Inhibition of Tumor Proteasome Activity by Gold-Dithiocarbamato Complexes via Both Redox-Dependent and -Independent Processes

The Prevention Program, Department of Pathology, Barbara Ann Karmanos Cancer Institute, School of Medicine, Wayne State University, Detroit, Michigan.
Journal of Cellular Biochemistry (Impact Factor: 3.26). 11/2009; 109(1):162-72. DOI: 10.1002/jcb.22394
Source: PubMed


We have previously reported on a gold(III) complex, namely [AuBr(2)(DMDT)] (N,N-dimethyldithiocarbamate) showing potent in vitro and in vivo growth inhibitory activities toward human cancer cells and identifying the cellular proteasome as one of the major targets. However, the importance of the oxidation state of the gold center and the involved mechanism of action has yet to be established. Here we show that both gold(III)- and gold(I)-dithiocarbamato species, namely [AuBr(2)(ESDT)] (AUL12) and [Au(ESDT)](2) (AUL15), could inhibit the chymotrypsin-like activity of purified 20S proteasome and 26S proteasome in human breast cancer MDA-MB-231 cells, resulting in accumulation of ubiquitinated proteins and proteasome target proteins, and induction of cell death, but at significantly different levels. Gold(I)- and gold(III)-compound-mediated proteasome inhibition and cell death induction were completely reversed by the addition of a reducing agent, dithiothreitol or N-acetyl-L-cysteine, suggesting the involvement of redox processes. Furthermore, treatment of MDA-MB-231 cells with gold(III) compound (AUL12), but not the gold(I) analog (AUL15), resulted in the production of significant levels of reactive oxygen species. Our study provides strong evidence that the cellular proteasome is an important target of both gold(I) and gold(III)-dithiocarbamates, but distinct cellular mechanisms of action are responsible for their different overall effect.

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    • "Interestingly, Auphen has been previously reported to possess anticancer properties in vitro (Messori et al., 2000; Casini et al., 2009). Indeed, in recent years several gold(III) compounds have shown promising anticancer effects related to the inhibition of different protein targets, such as the proteasome and specific zinc finger proteins (Zhang et al., 2010; Mendes et al., 2011). In this context, AQP3 might influence the biological effects of the compounds toward cancer cells. "
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    ABSTRACT: AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5-bromo-2′-deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12-AQP3, ≈15% in HEK-AQP3 and had no effect in wt-PC12 and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound's anti-proliferative effect correlates with its ability to inhibit AQP3 Pgly. Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 11/2014; 229(11). DOI:10.1002/jcp.24632 · 3.84 Impact Factor
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    • "Since first generation compounds were able to inhibit the CT-like (chymotrypsin-like) active site of the proteasome [7], [8], we hypothesized that these second generation complexes could target the tumor proteasome as well. To test this hypothesis, we investigated the effects of both compounds on the proteasomal CT-like, trypsin (T)-like and peptidyl-glutamyl-peptide-hydrolizing (PGPH)-like activities of MDA-MB-231 cell extracts. "
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    ABSTRACT: Since the serendipitous discovery of cisplatin, platinum-based drugs have become well-established antitumor agents, despite the fact that their clinical use is limited by many severe side-effects. In order to both improve the chemotherapeutic index and broaden the therapeutic spectrum of current drugs, our most recent anti-neoplastic agents, Au(III) complexes, were designed as carrier-mediated delivery systems exploiting peptide transporters, which are up-regulated in some cancers. Among all, we focused on two compounds and tested them on human MDA-MB-231 (resistant to cisplatin) breast cancer cell cultures and xenografts, discovering the proteasome as a major target both in vitro and in vivo. 53% inhibition of breast tumor growth in mice was observed after 27 days of treatment at 1.0 mg kg(-1) d(-1), compared to control. Remarkably, if only the most responsive mice are taken into account, 85% growth inhibition, with some animals showing tumor shrinkage, was observed after 13 days. These results led us to file an international patent, recognizing this class of gold(III) peptidomimetics as suitable candidates for entering phase I clinical trials.
    PLoS ONE 01/2014; 9(1):e84248. DOI:10.1371/journal.pone.0084248 · 3.23 Impact Factor
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    • "Interestingly, both Auphen and Audien have been previously reported to possess anticancer properties in vitro [28], [29]. Indeed, in recent years several gold(III) compounds have shown promising anticancer effects related to the inhibition of different protein targets, such as the proteasome and specific zinc finger proteins [57], [58], [59], [60]. In this context, we cannot exclude that inhibition of AQP3 might influence the biological effects of the compounds towards cancer cells, although other studies need to be performed to validate such a hypothesis. "
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    ABSTRACT: Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.
    PLoS ONE 05/2012; 7(5):e37435. DOI:10.1371/journal.pone.0037435 · 3.23 Impact Factor
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