Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients

Abstract

The thiocarbamate drug disulfiram has been used for decades in the treatment of alcohol abuse. Disulfiram induces apoptosis in a number of tumor cell lines and was recently by us proposed to act as a 26S proteasome inhibitor. In this work we characterized disulfiram in vitro with regard to tumor-type specificity, possible mechanisms of action and drug resistance and cell death in human tumor cell lines and in 78 samples of tumor cells from patients using the fluorometric microculture cytotoxicity assay and the automated fluorescence-imaging microscope ArrayScan^®. Disulfiram induced cytotoxicity in a biphasic pattern in both cell lines and patient tumor cells. Disulfiram induced apoptosis as measured by cell membrane permeability, nuclear fragmentation/condensation and caspase-3/7 activation using high content screening assays. For many of the cell lines tested disulfiram was active in sub-micromolar concentrations. When comparing the log IC₅₀ patterns with other cytotoxic agents, disulfiram showed low correlation (R < 0.5) with all drugs except lactacystin (R = 0.69), a known proteasome inhibitor, indicating that the two substances may share mechanistic pathways. Disulfiram was more active in hematological than in solid tumor samples, but substantial activity was observed in carcinomas of the ovary and the breast and in non-small cell lung cancer. Disulfiram also displayed higher cytotoxic effect in cells from chronic lymphocytic leukemia than in normal lymphocytes (p < 0.05), which may indicate some tumor selectivity. These results together with large clinical experience and relatively mild side effects encourage clinical studies of disulfiram as an anti-cancer agent.

Introduction

The dithiocarbamate drug disulfiram has been used for decades in the treatment of alcohol abuse since it inhibits aldehyde dehydrogenase. Several laboratories have investigated the antitumor activity of disulfiram, it has been shown to induce apoptosis in a number of cell lines [1], [2], [3], [4], [5], [6] as well as to reduce cell growth in glioma, lung carcinoma [7] and melanoma in mice [4]. However, the underlying mechanism has not been fully established. Disulfiram has before been shown to reduce angiogenesis [7], [8], inhibit DNA topoisomerases [9] and inhibit nuclear factor κB in hepatoma [3], colorectal cancer cell lines [2] and endothelial cells [10]. The cytotoxic activity has been attributed to pro-apoptotic redox-related mitochondrial membrane permeabilization [1], zinc complexation with subsequent inhibition of Zn²⁺-dependent matrix metalloproteinases [8], or Cu²⁺ complexation with inactivation of Cu/Zn superoxide dismutase [7] and consequently diminished cellular generation of reactive oxygen species. It has also been proposed that disulfiram itself has limited effect but must be in complex with Cu²⁺ to be active [11]. The NFκB inhibiting activity of the disulfiram analogue pyrrolidine dithiocarbamate (PDTC) has been attributed to its antioxidative effect and chelating properties [11], [12]. PDTC has been shown to inhibit proteasomal activity in combination with copper in human breast and prostate tumor cell lines [5], [6]. Recently we proposed a novel mechanism for the antitumor and NFκB inhibiting activity of disulfiram. Disulfiram and PDTC were identified as 26S proteasome inhibitors in a cell-based screening assay and the mechanism was confirmed with a NFκB translocation assay [13].

Indications of a potential tumor cell selectivity has been presented for disulfiram both for melanoma cells versus normal melanocytes [1] and for chronic lymphocytic leukemia (CLL) cells versus peripheral blood mononuclear cells [13].

In the present work we continue the characterization of disulfiram in vitro with respect to tumor-type specificity, possible mechanisms of drug resistance and cell death. This was carried out in human tumor cell lines and in primary cultures of human tumor cells from a broad spectrum of diagnoses and by use of the fluorometric microculture cytotoxicity assay (FMCA) and the automated fluorescence-imaging microscope ArrayScan^®.