Evaluation of the accumulation of disulfiram and its copper complex in A549 cells using mass spectrometry

Highlights

• A549 cell viability decreased with the increase of the DDC-Cu solution concentration.

• DDC-Cu could promote the production of intracellular ROS hence inducing apoptosis.

• Flow cytometry was used to analyze the influence of drug stimulation on A549 cell cycle.

• Metabolic kinetics of DSF/Cu and DDC-Cu in cancer cell were studied by mass spectrometry.

• Quantitative analysis of DDC-Cu inside and outside the cells were performed.

Abstract

The famous alcohol-aversion drug disulfiram (DSF) is a promising candidate for repurposing in cancer therapy, as indicated by many ongoing and completed clinical trials. Existing researches focus on demonstrating that the anti-cancer activity of DSF is enhanced by copper ions, or solving the problem that DSF is easily decomposed in the body to lose its activity. However, the metabolic kinetics of its ultimate anti-cancer metabolite DDC-Cu (bis-diethyldithiocarbamate-copper) in cells and how it exerts anti-cancer mechanisms remain unclear. In this work, mass spectrometric evaluation of the intracellular and extracellular accumulation of DSF and its copper complex DDC-Cu was performed. Combined with cytotoxicity assay, staining analysis and flow cytometry, we found that DDC-Cu could easily pass through the cell membrane of A549 cells, and accumulate intracellularly for a long time. This process can lead to cellular morphological changes, an increase in ROS content, cell cycle arrest in the G0/G1 phase and apoptosis. Besides, molecular cancer-relevant targets of DDC-Cu in cancer cells were further discussed. This work investigated the cytotoxic mechanism of DDC-Cu, which has important clinical significance for its application in cancer therapy.

Introduction

Cancer has always been one of the leading causes of death around the world over the last few decades. This disease has had a high global impact and continues to cause high morbidity and mortality. As medical technology develops and becomes increasingly more advanced, we are still struggling to fully understand cancer biology. Among them, lung cancer is a common malignant tumor with a high mortality rate and the five-year survival rate is less than 5% [1,2]. The most common type of lung cancer is a non-small cell lung cancer (NSCLC). It accounts for about 85% of all lung cancers [3]. Treatment for cancer is very difficult because there are many contributing factors and poor prognosis. In recent years, early diagnosis of lung cancer has made progress, but the results still remain gloomy [4]. 40% of newly diagnosed non-small cell lung cancer patients were in the terminal stages of cancer. The survival time of palliative chemotherapy is only about 8–11 months. This kind of cancer with metastasis is commonly considered to be incurable [5]. Therefore, the severe lung cancer situation highlights the importance of treatment innovation. Due to the high development cost of new drugs and the long test period [6], drug repositioning has become an attractive strategy for the development of anti-cancer drugs [7,8]. The famous alcohol-aversion drug Disulfiram (DSF) has been approved by the US Food and Drug Administration for the treatment of alcoholism, inhibiting the effects of acetaldehyde dehydrogenase on alcohol dependence, and has been used clinically for more than 60 years [[9], [10], [11]]. Preclinical data indicated that DSF has antitumor activity and is a promising anti-cancer drug [[12], [13], [14]]. It also exhibited anti-cancer activity in a variety of in vitro cancer cell lines [[15], [16], [17]]. According to reports, its anticancer activity is enhanced after complexation with copper ions [[16], [17], [18], [19]]. Copper is an essential trace metal in the body, and many key enzymes and transcription factors require copper for its activity [20]. Among many human cancers, including lung cancer and breast cancer, the copper content in serum or tissue is high [21,22], which is essential for the formation of copper complexes in vivo and for exerting anti-cancer effects. DSF quickly metabolized to diethyldithiocarbamate in the body, and the metabolite can firmly bind copper ions to form a complex DDC-Cu, DDC-Cu is more stable than DSF [23]. Furthermore, many drugs cannot distinguish between normal cells and cancer cells, and they have a killing effect on both of them [24], which demonstrate the selective action of drugs on cancer cells is particularly important. Researchers have found that inside the tumor of the mouse, the content of DDC-Cu complex was remarkably higher than other normal tissues, indicating that DSF can selectively enter tumor tissues to ensure the consequences of accidental injury to normal tissues [12]. Therefore, DDC-Cu is a promising anti-cancer drug candidate. The focus of existing research is to enhance the anticancer activity of DSF by copper ions [[16], [17], [18], [19]], or solving the problem that DSF is easily decomposed and inactivated in the body [[25], [26], [27]]. However, the metabolic kinetics of its ultimate anti-cancer metabolite DDC-Cu in cancer cells and how it exerts anti-cancer mechanisms remains unclear. We need an effective way to monitor drugs inside and outside the cell. Mass spectrometry has become one of the most powerful tools for identifying cellular metabolites [[28], [29], [30]]. Pharmacokinetics can be studied by realtime quantitative monitoring with mass spectrometry multiple reaction monitoring (MRM) technology. MRM has the advantages of high specificity, high sensitivity, high accuracy, good reproducibility, wide linear dynamic range, wide linear dynamic range, automation and high throughput. Therefore, the trace levels of drug in cells can be successfully detected by MRM.

In this work, in order to further investigate the cytotoxic mechanism of the drug action and accumulation effect of DDC-Cu in cancer cells, we focused on demonstrating the feasibility of performing cell apoptotic assay, drug absorption characterization and cell metabolites with ESI-MS. The NSCLC A549 cells were lysed after the drug stimulation to detect the level of drug absorption, MRM mode of mass spectrometry successfully detected trace amounts of the drug in cells. After stimulating A549 cells with different concentrations of drugs, the effect of DDC-Cu on the toxicity toward A549 cells was further observed by confocal laser scanning microscope, staining analysis and flow cytometry. Therefore, we found that DDC-Cu can accumulate intracellularly for a long time. In this process, cellular morphological changed, ROS content increased, cell cycle remained in the G0/G1 phase and finally induced apoptosis. ESI-MS technology has sufficient selectivity, sensitivity, high throughput, and small reagent consumption, making quantitative analysis of trace drugs in cells possible. Cytotoxicity assays and discussion of molecular cancer-relevant target will lead to a deeper exploration of cytotoxic mechanisms. This research has important reference value for the oral drug membrane permeability and the development of anticancer drugs.