A novel dithiocarbamate analogue with potentially decreased ALDH inhibition has copper-dependent proteasome-inhibitory and apoptosis-inducing activity in human breast cancer cells

Abstract

Dithiocarbamates are a class of sulfur-based metal-chelating compounds with various applications in medicine. We reported previously that certain members of dithiocarbamates, such as diethyldithiocarbamate, disulfiram (DSF) and pyrrolidine dithiocarbamate (PDTC), were able to bind with tumor cellular copper to inhibit tumor growth through the inhibition of proteasome activity and induction of cancer cell apoptosis. Since the DSF is an irreversible inhibitor of aldehyde dehydrogenase (ALDH), its ALDH-inhibitory activity might potentially affect its usefulness as an anti-cancer drug. For the purpose of selecting potent anti-cancer compounds that are not ALDH inhibitors and mapping out preliminary structure-activity relationship trends for these novel compounds, we synthesized a series of PDTC analogues and chose three novel compounds to study their ALDH-inhibitory activity, proteasome-inhibitory activity as well as the cancer cell apoptosis-inducing activity. The results showed that compared to DSF, compound 9 has less ALDH inhibition activity, and the in vitro results also proved the positive effects of 9-Cu in proteasome inhibition and apoptosis induction in breast cancer cells, suggesting that 9 as a lead compound could be developed into a novel proteasome inhibitor anti-cancer drug.

Fig. 1

Chemical structures of compound 1–9 and the Anti-proliferative effect of the copper mixtures with different DSF analogues. (A) Chemical structures of compound 1–9. (B, C) the Anti-proliferative effect of the copper mixtures with different DSF analogues. DCIS (B) and MDA-MB-231 (C) cells were treated with the copper mixtures of nine PDTC analogues at different concentrations as indicated. After 24 h, the cell viability was measured by MTT assay as described in Section 2. DMSO was used as negative control and DSF as positive control.

Fig. 2

Interactions between ALDH and compounds. (A) Details of the region of ALDH2 around the compounds, showing the hydrogen-bonding distance formed between the -SH of compound and Cys302. The side chain of the cysteine residues Cys302 is shown. (B) Details of the Cys302 of ALDH1 binding distance of the compound binding site. The backbone of ALDH is shown as a line model with the side chain of the Cys302 residues forming contact with compounds. The structures of compounds are shown as ball-and-stick model. The different atoms are marked as different colors: S (yellow), N (blue), C (gray), H (white), O (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 3

Copper complex formation and the binding of compound with copper. The UV spectra of complexes formed between PDTC analogues 1, 5 and 9 and copper and their binding constants. The concentration of PDTC analogue was 100 μM. The absorption band of the complex at 430 nm increases as Cu concentrations were 10, 20, 30, 40, 50 and 60 μM.

Fig. 4

Induction of apoptosis in cancer cells by 9-copper mixtures. (A) Dose-dependent poly(ADP-ribose) polymerase (PARP) cleavage and Bax accumulation in DCIS cells treated with 1, 5 and 9-Cu mixtures at increasing concentrations for 24 h. (B) Dose-dependent poly(ADP-ribose) polymerase (PARP) cleavage and Bax accumulation in MDA-MB-231 cells treated with 1, 5 and 9-Cu mixtures at increasing concentrations for 24 h. (C) Time-dependent poly(ADP-ribose) polymerase (PARP) cleavage and Bax accumulation in MDA-MB-231 cells treated with 2.5 μM of 5 and 9-Cu mixtures for up to 24 h. (D) Induction of apoptosis in MDA-MB-231 cells after treatment with 1.4 μM of DSF-Cu mixture, 9-Cu mixture or vehicle in the presence and absence of Z-VAD-FMK inhibitor for 24 h by Annexin V/PI staining (also see Fig. S2). (E) Detection of caspase-3/7 activity in MDA-MB-231 cells after treatment with 1.4 μM of DSF-Cu mixture, 9-Cu mixture or vehicle in the presence and absence of Z-VAD-FMK for 24 h using the Apo-ONE® homogeneous Caspase-3/7 Assay.

Fig. 5

Inhibition of proteasomal chymotrypsin-like activity by 1-Cu, 5-Cu and 9-Cu mixtures. (A) The DCIS cells were treated with 0.5, 1.0 and 2.0 μM of the indicated mixtures for 24 h, followed by the measurement of proteasomal CT-like activity. (B) Western blot analysis for the accumulation of ubiquitinated proteins and NOXA induction in the extracts prepared from the DCIS cells for 24 h. (C) The MDA-MB-231 cells were treated with 0.5, 1.0 and 2.0 μM of the indicated mixtures for 24 h, followed by the measurement of proteasomal CT-like activity. (D) Western blot analysis for the accumulation of ubiquitinated proteins and NOXA induction in the extracts prepared from the MDA-MB-231 cells for 24 h. (E) The MDA-MB-231 cells were exposed to a 2.5 μM mixture of 9-Cu and 5-Cu for the indicated times, followed by measurement of proteasomal CT-like activity. (F) Western blot analysis using specific antibodies to ubiquitinated proteins and NOXA in the extracts prepared form the MDA-MB-231 cells for the indicated times.

Scheme 1

Synthesis of PDTC analogues.