Jade B. Aitken

Parkinson's disease-linked human PARK9/ATP13A2 maintains zinc homeostasis and promotes α-Synuclein externalization via exosomes

α-Synuclein plays a central causative role in Parkinsons disease (PD). Increased expression of the P-type ATPase ion pump PARK9/ATP13A2 suppresses α-Synuclein toxicity in primary neurons. Our data indicate that ATP13A2 encodes a zinc pump; neurospheres from a compound heterozygous ATP13A2(-/-) patient and ATP13A2 knockdown cells are sensitive to zinc, whereas ATP13A2 over-expression in primary neurons confers zinc resistance. Reduced ATP13A2 expression significantly decreased vesicular zinc levels, indicating ATP13A2 facilitates transport of zinc into membrane-bound compartments or vesicles. Endogenous ATP13A2 localized to multi-vesicular bodies (MVBs), a late endosomal compartment located at the convergence point of the endosomal and autophagic pathways. Dysfunction in MVBs can cause a range of detrimental effects including lysosomal dysfunction and impaired delivery of endocytosed proteins/autophagy cargo to the lysosome, both of which have been observed in cells with reduced ATP13A2 function. MVBs also serve as the source of intra-luminal nanovesicles released extracellularly as exosomes that can contain a range of cargoes including α-Synuclein. Elevated ATP13A2 expression reduced intracellular α-Synuclein levels and increased α-Synuclein externalization in exosomes >3-fold whereas ATP13A2 knockdown decreased α-Synuclein externalization. An increased export of exosome-associated α-Synuclein may explain why surviving neurons of the substantia nigra pars compacta in sporadic PD patients were observed to over-express ATP13A2. We propose ATP13A2s modulation of zinc levels in MVBs can regulate the biogenesis of exosomes capable of containing α-Synuclein. Our data indicate that ATP13A2 is the first PD-associated gene involved in exosome biogenesis and indicates a potential neuroprotective role of exosomes in PD.

X-ray-induced photo-chemistry and X-ray absorption spectroscopy of biological samples

As synchrotron light sources and optics deliver greater photon flux on samples, X-ray-induced photo-chemistry is increasingly encountered in X-ray absorption spectroscopy (XAS) experiments. The resulting problems are particularly pronounced for biological XAS experiments. This is because biological samples are very often quite dilute and therefore require signal averaging to achieve adequate signal-to-noise ratios, with correspondingly greater exposures to the X-ray beam. This paper reviews the origins of photo-reduction and photo-oxidation, the impact that they can have on active site structure, and the methods that can be used to provide relief from X-ray-induced photo-chemical artifacts.

Studies of glutathione transferase P1-1 bound to a platinum(IV)-based anticancer compound reveal the molecular basis of its activation.

Platinum-based cancer drugs, such as cisplatin, are highly effective chemotherapeutic agents used extensively for the treatment of solid tumors. However, their effectiveness is limited by drug resistance, which, in some cancers, has been associated with an overexpression of pi class glutathione S-transferase (GST P1-1), an important enzyme in the mercapturic acid detoxification pathway. Ethacraplatin (EA-CPT), a trans-Pt(IV) carboxylate complex containing ethacrynate ligands, was designed as a platinum cancer metallodrug that could also target cytosolic GST enzymes. We previously reported that EA-CPT was an excellent inhibitor of GST activity in live mammalian cells compared to either cisplatin or ethacrynic acid. In order to understand the nature of the drug-protein interactions between EA-CPT and GST P1-1, and to obtain mechanistic insights at a molecular level, structural and biochemical investigations were carried out, supported by molecular modeling analysis using quantum mechanical/molecular mechanical methods. The results suggest that EA-CPT preferentially docks at the dimer interface at GST P1-1 and subsequent interaction with the enzyme resulted in docking of the ethacrynate ligands at both active sites (in the H-sites), with the Pt moiety remaining bound at the dimer interface. The activation of the inhibitor by its target enzyme and covalent binding accounts for the strong and irreversible inhibition of enzymatic activity by the platinum complex.

Biotransformations of Anticancer Ruthenium(III) Complexes: An X‐Ray Absorption Spectroscopic Study

An anti-metastatic drug, NAMI-A ((ImH)[Ru(III) Cl4 (Im)(dmso)]; Im=imidazole, dmso=S-bound dimethylsulfoxide), and a cytotoxic drug, KP1019 ((IndH)[Ru(III) Cl4 (Ind)2 ]; Ind=indazole), are two Ru-based anticancer drugs in human clinical trials. Their reactivities under biologically relevant conditions, including aqueous buffers, protein solutions or gels (e.g, albumin, transferrin and collagen), undiluted blood serum, cell-culture medium and human liver (HepG2) cancer cells, were studied by Ru K-edge X-ray absorption spectroscopy (XAS). These XAS data were fitted from linear combinations of spectra of well-characterised Ru compounds. The absence of XAS data from the parent drugs in these fits points to profound changes in the coordination environments of Ru(III) . The fits point to the presence of Ru(IV/III) clusters and binding of Ru(III) to S-donor groups, amine/imine and carboxylato groups of proteins. Cellular uptake of KP1019 is approximately 20-fold higher than that of NAMI-A under the same conditions, but it diminishes drastically after the decomposition of KP1019 in cell-culture media, which indicate that the parent complex is taken in by cells through passive diffusion.

Metabolism of selenite in human lung cancer cells: X-ray absorption and fluorescence studies

Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.

Studies on the Biotransformations and Biodistributions of Metal-Containing Drugs Using X-Ray Absorption Spectroscopy

Most metal-based drugs are pro-drugs; therefore, it is essential that methods are developed to follow their speciation in biological fluids, cells and tissues. This will lead to both a better understanding of the factors that affect their efficacies and toxicities and, consequently, to the design of new and superior drugs. The use of X-ray absorption spectroscopy on bulk samples, and X-ray microprobe techniques on cells and tissues, provides unprecedented information on the biotransformations and biodistributions of metal-containing drugs that is required for a better understanding of their pharmacology. Here the methodologies that have been used on a range of metal- or metalloid-containing drugs and dietary supplements are reviewed, with an emphasis on research conducted within our group. In particular, applications of these techniques to anti-cancer, anti-diabetic, and anti-inflammatory drugs are discussed.

Uptake, Distribution, and Speciation of Selenoamino Acids by Human Cancer Cells: X-ray Absorption and Fluorescence Methods

Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.

Selenium metabolism in cancer cells: The combined application of XAS and XFM techniques to the problem of selenium speciation in biological systems

Determining the speciation of selenium in vivo is crucial to understanding the biological activity of this essential element, which is a popular dietary supplement due to its anti-cancer properties. Hyphenated techniques that combine separation and detection methods are traditionally and effectively used in selenium speciation analysis, but require extensive sample preparation that may affect speciation. Synchrotron-based X-ray absorption and fluorescence techniques offer an alternative approach to selenium speciation analysis that requires minimal sample preparation. We present a brief summary of some key HPLC-ICP-MS and ESI-MS/MS studies of the speciation of selenium in cells and rat tissues. We review the results of a top-down approach to selenium speciation in human lung cancer cells that aims to link the speciation and distribution of selenium to its biological activity using a combination of X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM). The results of this approach highlight the distinct fates of selenomethionine, methylselenocysteine and selenite in terms of their speciation and distribution within cells: organic selenium metabolites were widely distributed throughout the cells, whereas inorganic selenium metabolites were compartmentalized and associated with copper. New data from the XFM mapping of electrophoretically-separated cell lysates show the distribution of selenium in the proteins of selenomethionine-treated cells. Future applications of this top-down approach are discussed.

Selective Aggregation of a Platinum-Gadolinium Complex Within a Tumor-Cell Nucleus**

Gadolinium(III) complexes are widely used in magnetic resonance imaging (MRI) as water relaxation agents to improve image contrast. Therapeutic gadolinium-containing agents are also known in which the metal complex enhances tumor response to chemotherapeutics such as cisplatin, or, more commonly, acts as a radiosensitizer in the treatment of diseases, such as cancer. Gadoliniummay also play an important role in therapeutic techniques, such as synchrotron stereotactic radiotherapy (SSR), in which the selective delivery of gadolinium to the cell nucleus would significantly enhance the efficacy of the treatment. Indeed, De Stasio and co-workers have demonstrated that motexafinGd, a gadolinium(III) complex of the pentadentate texaphyrin ligand, was accumulated by approximately 90% of glioblastoma cell nuclei in vitro, and its potential exploitation as a GdSSR agent is warranted. In recent years, gadolinium complexes have also been explored as potential agents in an experimental anti-cancer treatment known as gadolinium neutron-capture therapy (GdNCT), which is closely related to the well-established boron neutron-capture therapy (BNCT). GdNCTutilizes the non-radioactive Gd isotope (natural abundance 15.7%) in a highly effective thermal neutron-capture reaction to destroy tumor cells. Gd possesses the largest effective nuclear cross-section of all naturally-occurring elements (2.55 10 barns); this value is approximately 66 times greater than that of the B nucleus. Gd undergoes neutron capture to give the products of internal conversion, accompanying Auger and Coster–Kronig (ACK) electron emission and 7.94 MeVof energy. However, the very limited range of ACK electrons means that the gadolinium complex must be localized in close proximity to critical cellular components, such as the cell nucleus, if the neutron capture reaction is to be exploited effectively. The use of gadolinium(III) complexes as potential GdNCT delivery agents to brain tumors has been described, although the feasibility of using archetypal MRI agents such as Gd-DTPA (DTPA= diethylenetriaminepentaacetic acid) in a clinical context for GdNCT is considered unlikely owing to the limited number of tumor-cell nuclei that have been shown to incorporate gadolinium. Indeed, the number of gadolinium compounds reported to date that have a capacity to aggregate selectively in tumor-cell nuclei, for example, is very limited, and the search for new types of gadolinium(III) complexes with high nuclear affinity has recently been proposed. Herein, we present a new Pt-Gd complex that can effectively target the nuclei of tumor cells by means of a functionalized dtpa ligand linked to two {Pt(terpy)} (terpy= 2,2’:6’,2’’-terpyridine) units that have the capacity to bind DNA in an intercalative manner. Based on prior work with analogous Pt–Ln complexes (Ln=La, Nd, Eu), which were designed to act as luminescent probes for DNA recognition, we reasoned that the related Pt-Gd species 1 would have the capacity to deliver gadolinium to this important biomolecule. In this work, we report the first unequivocal example of gadolinium delivery to a tumor-cell nucleus by a platinum complex. Complex 1was prepared in good yield by a similar manner to that described for the analogous Pt-Ln species (Ln=La, Nd, Eu; Scheme 1). The convenient one-pot synthesis of 1 demonstrates the high affinity of the soft Pt and hard Gd cations for the soft and hard Lewis bases (S and N/O, respectively) that are present in the functionalized DTPA ligand. The purple Pt-Gd complex has excellent solubility and stability in aqueous solution, and no evidence was found for the loss of Pt or Gd ions from 1, even after 24 h of standing in a buffered pH 7.4 solution at room temperature. Preliminary DNA thermal denaturation (DNA melting) experiments involving calf-thymus DNAwere performed on 1 at pH 7.4 (Supporting Information, Figure S1). There exists a significant difference in the melting temperatures between the freeand drug-treated DNA samples (DTm= 4.5 0.5 8C), [*] Dr. E. L. Crossley, Dr. J. B. Aitken, Prof. L. M. Rendina School of Chemistry, The University of Sydney Sydney, NSW 2006 (Australia) Fax: (+61)2-9351-3329 E-mail: rendina@chem.usyd.edu.au

Reactivity and Speciation of Anti-Diabetic Vanadium Complexes in Whole Blood and Its Components: The Important Role of Red Blood Cells

Reactions with blood components are crucial for controlling the antidiabetic, anticancer, and other biological activities of V(V) and V(IV) complexes. Despite extensive studies of V(V) and V(IV) reactions with the major blood proteins (albumin and transferrin), reactions with whole blood and red blood cells (RBC) have been studied rarely. A detailed speciation study of Na3[V(V)O4] (A), K4[V(IV)2O2(citr)2]·6H2O (B; citr = citrato(4-)); [V(IV)O(ma)2] (C; ma = maltolato(-)), and (NH4)[V(V)(O)2(dipic)] (D; dipic = pyridine-2,6-dicarboxylato(2-)) in whole rat blood, freshly isolated rat plasma, and commercial bovine serum using X-ray absorption near-edge structure (XANES) spectroscopy is reported. The latter two compounds are potential oral antidiabetic drugs, and the former two are likely to represent their typical decomposition products in gastrointestinal media. XANES spectral speciation was performed by principal component analysis and multiple linear regression techniques, and the distribution of V between RBC and plasma fractions was measured by electrothermal atomic absorption spectroscopy. Reactions of A, C, or D with whole blood (1.0 mM V, 1-6 h at 310 K) led to accumulation of ∼50% of total V in the RBC fraction (∼10% in the case of B), which indicated that RBC act as V carriers to peripheral organs. The spectra of V products in RBC were independent of the initial V complex, and were best fitted by a combination of V(IV)-carbohydrate (2-hydroxyacid moieties) and/or citrate (65-85%) and V(V)-protein (15-35%) models. The presence of RBC created a more reducing environment in the plasma fraction of whole blood compared with those in isolated plasma or serum, as shown by the differences in distribution of V(IV) and V(V) species in the reaction products of A-D in these media. At physiologically relevant V concentrations (<50 μM), this role of RBC may promote the formation of V(III)-transferrin as a major V carrier in the blood plasma. The results reported herein have broad implications for the roles of RBC in the transport and speciation of metal pro-drugs that have broad applications across medicine.