Anticancer effects of disulfiram: a systematic review of in vitro, animal, and human studies

Background and objectives Cancer morbidity and mortality rates remain high, and thus, at present, considerable efforts are focused on finding drugs with higher sensitivity against tumor cells and fewer side effects. Disulfiram (DSF), as an anti-alcoholic drug, kills the cancer cells by inducing apoptosis. Several preclinical and clinical studies have examined the potential of repurposing DSF as an anticancer treatment. This systematic review aimed to assess evidence regarding the antineoplastic activity of DSF in in vitro and in vivo models, as well as in humans. Methods Two authors independently conducted this systematic review of English and Chinese articles from the PubMed, Embase, and the Cochrane Library databases up to July 2019. Eligible in vitro studies needed to include assessments of the apoptosis rate by flow cytometry using annexin V/propidium iodide, and studies in animal models and clinical trials needed to examine tumor inhibition rates, and progression-free survival (PFS) and overall survival (OS), respectively. Data were analyzed using descriptive statistics. Results Overall, 35 studies, i.e., 21 performed in vitro, 11 based on animal models, and three clinical trials, were finally included. In vitro and animal studies indicated that DSF was associated with enhanced apoptosis and tumor inhibition rates, separately. Human studies showed that DSF prolongs PFS and OS. The greatest anti-tumor activity was observed when DSF was used as combination therapy or as a nanoparticle-encapsulated molecule. There was no noticeable body weight loss after DSF treatment, which indicated that there was no major toxicity of DSF. Conclusions This systematic review provides evidence regarding the anti-tumor activity of DSF in vitro, in animals, and in humans and indicates the optimal forms of treatment to be evaluated in future research. Supplementary Information The online version contains supplementary material available at 10.1186/s13643-021-01858-4.


Introduction
Cancer is expected to be the leading cause of death and the foremost contributor to decreased life expectancy in every country worldwide during the twenty-first century and beyond [1]. Although comprehensive therapies prolong survival and improve the quality of life of cancer patients, approximately 96,000,000 cancer deaths occurred in 2018 worldwide [1]. The global community is well aware that new drug development, discovery, and synthesis are a time-consuming process, which involves intensive work and appraisal of the cost-effectiveness of the drug under development [2]. As a result, researchers are allocating considerable efforts for repurposing existing drugs such as disulfiram (DSF).
In the 1800s, DSF was used as an industrial catalyst in the production of rubber [3]. In 1948, DSF was approved by the Food and Drug Administration for treating alcoholism [4]. In 1988, DSF was associated with a decrease in the occurrence of occasional infections in symptomatic patients with human immunodeficiency virus infection [5], prompting the conduct of a wealth of clinical trials, some of which are still ongoing (www. clini caltr ials. gov).

Open Access
*Correspondence: liym@lzu.edu.com 3 Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou University Second Hospital, Lanzhou, Gansu 730030, P.R. China Full list of author information is available at the end of the article The antineoplastic activity of DSF was first recorded in 1977 by Dr. Lewison in a 35-year-old female breast cancer patient with systemic metastases who received DSF for her severe alcoholic syndrome and remained clinically free of cancer for 10 years without receiving any form of anticancer therapy [6]. This observation was noted in an era in which the anticancer effect of DSF was being researched. In recent years, a large number of preclinical studies and clinical trials (www. clini caltr ials. gov) of DSF have been conducted to explore the anticancer activities of this drug. Nonetheless, the antitumor effectiveness of DSF remains uncertain owing to existing heterogeneity across different studies with cell lines, animals, and humans. Currently, a systematic review of these studies to assess and clarify the anticancer potential of DSF is lacking.
It is worthy to explore whether there are substantial differences and are appropriate for clinical proposals. Therefore, this study aimed to perform a systematic review of published data on the antitumor activity of DSF. Specifically, this review aimed to assess the apoptosis and tumor inhibition rates of DSF based on data from studies in cell lines and animal models, respectively, and examine the benefit of DSF on progression-free survival (PFS) and overall survival (OS) based on results from clinical studies, regardless of the study design or type of cancer investigated. Meanwhile, it is important for evaluating the anti-tumor effect of disulfiram to include in the side effects. The side effects of disulfiram will be covered in this article.

Materials and methods
The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed to conduct this systematic review [7].

Search strategy
PubMed, Embase, and Cochrane Library databases were searched for relevant studies from their inception to the end of July 2019. The search was performed with a combination of Medical Subject Headings and free words as follows: (neoplasia OR neoplasm OR tumor OR cancer OR malignancy OR malignant neoplasm), and [disulfiram OR bis (diethylthiocarbamoyl) disulfide OR tetraethylthioperoxydicarbonic diamide OR tetraethylthiuram disulfide OR tetraethylthiuram OR antabus OR antabuse teturam OR dicupral OR esperal OR alcophobin OR anticol]. The details of the search strategy are presented in the supplement.

Study selection
Studies that implemented the below criteria were included: (1) solid cancer cell lines, animals, or patients treated with DSF; (2) in vitro studies focusing on parameters of the apoptosis index (early apoptosis or early apoptosis plus late apoptosis) using annexin V-fluorescein isothiocyanate/propidium iodide double-staining analysis by flow cytometry, in vivo studies evaluating the tumor inhibition rate in cell-line-derived xenograft animal models, or studies in humans, which included OS and PFS as endpoints, to assess the effect of DSF in cancer patients; and (3) studies published in the English and Chinese language. There were no restrictions on the type of cancer studied. To avoid duplication of data, only the most recent and most comprehensive articles were included. Studies with incomplete data or conference abstracts were excluded. Two investigators (Ling Wang, Run Wan) independently screened the databases for studies based on the eligibility criteria. Any discrepancies were resolved by consulting a third researcher (Cong Zhou).

Data derivation
Two investigators (Ling Wang, Cong Zhou) independently extracted data from the inclusive studies. Inconsistencies between the two investigators were resolved by consulting a third reviewer (Run Wan). When required, we contacted the authors of the research for further information. A pre-designed structured outline was used to abstract data. The outline included the following fields: study type (in vitro, in vivo, clinical study, or case series); general information (first author, publication year, country, and study design); supplement used; anticancer treatment used; and outcomes (i.e., apoptosis rate, tumor inhibition rate, OS and PFS, as applicable). The results of each study included were summarized. Descriptive statistics were used for data analysis. Meta-analysis was not performed owing to substantial heterogeneity across studies.

Study characteristics
The initial search yielded a total of 1278 studies. After excluding 274 irrelevant and duplicate studies, the full texts of 1004 studies were screened. Of these, 148 were considered eligible based on the availability of full texts as well as the description of target outcomes. Ultimately, 113 articles were removed (no full texts, n = 43; no target outcomes, n = 70), and 35 studies were selected. A detailed description of the steps followed during the retrieval process is provided in Fig. 1.
The three human studies included participants with differing characteristics and cancer types. All three clinical trials investigated DSF as a combination therapy with chemotherapy or/and radiation therapy [39][40][41], while two studies reported on adverse events [39,40].

Outcomes
Three cell lines and one animal study showed that treatment with DSF as a single agent induced apoptosis and increased the rate of tumor inhibition [17][18][19]35]. Although the sensitivity between the various cell lines varied, dose-dependency was consistently observed.
Compared with free molecule, DSF encapsulated with nanomaterials significantly induced selective deathdependent apoptosis, especially in acidic conditions (pH = 6.5) in cancer cell lines. Eleven animal studies demonstrated that DSF modified by particular nanomaterials increased the tumor inhibition rate and that the anticancer activity was more obvious when chemotherapy (Cis) was combined with nanoencapsulated DSF [32].
Changes in body weight during the whole study period were analyzed in nine animal studies. With the exception of three reports of weight changes in DSF-treated or DSF-modified groups [30,33,36], other studies recorded that there was no noticeable body weight loss after DSF treatment or no significant difference in body weight changes across different groups [28-32, 34, 36, 38], which       indicated that there was no major toxicity of DSF [28,29,[32][33][34][35].
Many clinical trials have mentioned the use of DSF for solid tumors (www. clini caltr ials. gov). One study clearly analyzed the difference in PFS (5.9 versus 4.9 months) and OS (10.0 versus 7.1 months) between control and experimental groups [42]. PFS and OS both improved in the experimental groups. Two studies described PFS and OS of the entire research cohort, and the treatment efficacy seemed to be in contrast to historical data [39,40]. Our systemic review included two single-arm trials in glioblastoma (GBM) patients and a randomized controlled trial in NSCLC patients. Although the two single-arm clinical trials did not compare treatment with a control group, positive effects were observed; e.g., a 40-year-old woman with unmethylated isocitrate dehydrogenase wild-type GBM had good health without any signs of tumor recurrence 33 months after study initiation.
Among the reported adverse effects, none were serious, and they were of grades 2-3. Adverse effects were reported in two studies and included diarrhea, nausea, dizziness, vomiting, motor neuropathy, and elevated alanine transaminase levels. Symptoms resolved quickly when the dose was reduced [39,40].
All three studies show that DSF is safe and seems to prolong survival of cancer patients. Because of individual differences in patients, the response to DSF was also varied [39,40,42]. The optimal concentration and sensitivity type should be further explored by in vitro and animal studies.

Discussion
DSF is decomposed into diethyldithiocarbamate in the body and exhibits anticancer activities [43]. Considering that the loss of cellular proliferation control leads to the development of cancer, effective clinical therapies of cancer have been developed based on the principle of inducing apoptosis [44]. In the included animal studies, the tumor inhibition rate was utilized to evaluate antitumor efficiency by calculating tumor volume. Most studies included in this review revealed enhanced apoptosis and tumor inhibition rates with DSF treatment (Table 4).
In recent years, metal-based complexes have been reported to exhibit anticancer activity [45]. Silver complexes demonstrate anti-tumor activity and display low toxicity in humans. The mechanism of action is related to their interaction with nucleic acids and proteins [46]. Metabolites of DSF chelate with metal ions, leading to alterations in the intracellular levels of metal ions, enhancement of oxidative stress, inhibition of the activities of superoxide dismutase or matrix metalloproteinases, inactivation of essential sulfhydryl groups by protein carbamoylation, and alteration of cancer cell invasion, tumor angiogenesis, and metastasis [47,48]. The observation that the combination of DSF with metal ions (Cu, Ag) leads to enhanced anticancer effectiveness is in accordance with the observations of in vitro and animal experiments [11,14].
In different cancer cell lines, the lethal concentration of DSF was different. The lethal concentration was reduced when DSF combined with metal ions or nano-reconstructed DSF.
The additive/synergistic action of DSF with other chemotherapy agents in inhibiting tumor cell growth and cytotoxicity is mediated through the enhancement of cellular oxidative stress, inhibition of P glycol-protein (P-gp) activity, and dysregulation of the NF-κB signaling pathway [8,49,50].
In the examined studies, anti-tumor activity, as evidenced by higher apoptosis and tumor inhibition rates, was enhanced with DSF-NPs in various ways. At the pH of 7.4, the half-life of DSF is 1-1.5 min [47]. The half-life was improved by nanomaterial packaging of DSF, with the anti-tumor effects increasing under acidic conditions (pH = 6.5) [51]. DSF-NPs enhanced cellular uptake, induced high levels of reactive oxygen species, activated the MAP-kinase pathway, sustained drug supply, and blocked copolymer micelles, such as the P-gp inhibitor [14,20,52]. Evidence supports that DSF-NPs ameliorate the instability and low treatment efficacy of free DSF.
Event-free survival (EFS) means that there are no adverse events since the start of treatment, including change of regimen, adverse side effects, intolerance, disease progression, and patient death. EFS represents a direct measure of the ability of the treatment to achieve a response, the durability of the response achieved, and its capacity to prolong life [53]. It was found that the doses of disulfiram significantly increased EFS [39].