Memo1 binds reduced copper ions, interacts with copper chaperone Atox1, and protects against copper-mediated redox activity in vitro.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

Acetylcholinesterase and Aß Aggregation Inhibition by Heterometallic Ruthenium(II)-Platinum(II) Polypyridyl Complexes.

Two heteronuclear ruthenium(II)-platinum(II) complexes [Ru(bpy)2(BPIMBp)PtCl2]2+ (3) and [Ru(phen)2(BPIMBp)PtCl2]2+ (4), where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, and BPIMBp = 1,4'-bis[(2-pyridin-2-yl)-1H-imidazol-1-ylmethyl]-1,1'-biphenyl, have been designed and synthesized from their mononuclear precursors [Ru(bpy)2(BPIMBp)]2+ (1) and [Ru(phen)2(BPIMBp)]2+ (2) as multitarget molecules for Alzheimer's disease (AD). The inclusion of the cis-PtCl2 moiety facilitates the covalent interaction of Ru(II) polypyridyl complexes with amyloid ß (Aß) peptide. These multifunctional complexes act as inhibitors of acetylcholinesterase (AChE), Aß aggregation, and Cu-induced oxidative stress and protect neuronal cells against Aß-toxicity. The study highlights the design of metal based anti-Alzheimer's disease (AD) systems.

Fluorescent Copper Probe Inhibiting Aß1-16-Copper(II)-Catalyzed Intracellular Reactive Oxygen Species Production.

A variety of fluorescent probes are proposed to monitor the intracellular copper content. So far, none of the probes have been evaluated for their potential to inhibit copper-associated intracellular oxidative stress. Herein, we studied the ability of a fluorescent copper probe, OBEP-CS1, to inhibit intracellular oxidative stress associated with an amyloid ß (Aß) peptide-copper complex. The data showed that OBEP-CS1 completely inhibits the copper-catalyzed oxidation as well as decarboxylation/deamination of Aß1-16. Moreover, the cell imaging experiments confirmed that OBEP-CS1 can inhibit Aß-CuII-catalyzed reactive oxygen species production in SH-SY5Y cells. We also demonstrated that Aß1-16 peptide can bind intracellular copper and thereby exert oxidative stress.

Cisplatin inhibits the formation of a reactive intermediate during copper-catalyzed oxidation of amyloid ß peptide.

Cisplatin was studied for its effect on the copper-catalyzed oxidation of amyloid ß (Aß) peptide. The interaction of cisplatin with Aß1-16 in the presence of Cu(II) was investigated using cyclic voltammetry and mass spectrometry. The positive shift in the E1/2 value of Aß1-16-Cu(II) suggests that the interaction of cisplatin alters the copper-binding properties of Aß1-16. The mass spectrometry data show complete inhibition of copper-catalyzed decarboxylation/deamination of the Asp1 residue of Aß1-16, while there is a significant decrease in copper-catalyzed oxidation of Aß1-16 in the presence of cisplatin. Overall, our results provide a novel mode by which cisplatin inhibits copper-catalyzed oxidation of Aß. These findings may lead to the design of better platinum complexes to treat oxidative stress in Alzheimer's disease and other related neurological disorders.

Cellular Uptake of the ATSM-Cu(II) Complex under Hypoxic Conditions.

The Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone) complex (ATSM-Cu(II)) has been suggested as a promising positron emission tomography (PET) agent for hypoxia imaging. Several in-vivo studies have shown its potential to detect hypoxic tumors. However, its uptake mechanism and its specificity to various cancer cell lines have been less studied. Herein, we tested ATSM-Cu(II) toxicity, uptake, and reduction, using four different cell types: (1) mouse breast cancer cells (DA-3), (2) human embryonic kidney cells (HEK-293), (3) breast cancer cells (MCF-7), and (4) cervical cancer cells (Hela) under normoxic and hypoxic conditions. We showed that ATSM-Cu(II) is toxic to breast cancer cells under normoxic and hypoxic conditions; however, it is not toxic to normal HEK-293 non-cancer cells. We showed that the Cu(I) content in breast cancer cell after treatment with ATSM-Cu(II) under hypoxic conditions is higher than in normal cells, despite that the uptake of ATSM-Cu(II) is a bit higher in normal cells than in breast cancer cells. This study suggests that the redox potential of ATSM-Cu(II) is higher in breast cancer cells than in normal cells; thus, its toxicity to cancer cells is increased.

Does the ATSM-Cu(II) Biomarker Integrate into the Human Cellular Copper Cycle?

Hypoxia is commonly encountered in the tumor microenvironment and drives proliferation, angiogenesis, and resistance to therapy. Imaging of hypoxia is important in many disease states in oncology, cardiology, and neurology. Finding clinically approved imaging biomarkers for hypoxia has proved challenging. Candidate biomarkers have shown low uptake into tumors and low signal to background ratios that adversely affect imaging quality. Copper complexes have been identified as potential biomarkers for hypoxia owing to their redox ability. Active uptake of copper complexes into cells could ensure selectivity and high sensitivity. We explored the reactivity and selectivity of the ATSM-Cu(II) biomarker to proteins that are involved in the copper cycle using electron paramagnetic resonance (EPR) spectroscopy and UV-vis measurements. We show that the affinity of the ATSM-Cu(II) complex to proteins in the copper cycle is low and the cell probably does not actively uptake ATSM-Cu(II).

Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aß1-16 peptide.

The binding of metal ions to Aß peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu(2+) on redox properties and cytotoxicity of Aß peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aß peptide has higher propensity of H2O2 generation. The oxidation of Aß1-16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aß1-16-Cu(2+) (1:2) complex.