Cancer Letters

Cancer Letters

Volume 300, Issue 1, 1 January 2011, Pages 87-95
Cancer Letters

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

https://doi.org/10.1016/j.canlet.2010.09.010Get rights and content

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.

Introduction

In multi-cellular organisms, apoptosis is a conserved cellular death program [1]. It plays an important role in tissue homeostasis, development, and defense against infections and mutations. Genes controlling the apoptotic process are suppressed or over expressed in many human diseases, especially in cancer [2], [3].

Copper is an essential trace element that can affect many biological pathways in human body [4], [5]. It can activate some critical proteins such as Cu–Zn superoxide dismutase, tyrosinase, ceruloplasmin and cytochrome oxidase, which are important in fundamental biological pathways [6]. Therefore, the copper concentration in human body is tightly regulated [7]. However, the concentration of copper in cancerous tissues, such as breast, prostate, lung, and brain, is higher than that of the normal tissues [8], [9], [10], [11]. In the serum of breast cancer patients, the level of copper can reach 1.67 μg/ml. This copper level is much higher than the healthy controls (0.98 μg/ml) [12]. For now, there is no explanation for this cancer-associated copper elevation. It was speculated that this accumulation might play a vital role in angiogenesis, a process critical for tumor growth [13], [14]. Copper can stimulate the production of cytokine, degradation of extracellular matrix, proliferation and migration of endothelial cell. In recent years, the methods of controlling the growth of tumor by eliminating the copper in serum of human body have been developed [15]. Tetrathiomolybdate (TM), which has the ability of copper-chelating was effective at inhibiting the growth of breast, prostate and lung tumors in various mouse models [16]. However, cancer kept developing in patients before the concentration of copper was lowered also suggesting that just passively chelating free copper is not enough to eliminate tumors.

The ubiquitin–proteasome pathway is essential for many fundamental cellular processes, such as cell cycle, apoptosis, angiogenesis and differentiation [17]. This pathway contributes to the pathological state of several human diseases including cancer, in which some regulatory proteins are either stabilized due to decreased degradation or lost due to accelerated degradation [18]. The 20S proteasome, the core of 26S proteasome complex, contains multiple peptidase activities (including the chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolyzing-like/PGPH-like) [19]. It has been reported that all three types of activities contributed significantly to protein breakdown and their relative importance varied widely with the substrate [20]. However, only the inhibition of chymotrypsin-like but not other proteasomal activities is a strong stimulus that induces apoptosis [21], [22].

Small molecule proteasome inhibitors can be developed as anti-cancer agents. These inhibitors can block proteasome function without, or slightly affecting other normal biological processes in the cell. The fact that cancer cells are more sensitive to the proteasome inhibitors than normal or untransformed cells makes these small molecules even more attractive potential anti-cancer drugs [22], [23], [24].

Dithiocarbamates are a class of copper-chelating compounds with various applications in medicine for the treatment of bacterial and fungal infections, and possible treatment of AIDS [25], [26]. One of the dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), is a synthetic antioxidant that has a potential to inhibit NFκB activation [27], [28]. We have previously found that PDTC formed a complex with copper and that PDTC-copper complex inhibited cell proliferation and induces apoptosis, and ubiquitin–proteasome pathway inhibition in cultured breast and prostate cancer cells [29].

Aldehyde dehydrogenases (ALDH) are NADP+ (NAD+) dependent enzymes. They catalyze the oxidation of endogenous and exogenous aldehydes [30], [31]. The cytosolic (ALDH1) and mitochondrial isozymes (ALDH2) are the most abundant in human body. Disulfiram (DSF), a member of the dithiocarbamate family, is the first drug that is developed for the treatment of alcoholism by inhibiting the ALDH1 activity. DSF bound tumor cellular copper and induced apoptotic cell death in human breast cancer cells in vitro and in vivo through proteasome inhibition [32]. Thus, DSF may have an important useful role in the treatment of human cancers. However, the ALDH inhibitor property of DSF may decrease the concentration that function as drug in the treatment of cancer. Thus, discovering novel cancer inhibitory compounds without the inhibition of ALDH activity is essential. Here we report the cancer inhibitory activity of potential non-ALDH binding PDTC analogues in human breast cancer cells through the inhibition of proteasomal chymotrypsin-like activity.

Section snippets

Reagents

CuCl2, disulfuram (DSF), 3-[4,5-dimethyltiazol-2-yl]-2.5-diphenyl-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO) and other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM)/F12, fetal bovine serum, horse serum, sodium bicarbonate, N-(2-hydroxyethyl) piperazine-N′-ethanesulfonic acid (HEPES) buffer solution, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Fluorogenic peptide substrate (Suc-LLVY-AMC)

Synthesis and characterization of PDTC analogues

Nine secondary amines (Fig. 1A) were used for synthesis of PDTC analogues. Since these compounds have different nucleophilicity, the reactions were carried out under different conditions. Aliphatic amines could easily react with CS2 in NaOH at room temperature with a yield of 90%. Secondary amines containing aromatic groups were less reactive and were treated at higher temperature (50 °C) and yielded about 50% of final product. Products were characterized by NMR and LC/MS.

Mixtures of 9 with copper inhibits proliferation of human breast cancer MDA-MB-231 and DCIS Cells

In order to discover

Discussion

High levels of copper are found in serum and tumors of cancer patients [8], [9], [10], [11]. The dramatic differences in Cu accumulation between normal and tumor tissues, and the requirement of Cu in tumor angiogenesis suggest that targeting cellular Cu can be a strategy for the treatment of breast caner. Previously, Cu depletion by TM has been suggested as a means to control angiogenesis and inhibit the tumor growth [16], [43], [44]. In clinical trials, TM treatment successfully blocked

Conflict of interest

None declared.

Acknowledgments

This work is supported by Grants from the National Natural Science Foundation of China (20902054 to S.M.Z. and 90913006 to B.Y.), Karmanos Cancer Institute 2009 Pilot project funding (to Q.P.D.) and the National Cancer Institute (1R01CA120009, 3R01CA120009-04S1, 1R21CA139386-01, to Q.P.D.), the American Lebanese Syrian Associated Charities (ALSAC), and St. Jude Children’s Research Hospital. We thank Yanwei Li for technical assistance in the computational docking analysis.

References (50)

  • Y. Zhang

    Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action

    Mutat. Res. – Fund. Mol. Mech. Mutagen.

    (2004)
  • M. Shen et al.

    Determination of in vivo adducts of disulfiram with mitochondrial aldehyde dehydrogenase

    Biochem. Pharmacol.

    (2001)
  • J. Adams

    The development of proteasome inhibitors as anticancer drugs

    Cancer Cell

    (2004)
  • J. Adams

    Preclinical and clinical evaluation of proteasome inhibitor PS-341 for the treatment of cancer

    Curr. Opin. Chem. Biol.

    (2002)
  • S.H. Chen et al.

    Involvement of activating transcription factors JNK, NF-κB, and AP-1 in apoptosis induced by pyrrolidine dithiocarbamate/Cu complex

    Eur. J. Pharmacol.

    (2008)
  • U. Thatte et al.

    Apoptosis: clinical relevance and pharmacological manipulation

    Drugs

    (1997)
  • Z. Song et al.

    Death by design: mechanism and control of apoptosis

    Trends Cell Biol.

    (1999)
  • G. Gregoriadis et al.

    A comparative study of trace elements in normal and cancerous colorectal tissues

    Cancer

    (1983)
  • M. Diez et al.

    Serum and tissue trace metal levels in lung cancer

    Oncology

    (1989)
  • K. Geraki et al.

    Concentrations of Fe, Cu and Zn in breast tissue: a synchrotron XRF study

    Phys. Med. Biol.

    (2002)
  • D. Yoshida et al.

    Quantitative analysis of copper, zinc and copper/zinc ratio in selected human brain tumors

    J. Neuro-Oncol.

    (1993)
  • S. Nayak et al.

    Copper and ceruloplasmin status in serum of prostate and colon cancer patients

    Indian J. Physiol. Pharmacol.

    (2003)
  • S.K. Gupta et al.

    Serum trace elements and Cu/Zn ratio in breast cancer patients

    J. Surg. Res.

    (1991)
  • G. Brewer

    Copper control as an antiangiogenic anticancer therapy: lessons from treating Wilson’s disease

    Exp. Biol. Med.

    (2001)
  • S. Adsule et al.

    Novel Schiff base copper complexes of quinoline-2 carboxaldehyde as proteasome inhibitors in human prostate cancer cells

    J. Med. Chem

    (2006)
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