Advanced metastatic colorectal cancer (CRC) with deficient DNA mismatch repair (MMR-d), or immune-hot CRCs, show significantly improved clinical outcomes compared to MMR-proficient (MMR-p), or immune-cold CRCs. While the prior represents about 5% of all CRCs, the latter represent 95% and are characterized by low immunogenicity. This study investigates bis-diethyldithiocarbamate (CuET), a novel anticancer compound, and its impact on the colorectal cancer tumor microenvironment (TME). CuET is shown to convert immunologically inactive tumors into hotbeds of antitumor immune responses, marked by increased lymphocyte infiltration, heightened cytotoxicity of natural killer (NK) and T cells, and enhanced non-self recognition by lymphocytes. The potent anticancer cytotoxicity and in vivo safety and efficacy of CuET are established. In summary, CuET transforms the colorectal cancer TME, bolstering NK and T cell cytotoxicity and refining tumor cell recognition through non-classical activation via the NKG2D/NKG2DL axis. This study unveils a novel mechanism of action for CuET: a potent immunomodulator capable of turning cold tumors hot.
- MeSH
- Killer Cells, Natural immunology drug effects metabolism MeSH
- Ditiocarb * pharmacology MeSH
- Colorectal Neoplasms * drug therapy immunology metabolism pathology MeSH
- NK Cell Lectin-Like Receptor Subfamily K * metabolism MeSH
- Humans MeSH
- Copper MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Tumor Microenvironment * drug effects immunology MeSH
- Antineoplastic Agents pharmacology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
PURPOSE: This phase 1/2 study aimed to evaluate the safety and preliminary efficacy of combining disulfiram and copper (DSF/Cu) with radiation therapy (RT) and temozolomide (TMZ) in patients with newly diagnosed glioblastoma (GBM). METHODS AND MATERIALS: Patients received standard RT and TMZ with DSF (250-375 mg/d) and Cu, followed by adjuvant TMZ plus DSF (500 mg/d) and Cu. Pharmacokinetic analyses determined drug concentrations in plasma and tumors using high-performance liquid chromatography-mass spectrometry. RESULTS: Thirty-three patients, with a median follow-up of 26.0 months, were treated, including 12 IDH-mutant, 9 NF1-mutant, 3 BRAF-mutant, and 9 other IDH-wild-type cases. In the phase 1 arm, 18 patients were treated; dose-limiting toxicity probabilities were 10% (95% CI, 3%-29%) at 250 mg/d and 21% (95% CI, 7%-42%) at 375 mg/d. The phase 2 arm treated 15 additional patients at 250 mg/d. No significant difference in overall survival or progression-free survival was noted between IDH- and NF1-mutant cohorts compared with institutional counterparts treated without DSF/Cu. However, extended remission occurred in 3 BRAF-mutant patients. Diethyl-dithiocarbamate-copper, the proposed active metabolite of DSF/Cu, was detected in plasma but not in tumors. CONCLUSIONS: The maximum tolerated dose of DSF with RT and TMZ is 375 mg/d. DSF/Cu showed limited clinical efficacy for most patients. However, promising efficacy was observed in BRAF-mutant GBM, warranting further investigation.
- MeSH
- Antineoplastic Agents, Alkylating therapeutic use pharmacokinetics MeSH
- Chemoradiotherapy * methods MeSH
- Disulfiram * therapeutic use pharmacokinetics administration & dosage MeSH
- Progression-Free Survival MeSH
- Adult MeSH
- Glioblastoma * radiotherapy genetics mortality therapy drug therapy MeSH
- Isocitrate Dehydrogenase genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Copper * blood therapeutic use MeSH
- Brain Neoplasms * radiotherapy mortality genetics therapy MeSH
- Proto-Oncogene Proteins B-raf genetics MeSH
- Aged MeSH
- Temozolomide * therapeutic use pharmacokinetics administration & dosage MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Clinical Trial, Phase I MeSH
- Clinical Trial, Phase II MeSH
Dithiocarbamates (DTCs) are simple organic compounds with many applications in industry and medicine. They are potent metal chelators forming complexes with various metal ions, including copper. Recently, bis(diethyldithiocarbamate)-copper complex (CuET) has been identified as a metabolic product of the anti-alcoholic drug Antabuse (disulfiram, DSF), standing behind DSF's reported anticancer activity. Mechanistically, CuET in cells causes aggregation of NPL4 protein, an essential cofactor of the p97 segregase, an integral part of the ubiquitin-proteasome system. The malfunction of p97/NPL4 caused by CuET leads to proteotoxic stress accompanied by heat shock and unfolded protein responses and cancer cell death. However, it is not known whether the NPL4 inhibition is unique for CuET or whether it is shared with other dithiocarbamate-copper complexes. Thus, we tested 20 DTCs-copper complexes in this work for their ability to target and aggregate NPL4 protein. Surprisingly, we have found that certain potency against NPL4 is relatively common for structurally different DTCs-copper complexes, as thirteen compounds scored in the cellular NPL4 aggregation assay. These compounds also shared typical cellular phenotypes reported previously for CuET, including the NPL4/p97 proteins immobilization, accumulation of polyubiquitinated proteins, the unfolded protein, and the heat shock responses. Moreover, the active complexes were also toxic to cancer cells (the most potent in the nanomolar range), and we have found a strong positive correlation between NPL4 aggregation and cytotoxicity, confirming NPL4 as a relevant target. These results show the widespread potency of DTCs-copper complexes to target NPL4 with subsequent induction of lethal proteotoxic stress in cancer cells with implications for drug development.
Disulfiram je jedným z preparátov účinných a odporúčaných na prevenciu relapsu pri liečbe závislosti od alkoholu. Centrum pre liečbu drogových závislostí v Bratislave od roku 2019 zaviedlo inovovaný liečebný program s využitím tohto lieku. Cieľom našej práce bolo preskúmať účinnosť komplexného terapeutického programu s využitím disulfiramu. Výskumnú vzorku tvorilo dohromady 159 probandov, ktorí sa liečili pre diagnózu závislosť od alkoholu (F10.2) v našom centre. Porovnávali sme výskumný súbor 35 probandov v disulfiramovom programe s kontrolnou skupinou 124 probandov v obvyklej liečbe. Sledovali sme komplianciu a počet relapsov v období 12 mesiacov od vstupu do liečby v disulfiramovom programe a v obvyklej liečbe. Komplianciou v našom výskume rozumieme účasť pacienta na liečbe v dvojtýždňových intervaloch počas 1 roka od vstupu do liečby. Pacienti v disulfiramovom programe dosiahli priemernú komplianciu 20,83 z 24 dvojtýždňových intervalov. Pacienti v obvyklej liečbe sa liečby zúčastnili priemerne 7,52 z 24 intervalov. Pacienti v disulfiramovom programe mali v sledovanom období priemerne 0,32 relapsu. Pacienti v obvyklej liečbe mali priemerne 0,91 relapsu. Rozdiely oboch premenných boli štatisticky významné. Naše skúsenosti a výskumné zistenia naznačujú vysokú účinnosť liečby s využitím disulfiramu a podporujú odôvodnenosť obnovenia regulárnej disulfiramovej liečby na Slovensku v inovovanom komplexnom terapeutickom usporiadaní.
Disulfiram is one of the medicines that are effective and recommended for relapse prevention in alcohol dependence treatment. Centre for treatment of drug dependencies in Bratislava since January 2019 started to implement innovated treatment programme utilizing disulfiram. The aim of our study was to explore the effectiveness of a complex therapeutic disulfiram programme. The study involved 159 participants treated for alcohol dependence (F10.2) in our centre. We have compared disulfiram group consisting of 35 subjects with control group consisting of 124 subjects in treatment as usual. The groups were compared in compliance and relapse rate over 12 months period after entering the treatment in either disulfiram programme or treatment as usual. Compliance in our study is defined as participation of patient in treatment in 2-week intervals for the first year after entering the treatment. Patients in disulfiram programme have average compliance rate 20,83 out of 24 two-week intervals. Patients in treatment as usual have participated 7,52 out of 24 intervals on average. Patients in disulfiram programme have average relapse rate 0,32 times per year. Patients in treatment as usual have relapsed 0,91 times on average. We have found statistically significant differences in both variables. Our experiences and study results indicate high efficacy of disulfiram and support justification of disulfiram treatment renewal in Slovakia, as a part of innovated complex therapeutic programme.
Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.
- MeSH
- Zebrafish metabolism MeSH
- Disulfiram * metabolism MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 genetics metabolism MeSH
- Neoplasms * metabolism MeSH
- Ribosomes metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Disulfiram (DSF), an established alcohol-aversion drug, is a candidate for repurposing in cancer treatment. DSF's antitumor activity is supported by preclinical studies, case reports, and small clinical trials; however, ongoing clinical trials of advanced-stage cancer patients encounter variable results. Here, we show that one reason for the inconsistent clinical effects of DSF may reflect interference by other drugs. Using a high-throughput screening and automated microscopy, we identify cannabidiol, an abundant component of the marijuana plant used by cancer patients to mitigate side effects of chemotherapy, as a likely cause of resistance to DSF. Mechanistically, in cancer cells, cannabidiol triggers the expression of metallothioneins providing protective effects by binding heavy metal-based substances including the bis-diethyldithiocarbamate-copper complex (CuET). CuET is the documented anticancer metabolite of DSF, and we show here that the CuET's anticancer toxicity is effectively neutralized by metallothioneins. Overall, this work highlights an example of undesirable interference between cancer therapy and the concomitant usage of marijuana products. In contrast, we report that insufficiency of metallothioneins sensitizes cancer cells toward CuET, suggesting a potential predictive biomarker for DSF repurposing in oncology.
Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.
- MeSH
- Spheroids, Cellular pathology MeSH
- Disulfiram pharmacology MeSH
- Fluorouracil * pharmacology therapeutic use MeSH
- Colorectal Neoplasms * drug therapy pathology MeSH
- Humans MeSH
- Copper pharmacology therapeutic use MeSH
- Cell Line, Tumor MeSH
- Tumor Microenvironment MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The current Coronavirus 2019 (COVID-19) pandemic has shown us that the pharmaceutical research community can organize and administer large nonprofit clinical trials (RECOVERY and SOLIDARITY) and achieve the swift development of common, unpatentable drugs for a new indication: in this case an old, inexpensive drug, dexamethasone, for COVID-19. Why is it that such nonprofit efforts are so rare and are not organized as a systemic, routine part of drug development in the public interest? Based on my own experience with repurposing the alcohol-abuse drug disulfiram (Antabuse) for cancer, I identify at least four serious deadlocks to development of nonprofit drugs. All of these obstacles should be addressed to leverage the potential of the COVID-19 pandemic for better future healthcare systems in all countries around the world.
- MeSH
- COVID-19 * MeSH
- Disulfiram MeSH
- Humans MeSH
- Organizations, Nonprofit MeSH
- Pandemics * MeSH
- Delivery of Health Care MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
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.
- MeSH
- Bortezomib pharmacology therapeutic use MeSH
- Drug Resistance, Neoplasm MeSH
- Disulfiram pharmacology MeSH
- Proteasome Inhibitors pharmacology therapeutic use MeSH
- Humans MeSH
- Neoplasm Recurrence, Local drug therapy MeSH
- Multiple Myeloma * pathology MeSH
- Cell Line, Tumor MeSH
- Drug Repositioning MeSH
- Proteasome Endopeptidase Complex metabolism MeSH
- Antineoplastic Agents * pharmacology therapeutic use MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The expression of genes related to the Toll-like receptors (TLRs) signaling pathway were determined. Group A, B and C fed with basal diet and group D, E and F induced TD by feeding a basal diet containing 100 mg·kg-1 thiram. rGSTA3 protein was injected at 20 μg·kg-1 in group B, E and at 50 μg·kg-1 in C, F. Results suggested that lameness and death of chondrocytes were significant on day 14. TLRs signaling pathway related genes were screened based on the transcriptome enrichment, and validated on qPCR. IL-7, TLR2, 3, 4, 5, 7, 15, MyD88, MHC-II, MDA5 and TRAF6 were significantly (p < 0.05) expressed in group E and F as compared to group D on day 14 and 23. IL-7, MHCII, TRAF6, TLR3, TLR5, TLR7, and TLR15 determined insignificant in group D compared to group A on day 23. TD occur in an early phase and alleviated in the later period. rGSTA3 protein can prevent apoptosis and repair degraded chondrocytes.
- MeSH
- Apoptosis MeSH
- Chondrocytes physiology MeSH
- Erythrocytes physiology MeSH
- Glutathione Transferase genetics metabolism MeSH
- Chickens immunology MeSH
- Poultry Diseases immunology MeSH
- Osteochondrodysplasias immunology MeSH
- Immunity, Innate MeSH
- Avian Proteins genetics metabolism MeSH
- Recombinant Proteins metabolism MeSH
- Signal Transduction genetics MeSH
- Thiram metabolism MeSH
- Toll-Like Receptors metabolism MeSH
- Transcriptome MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
