Copper-binding Compound Research Articles

Heterologous Biosynthesis of Methanobactin from Methylocystis sp. Strain SB2 in Methylosinus trichosporium OB3b.

Aerobic methanotrophs, or methane-consuming microbes, are strongly dependent on copper for their activity. To satisfy this requirement, some methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin (MB). In addition to playing a critical role in methanotrophy, MB has also been shown to have great promise in treating copper-related human diseases, perhaps most significantly Wilson's disease. In this congenital disorder, copper builds up in the liver, leading to irreversible damage and, in severe cases, complete organ failure. Remarkably, MB has been shown to reverse such damage in animal models, and there is a great deal of interest in upscaling MB production for expanded clinical trials. Such efforts, however, are currently hampered as (1) the natural rate of MB production rate by methanotrophs is low, (2) the use of methane as a substrate for MB production is problematic as it is explosive in air, (3) there is limited understanding of the entire pathway of MB biosynthesis, and (4) the most attractive form of MB is produced by Methylocystis sp. strain SB2, a methanotroph that is genetically intractable. Herein, we report heterologous biosynthesis of MB from Methylocystis sp. strain SB2 in an alternative methanotroph, Methylosinus trichosporium OB3b, not only on methane but also on methanol. As a result, the strategy described herein not only facilitates enhanced MB production but also provides opportunities to construct various mutants to delineate the entire pathway of MB biosynthesis, as well as the creation of modified forms of MB that may have enhanced therapeutic value.

Antioxidative effect of DJ-1 is enhanced in NG108-15 cells by DPMQ-induced copper influx.

Disruption of copper homeostasis is closely involved in neurodegenerative disorders. This study examined whether a hybrid copper-binding compound, (E)-2-(4-(dimethylamino)phenylimino)methyl)quinolin-8-ol (DPMQ), is able to protect NG108-15 cells against oxidative stress. We found that treatment of cells with rotenone or hydrogen peroxide increased cellular oxidative stress and resulted in mitochondrial dysfunction and apoptosis. The cellular levels of Nrf2 and the Cu2+ chaperone DJ-1 were also decreased. These oxidative detrimental effects were all inhibited when cells were cotreated with DPMQ. DPMQ increased cellular Cu2+ content, DJ-1 protein level, superoxide dismutase (SOD) activity, and Nrf2 nuclear translocation under basal state. The activity of SOD decreased under redox imbalance and this decrease was blocked by DPMQ treatment, while the protein level of SOD1 remained unaltered regardless of the oxidative stress and DPMQ treatment. Using endogenous proteins, coimmunoprecipitation showed that DJ-1 bound with SOD1 and Nrf2 individually. The amount of Nrf2, bound to DJ-1, consistently reflected its cellular level, while the amount of SOD1, bound to DJ-1, was potentiated by DPMQ, being greater in the basal state than under redox imbalance. Simultaneous inclusion of nonpermeable Cu2+ chelator tetrathiomolybdate or triethylenetetramine during DPMQ treatment blocked all aforementioned effects of DPMQ, showing that the dependency of the effect of DPMQ on extracellular Cu2+. In addition, silencing of DJ-1 blocked the protection of DPMQ against oxidative stress. Taken all together, our results suggest that DPMQ stabilizes DJ-1 in a Cu2+-dependent manner, which then brings about SOD1 activation and Nrf2 nuclear translocation; these together alleviate cellular oxidative stress.

Oxidation of L-Ascorbic Acid in the Presence of the Copper-Binding Compound from Methanotrophic Bacteria Methylococcus capsulatus (M).

The oxidation of ascorbic acid by air oxygen and hydrogen peroxide in the presence of the copper-binding compound (cbc) from bacteria Methylococcus capsulatus (M) was studied. The rate constant of ascorbic acid oxidation by air oxygen in the presence of the copper complex with cbc from M. capsulatus (M) was shown to be 1.5 times higher than that of the noncatalytic reaction. The rate constant of ascorbic acid oxidation by hydrogen peroxide in the presence of the copper complex with cbc from M. capsulatus (M) decreased by almost one-third compared to the reaction in the absence of the copper complex with cbc. It was assumed that cbc can be involved in a multilevel system of antioxidant protection and can protect a bacterial cell from oxidation stress. Thus, the cbc is mimetic ascorbate oxidase in the oxidation of ascorbic acid by molecular oxygen.

Synergistic Effects of a Chalkophore, Methanobactin, on Microbial Methylation of Mercury.

Microbial production of the neurotoxin methylmercury (MeHg) is a significant health and environmental concern, as it can bioaccumulate and biomagnify in the food web. A chalkophore or a copper-binding compound, termed methanobactin (MB), has been shown to form strong complexes with mercury [as Hg(II)] and also enables some methanotrophs to degrade MeHg. It is unknown, however, if Hg(II) binding with MB can also impede Hg(II) methylation by other microbes. Contrary to expectations, MB produced by the methanotroph Methylosinus trichosporium OB3b (OB3b-MB) enhanced the rate and efficiency of Hg(II) methylation more than that observed with thiol compounds (such as cysteine) by the mercury-methylating bacteria Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. Compared to no-MB controls, OB3b-MB decreased the rates of Hg(II) sorption and internalization, but increased methylation by 5- to 7-fold, suggesting that Hg(II) complexation with OB3b-MB facilitated exchange and internal transfer of Hg(II) to the HgcAB proteins required for methylation. Conversely, addition of excess amounts of OB3b-MB or a different form of MB from Methylocystis strain SB2 (SB2-MB) inhibited Hg(II) methylation, likely due to greater binding of Hg(II). Collectively, our results underscore the complex roles of microbial exogenous metal-scavenging compounds in controlling net production and bioaccumulation of MeHg in the environment.IMPORTANCE Some anaerobic microorganisms convert inorganic mercury (Hg) into the neurotoxin methylmercury, which can bioaccumulate and biomagnify in the food web. While the genetic basis of microbial mercury methylation is known, factors that control net methylmercury production in the environment are still poorly understood. Here, it is shown that mercury methylation can be substantially enhanced by one form of an exogenous copper-binding compound (methanobactin) produced by some methanotrophs, but not by another. This novel finding illustrates that complex interactions exist between microbes and that these interactions can potentially affect the net production of methylmercury in situ.

Methanotrophy - Environmental, Industrial and Medical Applications.

Aerobic methanotrophs are an intriguing group of microbes with the singular ability to consume methane as their sole source of carbon and energy. As such, methanotrophs are receiving increased attention to control methane emissions to limit future climate change. Methanotrophs have a wide range of other applications, including pollutant remediation and methane valorization (e.g. conversion of methane to protein, bioplastics, and biodiesel amongst other products). Methanotrophs also produce a novel copper-binding compound, methanobactin, that has significant potential for the treatment of copper-related human pathologies. Here we provide an overview of aerobic methanotrophy, describe current and future applications of these unique microbes, as well as discuss various strategies one can consider to better realize the opportunities these microbes present.

An Aminotransferase Is Responsible for the Deamination of the N-Terminal Leucine and Required for Formation of Oxazolone Ring A in Methanobactin of Methylosinus trichosporium OB3b.

Methanotrophs, microbes that play a critical role in the carbon cycle, are influenced by copper, with gene expression and enzyme activity changing as copper levels change. Methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin for copper uptake, and methanobactin plays a key role in controlling methanotrophic activity. Methanobactin has also been shown to be effective in the treatment of Wilson disease, an autosomal recessive disorder where the human body cannot correctly assimilate copper. It is important to characterize the methanobactin biosynthesis pathway to understand how methanotrophs respond to their environment as well as to optimize the use of methanobactin for the treatment of copper-related diseases such as Wilson disease. Here we show that mbnN, encoding an aminotransferase, is involved in the deamination of the N-terminal leucine and necessary for the formation of one but not both of the heterocyclic rings in methanobactin that are responsible for copper binding.

Mercury binding by methanobactin from Methylocystis strain SB2

Methanobactin (mb) is a post-translationally modified copper-binding compound, or chalkophore, secreted by many methane-oxidizing bacteria or methanotrophs in response to copper limitation. In addition to copper, methanobactin from Methylosinus trichosporium OB3b (mb-OB3b) has been shown to bind a variety of metals including Hg2+. In this report, Hg binding by the structurally unique methanobactin from Methylocystis strain SB2 (mb-SB2) was examined and compared to mb-OB3b. Mb-SB2 is shown to bind the common forms of Hg found in aqueous environments, Hg2+, Hg(CN)2 and CH3Hg+. The spectral and thermodynamic properties of binding for each form of mercury differed. UV-visible absorption spectra suggested that Hg2+ binds to both the oxazolone and imidazolone rings of mb-SB2, whereas CH3Hg+ appeared to only bind to the oxazolone ring. Hg(CN)2 showed spectral properties between Hg2+ and CH3Hg+. Isothermal titration calorimetry (ITC) showed both Hg(CN)2 and CH3Hg+ fit into two-site binding models. For Hg(CN)2 the first site was exothermic and the second endothermic. Both binding sites in CH3Hg+ were exothermic, but at equilibrium the reaction never moved back to the baseline, suggesting a slow residual reaction. ITC results for Hg2+ were more complex and suggested a 3- or 4-site model. The spectral, kinetic and thermodynamic changes following Hg binding by mb-SB2 also differed from the changes associated with mb-OB3b. Like mb-OB3b, copper did not displace Hg bound to mb-SB2. In contrast to mb-OB3b Hg2+ could displace Cu from Cu-containing mb-SB2 and preferentially bound Hg2+ over Cu2+ at metal to mb-SB2 molar ratios above 1.0.

Abstract 4382: N-acetylcysteine interacts with Cu++ to induce cancer cell death through the generation of hydrogen peroxide

Abstract Metal ionophores that transport copper or zinc into cells are toxic to cancer cells. Loading of metals into cells results in loss of protective intracellular thiols, which can contribute to cell death. We postulated that the metal-thiol interaction might also lead to the generation of cytotoxic compounds. We tested this hypothesis by adding N-acetylcysteine (NAC) to cancer cell lines in the presence of copper or zinc. NAC itself, at concentrations up to 10 mM, was not toxic to a variety of human cancer cell lines, including A2780 (ovarian), MCF-7 (breast), Panc-1 (pancreas), and A375 (melanoma). When added to cells in the presence of Cu++ (3-30 µM), NAC significantly enhanced the cytotoxicity induced by CuCl2 alone. The cytotoxicity of the NAC/copper combination was blocked by the copper-binding compounds tetrathiomolybdate and bathocuproinedisulfonic acid. NAC was not cytotoxic in the presence of zinc. This finding suggested that the redox activity of copper was an important determinant of cytotoxicity and raised the possibility that H2O2 was being generated. This was confirmed in vitro using a colorimetric assay (Biovision). The ability of other thiols to promote H2O2 generation when exposed to Cu++ was then examined. Reduced lipoic acid and L-cysteine generated higher level of H2O2 than NAC did; reduced glutathione was able to generate comparable levels of H2O2 as NAC, while oxidized glutathione generated significantly lower levels of H2O2; oxidized cysteine did not generate H2O2. To determine if H2O2 played a role in the observed cytotoxicity, catalase was added to cells treated with NAC/copper. Its addition completely blocked cell killing, indicating that significant amounts of extracellular H2O2 were being generated. These findings indicate that the interaction of copper with thiols leads to cytotoxic concentrations of H2O2. Thiols may differ in their reactivity with redox active compounds such as copper. Since the copper concentration of plasma is greater than that of tissue culture media, NAC's effects in vivo may differ from those seen in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4382.

Relevant activities of extracts and constituents of animals used in traditional Chinese medicine for central nervous system effects associated with Alzheimer's disease

The centipede Scolopendra subspinipes mutilans L. Koch ('Wugong'), the beetle Mylabris phalerata Pallas ('Ban mao') and the earthworm Pheretima aspergillum Chen ('DiLong') have a reputation in traditional Chinese medicine for reducing symptoms of central nervous system decline, including memory loss. A series of extracts of all three organisms was tested for acetylcholinesterase (AChE) inhibition and copper ion binding effects, the latter likely to reduce oxidative damage caused by excess copper. The beetle and centipede chloroform extracts showed the strongest AChE inhibitory effects (30.6% inhibition at 105 microg mL(-1) and 32.3% inhibition at 167 microg mL(-1), respectively) and, in the case of the centipede, this was traced to the unsaturated fatty acids present using bioassay-guided fractionation. Cantharidin from the beetle was shown to have AChE activity (31% inhibition at 1 muM, 0.196 microg mL(-1)), making it a major contributor to the activity of the beetle extract. The earthworm showed no AChE inhibitory activity. Since unsaturated fatty acids have not been previously reported to have AChE inhibitory activity, a series of related compounds was tested to determine structure-activity relationships. It was found that activity existed where there was a chain length of more than 16 C atoms with at least one unsaturated bond in the chain. The carboxylic acid group was also necessary for activity. The fatty acids present in the centipede also showed the ability to bind copper ions when tested using a novel thin layer chromatography method designed to detect copper-binding compounds. The activities reported give some support to the use of the beetle and centipede in traditional Chinese medicine for improving cognitive function.

Copper methanobactin: a molecule whose time has come

Copper plays a key role in the physiology of methanotrophs. One way that these bacteria meet their high copper requirement is by the biosynthesis and release of high affinity copper binding compounds called methanobactins. Recent advances in methanobactin characterization include the first crystal structure, detailed spectroscopic analyses, and studies of metal ion specificity. Methanobactin may function in copper uptake, regulation of methane monooxygenase expression, protection against copper toxicity, and particulate methane monooxygenase activity. Methanobactin can extract copper from insoluble minerals and could be important for mineral weathering. Many methanobactin properties are reminiscent of iron siderophores, suggesting a similar mechanism of handling. Methanobactin-like compounds have also been identified in yeast mitochondria, suggesting that these molecules are a more universal phenomenon.

Early Biomarkers for the Anti-Angiogenic and Anti-Tumor Activity of the Superoxide Dismutase 1 (SOD1) Inhibitor Tetrathiomolybdate.

Background: Identifying biomarkers that monitor clinical response to cancer therapies is crucial. Tetrathiomolybdate (TM) is a highly specific, high-affinity, orally available copper-binding compound that inhibits CuZn SOD1, leading to robust anti-angiogenic and anti-tumor effects. TM is currently being evaluated in Phase II cancer trials including for relapsed and refractory multiple myeloma (MM). TM's inhibition of angiogenesis has been attributed to depletion of systemic Cu, which is known to affect multiple key angiogenesis regulators; however, in mice, inhibition of angiogenesis by TM occurred before a measurable decrease in systemic Cu. We have recently shown that in MM, circulating endothelial progenitor cell (EPC) levels serve as a reliable biomarker of disease severity which covaries with tumor load and disease progression. Furthermore, recent evidence from our laboratory and by others indicates that EPCs display evidence of clonality and are genetically related to MM cells. In the present study, the relationship between anti-angiogenic and anti-SOD1 activities of TM were compared by determining its effects on:1.circulating endothelial progenitor cells (EPCs) in bonnet macaques (M. radiata);2.on angiogenesis and SOD1 activity in blood cells from mice and humans.Methods: TM was manufactured with >99% purity using a proprietary process (choline salt: ATN-224). For animal studies, three female and three male young adult monkeys were treated with TM (0.5 mg/kg, s.c.) daily for 22 days, followed by a 26 day drug holiday, and a second course of drug. Blood was drawn from the cephalic vein, separated by Ficoll, and red cell lysis. EPCs were identified as CD31+/CD133+/CD45− weak cells using 3-color flow cytometry. For SOD activity, blood from normal control subjects was incubated with TM for 5.5 hr at 37°C. SOD activity was measured in 30 μg of protein from treated blood pellets using an SOD Activity Kit.Results: Administration of TM to the monkeys resulted in a rapid, profound, and reversible decrease in circulating EPCs without significant toxicity. The levels of EPCs increased initially at day 6 in 5 of the 6 subjects, and then decreased to 7% of baseline at day 21. The EPC levels rebounded to normal levels once treatment was discontinued and dropped again after treatment was reinstated. Other hematopoietic parameters (RBC, WBC, platelet counts, and hemoglobin) were not affected by treatment. Although we did not measure blood cell SOD in the macaque study, substantial inhibition of blood cell SOD is associated with hematologic toxicities which did not occur in these animals, suggesting that RBC SOD activity is inhibited at higher doses than those needed for anti-angiogenic activity. TM treatment inhibited SOD activity in blood cell pellets obtained from human controls in a dose dependent manner, with an IC50 3 μM.Conclusion: These results show, for the first time, that in vivo effects of TM include suppression of circulating EPC levels. Since circulating EPCs are genetically related to tumor cells in MM, at least the anti-MM effects of TM may depend on its suppressive effects on EPCs as well as on angiogenesis in general. Furthermore, EPCs are a sensitive indicator of TM actions since their reversible modulation correlated with drug therapy. Lastly, macaques may represent a reliable model of human response to determine drug effects on EPCs and angiogenesis.

Sodium diethyldithiocarbamate, an AIDS progression inhibitor and a copper-binding compound, has proteasome-inhibitory and apoptosis-inducing activities in cancer cells

Diethyldithiocarbamate (DDTC) is a member of the dithiocarbamate family and a potent copper-chelating agent. DDTC was used in a clinical trial for patients with HIV-1 infection and showed a significant delay in progression to AIDS. In this study, we investigated the effects of DDTC-copper complex in human prostate and breast cancer cells. We found that DDTC was capable of binding copper and forming a new complex that potently inhibited the proteasomal chemotrypsin-like activity, decreased expression of androgen receptor (AR), estrogen receptor (ER) alpha and ERbeta proteins, and induced apoptosis in both prostate and breast cancer cells. Our data support the concept of using accumulated copper in cancer cells and tissues as a novel target for chemotherapy. This study provides a mechanistic interpretation for utilization of copper chelators in cancer treatment.

Copper-binding compounds as proteasome inhibitors and apoptosis inducers in human cancer

The trace element copper is vital to the healthy functioning of organisms. Copper is used in a multitude of cellular activities including respiration, angiogenesis, and immune responses. Recently, copper has become a focus in medical research ranging from Alzheimer's disease to cancer. Copper modulation has been suggested to be a potential modality for therapy in these diseases. Several copper-binding compounds have been found to spontaneously complex with copper and form active proteasome inhibitors and apoptosis inducers. This review examines compounds in the quinoline and dithiocarbamate families and from the National Cancer Institute (NCI) Diversity Set that bind with copper and act as anticancer agents. In each case, it is shown that these compounds can bind with copper, inhibit the proteasome activity, and induce apoptosis in cancer cells. These activities are absent when copper is not present. Compounds alone, clioquinol and pyrrolidinedithiocarbamate as examples, are shown to have no effects in normal breast cells. Current research suggests that a possible therapeutic modality for cancer may be developed using the difference of high copper load in tumors versus low copper load in normal cells. This strategy would convert tumor cellular copper into a potent, specific proteasome inhibitor and apoptosis inducer. Thus, this approach could pave the way for the development of nontoxic anticancer therapy.

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.

Nuclear Factor-κB Activated by Capacitative Ca2+ Entry Enhances Muscarinic Receptor-mediated Soluble Amyloid Precursor Protein (sAPPα) Release in SH-SY5Y Cells

G(q/11) protein-coupled muscarinic receptors are known to regulate the release of soluble amyloid precursor protein (sAPPalpha) produced by alpha-secretase processing; however, their signaling mechanisms remain to be elucidated. It has been reported that a muscarinic agonist activates nuclear factor (NF)-kappaB, a transcription factor that has been shown to play an important role in the Alzheimer disease brain, and that NF-kappaB activation is regulated by intracellular Ca2+ level. In the present study, we investigated whether NF-kappaB activation plays a role in muscarinic receptor-mediated sAPPalpha release enhancement and contributes to a changed capacitative Ca2+ entry (CCE), which was suggested to be involved in the muscarinic receptor-mediated stimulation of sAPPalpha release. Muscarinic receptor-mediated NF-kappaB activation was confirmed by observing the translocation of the active subunit (p65) of NF-kappaB to the nucleus by the muscarinic agonist, oxotremorine M (oxoM), in SH-SY5Y neuroblastoma cells expressing muscarinic receptors that are predominantly of the M3 subtype. NF-kappaB activation and sAPPalpha release enhancement induced by oxoM were inhibited by NF-kappaB inhibitors, such as an NF-kappaB peptide inhibitor (SN50), an IkappaB alpha kinase inhibitor (BAY11-7085), a proteasome inhibitor (MG132), the inhibitor of proteasome activity and IkappaB phosphorylation, pyrrolidine dithiocarbamate, the novel NF-kappaB activation inhibitor (6-amino-4-(4-phenoxyphenylethylamino) quinazoline), and by an intracellular Ca2+ chelator (TMB-8). Furthermore, both oxoM-induced NF-kappaB activation and sAPPalpha release were antagonized by CCE inhibitors (gadolinium or SKF96365) but not by voltage-gated Ca2+-channel blockers. On the other hand, treatment of cells with NF-kappaB inhibitors (SN50, BAY11-7085, MG132, or pyrrolidine dithiocarbamate) did not inhibit muscarinic receptor-mediated CCE. These findings provide evidence for the involvement of NF-kappaB regulated by CCE in muscarinic receptor-mediated sAPPalpha release enhancement.

Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells

IntroductionA physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison.MethodsBreast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage.ResultsWhen in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations similar to those found in patients, when treated with just CQ or PDTC alone, but not TM, undergo proteasome inhibition and apoptosis.ConclusionThe feature of breast cancer cells and tissues to accumulate copper can be used as a targeting method for anticancer therapy through treatment with novel compounds such as CQ and PDTC that become active proteasome inhibitors and breast cancer cell killers in the presence of copper.

Distribution of copper in the diatom Haslea ostrearia simonsen

Haslea ostrearia is a pennate diatom important for oysters culture. This species was selected to study experimentally the distribution of copper in algal cells since the phytoplankton exhibits a high capacity for metal accumulation and thus influences metal transfer to consumers. This alga was grown in batch culture and exposed to copper (30 μg/l) added in the medium on the first day of experiment. In order to separate the soluble and insoluble fractions binding copper algal cells were centrifuged after homogenization with Tris NaCl buffer (pH 8.6). A part of the cytosol was heat-denatured and then centrifuged again to separate heat-stable and heat denaturable compounds. Gel filtration Sephadex (G-75SF:10 3−7 × 10 4 Da) was used to determine the molecular weight of soluble compounds binding copper. In controls, insoluble compounds were preponderant (63%) versus soluble compounds whereas in contamined cells, soluble compounds became preponderant (66%). In the cytosol of both controls and contamined cells, copper was mainly associated with thermoresistant compounds (about 80%). In contamined cells, gel filtration have shown that copper binding compounds had low molecular weights less than 3 KDa associated with sulfhydrl groups represented 65% of total cytosolic copper and few minority (8%) compounds binding copper between 3 and 9 KDa. In conclusion, contamination of Haslea ostrearia by copper induces an increase of copper-binding to cytosolic, heat stable, thiol-rich compounds of low molecular weight with characteristics of phytochelatins.

Copper tolerance in Saccharomyces cerevisiae

A commercial strain of Saccharomyces cerevisiae was serially cultured in media containing copper up to a final concentration of 10 mmol l-1. This copper-tolerant subculture was assessed for its capacity to accumulate further quantities of copper. It was found that after Cu2+ accumulation the total copper content of this yeast was lower than the parent culture when exposed to similar conditions, indicating that the subculture was copper-resistant owing to reduced copper bioaccumulation properties. Although a low mass copper binding compound was isolated from the copper-tolerant subculture, no metallothionein was found. Scanning electron microscopy of S. cerevisiae showed the cell surface to be smooth except for bud scars. After exposure to copper ion-containing solutions the surface of copper-tolerant yeast became convoluted, the cell was generally more spherical and somewhat smaller.

Production of an extracellular copper-binding compound by the heterotrophic marine bacterium Vibrio alginolyticus

The heterotrophic marine bacterium Vibrio alginolyticus was shown to produce extracellular copper-binding compound (s) when exposed to copper in a seawater medium. Fractionation and analysis of copper, and methionine incorporation in culture supernatant fractions showed that the copper-binding compound coeluted with material which was radiolabeled with 35S methionine. This suggests that the copper-binding compound may be proteinaceous. Analysis of label incorporation from 14C glucose into the fraction containing the copper-binding compound indicated that it was produced actively by the cells during recovery from copper toxicity, and was not present because of non-specific release by lysed bacterial cells. Concentrated supernatants from control and copper-challenged cultures contained two compounds which could be marginally resolved by size exclusion HPLC (26 kD and 28 kD), and which were produced at about a ten-fold higher level in copper-challenged cultures than in controls. These data indicate that the bacteria respond to toxic copper levels by excretion of a ca. 28 kD compound (s), which serves to detoxify copper in the medium by formation of organic copper complexes. The data suggest a potential role for macromolecules excreted by heterotrophic bacteria in control of copper ion activity in seawater. The production of copper-complexing compounds in response to copper represents a mechanism through which bacteria may directly influence the speciation of metal ions in seawater.