Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

Abstract

The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF-Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 32 micromol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF-Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF-Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF-Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.

Fig. 1

Disulfiram–cadmium mixture (DSF–Cd) is a potent proteasome inhibitor and apoptosis inducer in human prostate cancer PC-3 cells. PC-3 cells were treated with indicated DSF–metal mixtures at 20 μM for 12 h, followed by the chymotrypsin-like activity assay (A), Western blot analysis of polyubiquitinated proteins, p27, PARP and Bax (B), and morphological evaluation (C). DMSO and DSF alone at 20 μM were used as controls. Molecular weight of intact PARP and the cleaved PARP fragment were 116 kDa and 65 kDa, respectively. Molecular weight of three forms of Bax, is 18, 21, 36 kDa. β-Actin was used as a loading control. Columns, mean of independent triplicate experiments; bars, SD;**, p<0.01.

Fig. 2

Inhibition of the chymotrypsin-like activity of 20S proteasome by DSF–Cd in vitro. Purified rabbit 20S proteasome (35 ng) was incubated with the peptide substrate for the proteasomal chymotrypsin (CT)-like activity in the presence of DSF, CdCl₂ (Cd) and their mixture DSF–Cd at indicated concentrations, as described in Materials and methods. Bars, SD;*, p<0.05.

Fig. 3

Comparison of proteasome-inhibitory effects between DSF–Cd and MG132. A, Inhibitory effects on purified 20S proteasome by DSF–Cd and MG132 were tested. B, Intact PC-3 cells containing 26S proteasome were incubated with indicated concentrations of DSF–Cd and MG132 for 6 h, followed by a co-incubation for 2 h with the fluorogenic substrate Suc-LLVY-AMC (at 40 μmol/L) for the proteasomal chymotrypsin-like activity. After incubation, production of hydrolyzed AMC groups was measured. C, Morphological changes induced by DSF–Cd and MG132 at different concentrations. Columns, mean of independent triplicate experiments; bars, SD.

Fig. 4

Kinetic studies on proteasome inhibition and apoptosis induction by disulfiram–cadmium in PC-3 cells. Exponentially grown PC-3 cells (0 h) were exposed to 20 μM DSF–Cd, DSF or cadmium chloride (Cd) for the indicated time points, followed by measuring inhibition of the proteasomal chymotrypsin-like activity using Suc-LLVY-AMC (A), Western blot analysis using specific antibodies to ubiquitin, Bax, and PARP (B, D), and apoptotic morphologic changes (C). β-Actin was used as a loading control. Columns, mean of independent triplicate experiments; bars, SD;**, p<0.01.

Fig. 5

Differential effects of DSF–Cd complex on malignant and non-tumorigenic breast cells. Malignant MCF10-DCIS cells (DCIS) and immortalized but non-tumorigenic MCF-10A cells (10A) were treated with DSF–Cd at 1–20 μM, DSF at 20 μM, cadmium chloride (Cd) at 20 μM, or DMSO for 10 h, followed by morphological changes evaluation (A, B) and the CT-like activity assay using whole cell lysates (C). Columns, mean of independent triplicate experiments; bars, SD;**, p<0.01.