data that point to dopamine beta-hydroxylase inhibition as a mechanism underlying the efficacy of disulfiram in the treatment of cocaine dependence.

Synergistic activity of 2-deoxyglucose, an endoplasmic reticulum stress inducer, and efrapeptins, dual inhibitors of proteasome and Hsp90, in breast cancer cells

The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF–Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value of 32 μmol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF–Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF–Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF–Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.

Effects of disulfiram and dopamine beta-hydroxylase knockout on cocaine-induced seizures

The antialcoholism drug disulfiram has shown recent promise as a pharmacotherapy for treating cocaine dependence, probably via inhibition of dopamine β-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE). We previously showed that DBH knockout ( Dbh −/−) mice, which lack NE, are susceptible to seizures and are hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, suggesting that disulfiram might exacerbate cocaine-induced seizures (CIS) by inhibiting DBH. To test this, we examined CIS in wild-type and Dbh −/− mice following administration of disulfiram or the selective DBH inhibitor nepicastat. We found that Dbh genotype had no effect on CIS probability or frequency, whereas disulfiram, but not nepicastat, increased the probability of having CIS in both wild-type and Dbh −/− mice. Both disulfiram and nepicastat increased CIS frequency in wild-type but not Dbh −/− mice. There were no genotype or treatment effects on serum cocaine levels, except for an increase in disulfiram-treated Dbh −/− mice at the highest dose of cocaine. These results suggest that disulfiram enhances CIS via two distinct mechanisms: it both increases CIS frequency by inhibiting DBH and increases CIS frequency in a DBH-independent manner.

Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF–Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC 50 value of 32 μmol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF–Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF–Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF–Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.

Inhibition of proteasome activity, nuclear factor-KB translocation and cell survival by the antialcoholism drug disulfiram

The proteasome pathway is an important target for anticancer drug development. Here, we identify the antialcoholism drug disulfiram and its analogue pyrrolidine dithiocarbamate (PDTC) as inhibitors of the 26S proteasome activity in a cell-based screening assay. As expected for proteasome inhibitors, these compounds also inhibited TNF-alpha-induced nuclear factor-KappaB (NF-KappaB) translocation and were cytotoxic. Disulfiram was more cytotoxic against chronic lymphocytic leukemia cells compared to peripheral blood mononuclear cells (PBMC) at clinically achievable concentrations. Proteasome and NF-KappaB inhibition were achieved with a potency in the same range as that of the clinically used proteasome inhibitor bortezomib. Disulfiram was also able to induce accumulation of p27(Kip1) and to prolong the half-life of c-Myc, both targets for proteasome-dependent degradation. It is concluded that the previously observed antitumoral and NF-KappaB inhibiting activity of disulfiram and PDTC could be attributed to their inhibition of the 26S proteasome.