LIFU/MMP-2 dual-responsive release of repurposed drug disulfiram from nanodroplets for inhibiting vasculogenic mimicry and lung metastasis in triple-negative breast cancer.

BACKGROUND: Vasculogenic mimicry (VM), when microvascular channels are formed by cancer cells independent of endothelial cells, often occurs in deep hypoxic areas of tumors and contributes to the aggressiveness and metastasis of triple-negative breast cancer (TNBC) cells. However, well-developed VM inhibitors exhibit inadequate efficacy due to their low drug utilization rate and limited deep penetration. Thus, a cost-effective VM inhibition strategy needs to be designed for TNBC treatment. RESULTS: Herein, we designed a low-intensity focused ultrasound (LIFU) and matrix metalloproteinase-2 (MMP-2) dual-responsive nanoplatform termed PFP@PDM-PEG for the cost-effective and efficient utilization of the drug disulfiram (DSF) as a VM inhibitor. The PFP@PDM-PEG nanodroplets effectively penetrated tumors and exhibited substantial accumulation facilitated by PEG deshielding in a LIFU-mediated and MMP-2-sensitive manner. Furthermore, upon exposure to LIFU irradiation, DSF was released controllably under ultrasound imaging guidance. This secure and controllable dual-response DSF delivery platform reduced VM formation by inhibiting COL1/pro-MMP-2 activity, thereby significantly inhibiting tumor progression and metastasis. CONCLUSIONS: Considering the safety of the raw materials, controlled treatment process, and reliable repurposing of DSF, this dual-responsive nanoplatform represents a novel and effective VM-based therapeutic strategy for TNBC in clinical settings.

Turning anecdotal irradiation-induced anticancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE); even combining IR with immune checkpoint inhibitors has shown only anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug, disulfiram (DSF), complexed with copper (DSF/Cu) to induce tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anticancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs relative to spontaneous lung metastasis. In addition, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anticancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral (i.t.) injection of DSF/Cu and IR(12Gy) demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8+ and CD4+ cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, dendritic cells (DC), and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8+ and CD4+ cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anticancer immune response that results in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300 g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75 mg/h) monotherapy or co-solvent only (50% hydroxypropyl-ß-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

Localized Sustained Release of Copper Enhances Antitumor Effects of Disulfiram in Head and Neck Cancer.

Drug repurposing uses approved drugs as candidate anticancer therapeutics, harnesses previous research and development efforts, and benefits from available clinically suitable formulations and evidence of patient tolerability. In this work, the drug used clinically to treat chronic alcoholism, disulfiram (DSF), was studied for its antitumor efficacy in a copper-dependent manner. The combination of DSF and copper could achieve a tumor cell growth inhibition effect comparable to those of 5-fluorouracil and taxol on head and neck cancer cells. Both bulk dendrimer hydrogel and microsized dendrimer hydrogel particles were utilized for the localized sustained release of copper in the tumor site. The localized sustained release of copper facilitated the tumor inhibition effect following intratumoral injection in a mouse's head and neck cancer model.

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism.

Antibody responses, involving B cells, CD4 + T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.

Disulfiram Inhibits Opsonin-Independent Phagocytosis and Migration of Human Long-Lived In Vitro Cultured Phagocytes from Multiple Inflammatory Diseases.

Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through its copper chelating derivatives and induction of oxidative stress in mitochondria. Since chronic/persistent borreliosis is characterized by increased amounts of pro-inflammatory macrophages, this study investigated opsonin-independent phagocytosis, migration, and surface marker expression of in vivo activated and in vitro cultured human monocyte-derived phagocytes (macrophages and dendritic cells) with and without DSF treatment. Phagocytosis of non-opsonized Dynabeads® M-450 and migration of macrophages and dendritic cells were monitored using live cell analyzer Juli™ Br for 24 h, imaging every 3.5 min. To simultaneously monitor phagocyte function, results were analyzed by a newly developed software based on the differential phase contrast images of cells before and after ingestion of Dynabeads. DSF decreased the phagocytic capacities exhibited by in vitro enriched and long-lived phagocytes. Although no chemotactic gradient was applied to the test system, vigorous spontaneous migration was observed. We therefore set up an algorithm to monitor and quantify both phagocytosis and migration simultaneously. DSF not only reduced phagocytosis in a majority of these long-lived phagocytes but also impaired their migration. Despite these selective effects by DSF, we found that DSF reduced the expression densities of surface antigens CD45 and CD14 in all of our long-lived phagocytes. In cells with a high metabolic activity and high mitochondrial contents, DSF led to cell death corresponding to mitochondrial oxidative stress, whereas metabolically inactive phagocytes survived our DSF treatment protocol. In conclusion, DSF affects the viability of metabolically active phagocytes by inducing mitochondrial stress and secondly attenuates phagocytosis and migration in some long-lived phagocytes.

Lipopolysaccharide-induced intestinal inflammation on AIM2-mediated pyroptosis in the brain of rats with cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is a cerebral vascular disease with insidious onset and poor clinical treatment effect, which is related to neuroinflammation. This study investigated whether lipopolysaccharide-induced intestinal inflammation enhanced the level of pyroptosis in the brain of rats with CSVD. The bilateral carotid artery occlusion (BCAO) model was selected as the object of study. Firstly, behavioral tests and Hematoxylin-eosin staining (HE staining) were performed to determine whether the model was successful, and then the AIM2 inflammasome and pyroptosis indexes (AIM2, ASC, Caspase-1, IL-1ß, GSDMD, N-GSDMD) in the brain were detected by Western blotting and Immunohistochemistry (IHC). Finally, a single intraperitoneal injection of lipopolysaccharide (LPS) was used to induce intestinal inflammation in rats, the expression of GSDMD and N-GSDMD in the brain was analyzed by Western blotting and to see if pyroptosis caused by intestinal inflammation can be inhibited by Disulfiram, an inhibitor of pyroptosis. The results showed that the inflammatory response and pyroptosis mediated by the AIM2 inflammasome in BCAO rats were present in both brain and intestine. The expression of N-GSDMD, a key marker of pyroptosis, in the brain was significantly increased and inhibited by Disulfiram after LPS-induced enhancement of intestinal inflammation. This study shows that AIM2-mediated inflammasome activation and pyroptosis exist in both brain and intestine in the rat model of CSVD. The enhancement of intestinal inflammation will increase the level of pyroptosis in the brain. In the future, targeted regulation of the AIM2 inflammasome may become a new strategy for the clinical treatment of CSVD.

FDA-approved disulfiram as a novel treatment for aggressive leukemia.

Acute leukemia continues to be a major cause of death from disease worldwide and current chemotherapeutic agents are associated with significant morbidity in survivors. While better and safer treatments for acute leukemia are urgently needed, standard drug development pipelines are lengthy and drug repurposing therefore provides a promising approach. Our previous evaluation of FDA-approved drugs for their antileukemic activity identified disulfiram, used for the treatment of alcoholism, as a candidate hit compound. This study assessed the biological effects of disulfiram on leukemia cells and evaluated its potential as a treatment strategy. We found that disulfiram inhibits the viability of a diverse panel of acute lymphoblastic and myeloid leukemia cell lines (n = 16) and patient-derived xenograft cells from patients with poor outcome and treatment-resistant disease (n = 15). The drug induced oxidative stress and apoptosis in leukemia cells within hours of treatment and was able to potentiate the effects of daunorubicin, etoposide, topotecan, cytarabine, and mitoxantrone chemotherapy. Upon combining disulfiram with auranofin, a drug approved for the treatment of rheumatoid arthritis that was previously shown to exert antileukemic effects, strong and consistent synergy was observed across a diverse panel of acute leukemia cell lines, the mechanism of which was based on enhanced ROS induction. Acute leukemia cells were more sensitive to the cytotoxic activity of disulfiram than solid cancer cell lines and non-malignant cells. While disulfiram is currently under investigation in clinical trials for solid cancers, this study provides evidence for the potential of disulfiram for acute leukemia treatment. KEY MESSAGES: Disulfiram induces rapid apoptosis in leukemia cells by boosting oxidative stress. Disulfiram inhibits leukemia cell growth more potently than solid cancer cell growth. Disulfiram can enhance the antileukemic efficacy of chemotherapies. Disulfiram strongly synergises with auranofin in killing acute leukemia cells by ROS induction. We propose testing of disulfiram in clinical trial for patients with acute leukemia.

Repurposing Disulfiram to Combat Acute Respiratory Distress Syndrome with Targeted Delivery by LET-Functionalized Nanoplatforms.

Acute respiratory distress syndrome (ARDS) is a serious respiratory condition characterized by a damaged pulmonary endothelial barrier that causes protein-rich lung edema, an influx of proinflammatory cells, and treatment-resistant hypoxemia. Damage to pulmonary endothelial cells and inflammation are pivotal in ARDS development with a key role played by endothelial cell pyroptosis. Disulfiram (DSF), a drug that has long been used to treat alcohol addiction, has recently been identified as a potent inhibitor of gasdermin D (GSDMD)-induced pore formation and can thus prevent pyroptosis and inflammatory cytokine release. These findings indicate that DSF is a promising treatment for inflammatory disorders. However, addressing the challenge posed by its intrinsic physicochemical properties, which hinder intravenous administration, and effective delivery to pulmonary vascular endothelial cells are crucial. Herein, we used biocompatible liposomes incorporating a lung endothelial cell-targeted peptide (CGSPGWVRC) to produce DSF-loaded nanoparticles (DTP-LET@DSF NPs) for targeted delivery and reactive oxygen species-responsive release facilitated by the inclusion of thioketal (TK) within the liposomal structure. After intravenous administration, DTP-LET@DSF NPs exhibited excellent cytocompatibility and minor systemic toxicity, effectively inhibited pyroptosis, mitigated lipopolysaccharide (LPS)-induced ARDS, and prevented cytokine storms resulting from excessive immune reactions in ARDS mice. This study presents a straightforward nanoplatform for ARDS treatment that potentially paves the way for the clinical use of this nanomedicine.

Orchestrating Precision within the Tumor Microenvironment by Biomimetic Nanoprodrugs for Effective Tumor Therapy.

Malignant tumors are still one of the most deadly diseases that threaten human life and health. However, developing new drugs is challenging due to lengthy trials, funding constraints, and regulatory approval procedures. Consequently, researchers have devoted themselves to transforming some clinically approved old drugs into antitumor drugs with certain active ingredients, which have become an attractive alternative. Disulfiram (DSF), an antialcohol medication, can rapidly metabolize in the physiological environment into diethyldithiocarbamate (DTC) which can readily react with Cu2+ ions in situ to form the highly toxic bis(N,N-diethyldithiocarbamate)-copper(II) (CuET) complex. In this study, DSF is loaded into mesoporous dopamine nanocarriers and surface-chelated with tannin and Cu2+ to construct M-MDTC nanoprodrugs under the camouflage of K7 tumor cell membranes. After intravenous injection, M-MDTC nanoprodrugs successfully reach the tumor sites with the help of mediated cell membranes. Under slightly acidic pH and photothermal stimulation conditions, DSF and Cu2+ are simultaneously released, forming a highly toxic CuET to kill tumor cells in situ. The generated CuET can also induce immunogenic cell death of tumor cells, increase the proportion of CD86+ CD80+ cells, and promote dendritic cell maturation. In vitro and in vivo studies of M-MDTC nanoprodrugs have shown excellent tumor-cell-killing ability and solid tumor suppression. This approach enables in situ amplification of chemotherapy in the tumor microenvironment, achieving an effective antitumor treatment.

Apoptosis and Paraptosis Induced by Disulfiram-Loaded Ca2+/Cu2+ Dual-Ions Nano Trap for Breast Cancer Treatment.

Regarded as one of the hallmarks of tumorigenesis and tumor progression, the evasion of apoptotic cell death would also account for treatment resistance or failure during cancer therapy. In this study, a Ca2+/Cu2+ dual-ion "nano trap" to effectively avoid cell apoptosis evasion by synchronously inducing paraptosis together with apoptosis was successfully designed and fabricated for breast cancer treatment. In brief, disulfiram (DSF)-loaded amorphous calcium carbonate nanoparticles (NPs) were fabricated via a gas diffusion method. Further on, the Cu2+-tannic acid metal phenolic network was embedded onto the NPs surface by self-assembling, followed by mDSPE-PEG/lipid capping to form the DSF-loaded Ca2+/Cu2+ dual-ions "nano trap". The as-prepared nanotrap would undergo acid-triggered biodegradation upon being endocytosed by tumor cells within the lysosome for Ca2+, Cu2+, and DSF releasing simultaneously. The released Ca2+ could cause mitochondrial calcium overload and lead to hydrogen peroxide (H2O2) overexpression. Meanwhile, Ca2+/reactive oxygen species-associated mitochondrial dysfunction would lead to paraptosis cell death. Most importantly, cell paraptosis could be further induced and strengthened by the toxic dithiocarbamate (DTC)-copper complexes formed by the Cu2+ combined with the DTC, the metabolic products of DSF. Additionally, the released Cu2+ will be reduced by intracellular glutathione to generate Cu+, which can catalyze the H2O2 to produce a toxic hydroxyl radical by a Cu+-mediated Fenton-like reaction for inducing cell apoptosis. Both in vitro cellular assays and in vivo antitumor evaluations confirmed the cancer therapeutic efficiency by the dual ion nano trap. This study can broaden the cognition scope of dual-ion-mediated paraptosis together with apoptosis via a multifunctional nanoplatform.

Tumor-Microenvironment-Responsive Cerium-Enriched Copper Nanozyme with O2 Supply and Oxidative Stress Amplification for In Situ Disulfiram Chemotherapy and Chemodynamic Therapy Intensification.

Traditional chemotherapy has faced tough challenges of systemic toxicity, hypoxia resistance, and inadequacy of monotherapy. Developing the tumor-specific O2-supply-enhanced chemotherapy without toxic drugs while combing other precise treatments can substantially improve therapeutic efficacy. Herein, a CeO2-enriched CuO nanozyme with O2 supply and oxidative stress amplification for tumor-specific disulfiram (DSF) chemotherapy and intensified chemodynamic therapy by synergistic in situ "nontoxicity-toxicity" activation is developed. Notably, CeO2 can not only act as a morphological "regulator," but also serve as a cascaded enzyme-mimetic catalyst via tumor-microenvironment-responsive cascaded-logical programmable valence conversion. Once internalized inside tumor cells, the nanozyme can be degraded by lysosomal acidity to release nontoxic DSF and Cu2+, which can trigger in situ "Cu2+-DSF" chelation, generating a highly toxic Cu(DTC)2 for in situ chemotherapy. Moreover, the enriched CeO2 with catalase-mimetic activity can decompose the endogenous H2O2 into O2, which can relieve the hypoxia to enhance the chemotherapeutic efficacy. Furthermore, the simultaneously generated Ce3+ can exert peroxidase-mimetic activity to catalyze H2O2 into hydroxyl radicals (•OH) for chemodynamic therapy. This Fenton-like chemistry is accompanied by the regeneration of Ce4+, which can deplete the intracellular overproduced GSH to amplify the oxidative stress. Therefore, this nanozyme can provide an alternative to precise cancer treatment.

Combination of the cuproptosis inducer disulfiram and anti­PD­L1 abolishes NSCLC resistance by ATP7B to regulate the HIF­1 signaling pathway.

Disulfiram (DSF) is used to treat non­small cell lung cancer (NSCLC). DSF significantly increases expression of programmed death­ligand 1 (PD­L1), which may enhance immunosuppression and immune escape of tumors. Therefore, the present study aimed to investigate the role of combined treatment of DSF and anti­PD­L1 in NSCLC resistance. The viability and apoptosis of A549 cells were detected by the Cell Counting Kit­8 assay and flow cytometry, respectively. The expression levels of ATPase copper­transporting ß (ATP7B) and PD­L1 in A549 cells were detected by reverse transcription­quantitative PCR and western blot analysis. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) in A549 cells were detected by respective assay kits. The expression levels of cuproptosis­associated proteins ferredoxin­1 (FDX1), ATP7B, solute carrier family 31 member 1 (SLC31A1), succinate dehydrogenase B (SDHB), PD­L1 and hypoxia inducible factor (HIF)­1A were analyzed by western blotting in A549 cells. DSF inhibited the viability of A549 cells and promoted expression levels of ATP7B and PD­L1 at both mRNA and protein levels in A549 cells. The viability of cisplatin (DPP)­treated A549 cells was increased following DSF treatment. JQ­1 (a PD­L1 inhibitor) suppressed the viability of DPP­treated A549 cells pretreated with DSF. DSF increased expression levels of ATP7B and PD­L1. The combination treatment of DSF and JQ­1 in A549 cells increased levels of ROS and MDA, as well as expression levels of FDX1 and SLC31A1; however, combination treatment decreased levels of SOD, as well as expression levels of ATP7B, SDHB, PD­L1, and HIF­1A. PX478 (an HIF­1 inhibitor) acted with DSF to enhance the inhibitory effects on the viability and on the induction of apoptosis of A549 cells. PX478 upregulated the levels of ROS and MDA, while it downregulated levels of SOD in DSF­treated A549 cells. PX478 promoted expression levels of FDX1 and SLC31A1, while it suppressed expression levels of ATP7B, PD­L1, and HIF­1A in DSF­treated A549 cells. In conclusion, the combined treatment of A549 cells with anti­PD­L1 and DSF enhanced the effect of cuproptosis on the inhibition of NSCLC cell viability.

In situ generation of copper(â ¡)/diethyldithiocarbamate complex through tannic acid/copper(â ¡) network coated hollow mesoporous silica for enhanced cancer chemodynamic therapy.

The Cu2+ complex formed by the coordination of disulfiram (DSF) metabolite diethyldithiocarbamate (DTC), Cu(DTC)2, can effectively inhibit tumor growth. However, insufficient Cu2+ levels in the tumor microenvironment can impact tumor-suppressive effects of DTC. In this study, we proposed a Cu2+ and DSF tumor microenvironment-targeted delivery system. This system utilizes hollow mesoporous silica (HMSN) as a carrier, after loading with DSF, encases it using a complex of tannic acid (TA) and Cu2+ on the outer layer. In the slightly acidic tumor microenvironment, TA/Cu undergoes hydrolysis, releasing Cu2+ and DSF, which further form Cu(DTC)2 to inhibit tumor growth. Additionally, Cu2+ can engage in a Fenton-like reaction with H2O2 in the tumor microenvironment to form OH, therefore, chemodynamic therapy (CDT) and Cu(DTC)2 are used in combination for tumor therapy. In vivo tumor treatment results demonstrated that AHD@TA/Cu could accumulate at the tumor site, achieving a tumor inhibition rate of up to 77.6 %. This study offers a novel approach, circumventing the use of traditional chemotherapy drugs, and provides valuable insights into the development of in situ tumor drug therapies.

Disulfiram ameliorates STING/MITA-dependent inflammation and autoimmunity by targeting RNF115.

STING (also known as MITA) is an adaptor protein that mediates cytoplasmic DNA-triggered signaling, and aberrant activation of STING/MITA by cytosolic self-DNA or gain-of-function mutations causes severe inflammation. Here, we show that STING-mediated inflammation and autoimmunity are promoted by RNF115 and alleviated by the RNF115 inhibitor disulfiram (DSF). Knockout of RNF115 or treatment with DSF significantly inhibit systemic inflammation and autoimmune lethality and restore immune cell development in Trex1-/- mice and STINGN153S/WT bone marrow chimeric mice. In addition, knockdown or pharmacological inhibition of RNF115 substantially downregulate the expression of IFN-α, IFN-γ and proinflammatory cytokines in PBMCs from patients with systemic lupus erythematosus (SLE) who exhibit high concentrations of dsDNA in peripheral blood. Mechanistically, knockout or inhibition of RNF115 impair the oligomerization and Golgi localization of STING in various types of cells transfected with cGAMP and in organs and cells from Trex1-/- mice. Interestingly, knockout of RNF115 inhibits the activation and Golgi localization of STINGN153S as well as the expression of proinflammatory cytokines in myeloid cells but not in endothelial cells or fibroblasts. Taken together, these findings highlight the RNF115-mediated cell type-specific regulation of STING and STINGN153S and provide potential targeted intervention strategies for STING-related autoimmune diseases.

Nrf2/HO-1 Alleviates Disulfiram/Copper-Induced Ferroptosis in Oral Squamous Cell Carcinoma.

Accumulating evidence indicates that the disulfiram/copper complex (DSF/Cu) has been shown to have potent antitumor activity against various cancers. This research evaluated the effects and probable mechanisms of DSF/Cu on oral squamous cell carcinoma (OSCC). In this study, we report the toxicity of the DSF/Cu to OSCC both in vitro and in vivo. Our study showed that DSF/Cu reduced the proliferation and clonogenicity of OSCC cells. DSF/Cu also induced ferroptosis. Importantly, we confirmed that DSF/Cu could increase the free iron pool, enhance lipid peroxidation, and eventually result in ferroptosis cell death. Inhibition of NRF2 or HO-1 enhances the sensitivity of OSCC cells to DSF/Cu-induced ferroptosis. DSF/Cu inhibited the xenograft growth of OSCC cells by suppressing the expression of Nrf2/HO-1. In conclusion, these results provide experimental evidence that Nrf2/HO-1 alleviates DSF/Cu-induced ferroptosis in OSCC. We propose that this therapy could be a novel strategy for treating OSCC.

Inhibiting the compensatory elevation of xCT collaborates with disulfiram/copper-induced GSH consumption for cascade ferroptosis and cuproptosis.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors and the fourth leading cause of cancer-related death globally, which is characterized by complicated pathophysiology, high recurrence rate, and poor prognosis. Our previous study has demonstrated that disulfiram (DSF)/Cu could be repurposed for the treatment of HCC by inducing ferroptosis. However, the effectiveness of DSF/Cu may be compromised by compensatory mechanisms that weaken its sensitivity. The mechanisms underlying these compensatory responses are currently unknown. Herein, we found DSF/Cu induces endoplasmic reticulum stress with disrupted ER structures, increased Ca2+ level and activated expression of ATF4. Further studies verified that DSF/Cu induces both ferroptosis and cuproptosis, accompanied by the depletion of GSH, elevation of lipid peroxides, and compensatory increase of xCT. Comparing ferroptosis and cuproptosis, it is interesting to note that GSH acts at the crossing point of the regulation network and therefore, we hypothesized that compensatory elevation of xCT may be a key aspect of the therapeutic target. Mechanically, knockdown of ATF4 facilitated the DSF/Cu-induced cell death and exacerbated the generation of lipid peroxides under the challenge of DSF/Cu. However, ATF4 knockdown was unable to block the compensatory elevation of xCT and the GSH reduction. Notably, we found that DSF/Cu induced the accumulation of ubiquitinated proteins, promoted the half-life of xCT protein, and dramatically dampened the ubiquitination-proteasome mediated degradation of xCT. Moreover, both pharmacologically and genetically suppressing xCT exacerbated DSF/Cu-induced cell death. In conclusion, the current work provides an in-depth study of the mechanism of DSF/Cu-induced cell death and describes a framework for the further understanding of the crosstalk between ferroptosis and cuproptosis. Inhibiting the compensatory increase of xCT renders HCC cells more susceptible to DSF/Cu, which may provide a promising synergistic strategy to sensitize tumor therapy and overcome drug resistance, as it activates different programmed cell death.

Microgel Encapsulated Nanoparticles for Intra-articular Disulfiram Delivery to Treat Osteoarthritis.

Osteoarthritis (OA) affects numerous patients worldwide, and there are no approved disease-modifying drugs. Repurposing FDA-approved small molecular drugs could be a promising alternative strategy to treat OA. Disulfiram (DSF), a clinically approved drug for treatment of alcoholism, inhibits inflammasome activation and exhibits a protective role in interleukin-1ß-induced cardiac injury. However, its efficacy in treating OA remains to be explored due to its poor water solubility and stability, which limit its use in OA treatment. Here, the anti-inflammatory effect of DSF is evaluated in vitro, and a double-layer encapsulation approach is developed for intra-articular delivery of DSF for OA treatment in vivo. DSF is loaded into poly(lactic-co-glycolic acid)-based nanoparticles and encapsulated in gelatin methacrylate microgels through a microfluidic device. Results show that DSF effectively inhibits the expression of key inflammatory cytokines in OA chondrocytes, and the double-layer encapsulation approach reduces the burst release of DSF and prolongs its retention time in the in vitro study. Sustained release of DSF from microgels mitigates cartilage inflammation and subchondral bone erosion in a monoiodoacetate-induced rat OA model. This work demonstrates the potential of repurposing FDA-approved drugs for OA treatment and provides a promising platform for intra-articular delivery of small molecules for superior therapeutic effect.

A cautionary note on the use of N-acetylcysteine as a reactive oxygen species antagonist to assess copper mediated cell death.

A new form of cell death has recently been proposed involving copper-induced cell death, termed cuproptosis. This new form of cell death has been widely studied in relation to a novel class of copper ionophores, including elesclomol and disulfiram. However, the exact mechanism leading to cell death remains contentious. The oldest and most widely accepted biological mechanism is that the accumulated intracellular copper leads to excessive build-up of reactive oxygen species and that this is what ultimately leads to cell death. Most of this evidence is largely based on studies using N-acetylcysteine (NAC), an antioxidant, to relieve the oxidative stress and prevent cell death. However, here we have demonstrated using inductively coupled mass-spectrometry, that NAC pretreatment significantly reduces intracellular copper uptake triggered by the ionophores, elesclomol and disulfiram, suggesting that reduction in copper uptake, rather than the antioxidant activity of NAC, is responsible for the diminished cell death. We present further data showing that key mediators of reactive oxygen species are not upregulated in response to elesclomol treatment, and further that sensitivity of cancer cell lines to reactive oxygen species does not correlate with sensitivity to these copper ionophores. Our findings are in line with several recent studies proposing the mechanism of cuproptosis is instead via copper mediated aggregation of proteins, resulting in proteotoxic stress leading to cell death. Overall, it is vital to disseminate this key piece of information regarding NAC's activity on copper uptake since new research attributing the effect of NAC on copper ionophore activity to quenching of reactive oxygen species is being published regularly and our studies suggest their conclusions may be misleading.

Photothermal performance of a novel carbon dot and its conjugate with disulfiram for prostate cancer PC3 cell therapy.

Aim: To develop and employ a copper, sulfur, nitrogen-carbon quantum dot (C,S,N-CQD) multifunctional platform for synergistic cancer therapy, combining chemotherapy and photothermal treatment with in vitro cancer cell imaging. Materials & methods: Cu,S,N-CQDs were synthesized hydrothermally, loaded with disulfiram (DSF), and characterized through UV-Vis spectrophotometry, photoluminescence, Fourier-transform infrared spectroscopy, high-resolution transmission electron microscopy, dynamic light scattering, x-ray diffraction and EDAX. Results: Cu,S,N-CQD exhibited 5.5% absolute fluorescence quantum yield, 46.0% photothermal conversion efficiency and excellent stability. The release of DSF-loaded Cu,S,N-CQD, photothermal performance, and IC50 on PC3 prostate cancer cells, were evaluated. The impact of cellular glutathione on nanocarrier performance was investigated. Conclusion: Cu,S,N-CQD as a photothermal agent and DSF carrier showed synergy (combination index: 0.71) between chemotherapy and photothermal therapy. The nanocarrier simultaneously employed for in vitro cancer cell imaging due to its unique fluorescence properties.

Nanometer-scale particles can be used to treat and detect cancer in many ways. A type of nanoparticle was designed to attack cancer in two different ways. These nanoparticles ­ copper, sulfur, nitrogen­carbon quantum dots (C,S,N­CQDs) ­ were designed to both deliver a chemotherapy drug to cancer cells and act as a photothermal agent. This means that when light of a particular energy is shone on these particles, they heat up and can kill cancer cells. These C,S,N­CQDs loaded with the chemotherapy drug disulfiram were then tested on the prostate cancer cell line PC3. When a laser was shone on these particles and they became excited, they reduced cancer cell viability both by releasing the drug and heating up and killing the surrounding cells. These Cu,S,N-CQDs are also fluorescent, meaning they can be used to image cancer cells in tests like these.