The ionophore thiomaltol induces rapid lysosomal accumulation of copper and apoptosis in melanoma.

In this report, we investigate the toxicity of the ionophore thiomaltol (Htma) and Cu salts to melanoma. Divalent metal complexes of thiomaltol display toxicity against A375 melanoma cell culture resulting in a distinct apoptotic response at submicromolar concentrations, with toxicity of Cu(tma)2 > Zn(tma)2 >> Ni(tma)2. In metal-chelated media, Htma treatment shows little toxicity, but the combination with supplemental CuCl2, termed Cu/Htma treatment, results in toxicity that increases with suprastoichiometric concentrations of CuCl2 and correlates with the accumulation of intracellular copper. Electron microscopy and confocal laser scanning microscopy of Cu/Htma treated cells shows a rapid accumulation of copper within lysosomes over the course of hours, concurrent with the onset of apoptosis. A buildup of ubiquitinated proteins due to proteasome inhibition is seen on the same timescale and correlates with increases of copper without additional Htma.

Characterization of Endogenous and Extruded H2S and Small Oxoacids of Sulfur (SOS) in Cell Cultures.

This report characterizes and quantifies endogenous hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS = HOSH, HOSOH) in a panel of cell lines including human cancer (A375 melanoma cells, HeLa cervical cells) and noncancer (HEK293 embryonic kidney cells), as well as E. coli DH5α and S. cerevisiae S288C. The methodology used is a translation of well-studied nucleophilic and electrophilic traps for cysteine and oxidized cysteines residues to target small molecular weight sulfur species; mass spectrometric analysis allows for species quantification. The observed intracellular concentrations of H2S and SOS vary in different cell types, from nanomolar to femtomolar, typically with H2S > HOSOH > HOSH. We propose the term sulfome, a subset of the metabolome, describing the nonproteinaceous metabolites of H2S; the sulfomic index is as a measure of the S-oxide redox status, which gives a profile of endogenous sulfur at different oxidation states. An important observation is that H2S and SOS were found to be continuously extruded into surrounding media against a concentration gradient, implying an active efflux process. Small molecule inhibition of several H2S generating enzymes suggest that SOS are not derived solely from H2S oxidation. Even after successful inhibition of H2S production, cells maintain constant efflux and repopulate H2S and SOS over time. This work proves that these small sulfur oxoacids are generated in cells of all types, and their efflux implies that they play a role in cell signaling and possibly other vascular physiology attributed to H2S.

Characterization of Polysulfides, Polysulfanes, and Other Unique Species in the Reaction between GSNO and H2S.

Glutathione-based products, GSnX, of the reaction of hydrogen sulfide, H2S, S-nitroso glutathione, and GSNO, at varied stoichiometries have been analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) and chemical trapping experiments. A wide variety of glutathione-based species with catenated sulfur chains have been identified including sulfanes (GSSnG), sulfides (GSSnH), and sulfenic acids (GSnOH); sulfinic (GSnO2H) and sulfonic (GSnO3H) acids are also seen in reactions exposed to air. The presence of each species of GSnX within the original reaction mixtures was confirmed using Single Ion Chromatograms (SICs), to demonstrate the separation on the LC column, and given approximate quantification by the peak area of the SIC. Further, confirmation for different GSnX families was obtained by trapping with species-specific reagents. Several unique GSnX families have been characterized, including bridging mixed di- and tetra-valent polysulfanes and internal trithionitrates (GSNHSnH) with polysulfane branches. Competitive trapping experiments suggest that the polysulfane chains are formed via the intermediacy of sulfenic acid species, GSSnOH. In the presence of radical trap vinylcyclopropane (VCP) the relative distributions of polysulfane speciation are relatively unaffected, suggesting that radical coupling is not a dominant pathway. Therefore, we suggest polysulfane catenation occurs via reaction of sulfides with sulfenic acids.

Amyloids, melanins and oxidative stress in melanomagenesis.

Melanoma has traditionally been viewed as an ultraviolet (UV) radiation-induced malignancy. While UV is a common inducing factor, other endogenous stresses such as metal ion accumulation or the melanin pigment itself may provide alternative pathways to melanoma progression. Eumelanosomes within melanoma often exhibit disrupted membranes and fragmented pigment which may be due to alterations in their amyloid-based striated matrix. The melanosomal amyloid can itself be toxic, especially in combination with reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated by endogenous NADPH oxidase (NOX) and nitric oxide synthase (NOS) enzymes, a toxic mix that may initiate melanomagenesis. Further understanding of the loss of the melanosomal organization, the behaviour of the exposed melanin and the induction of ROS/RNS in melanomas may provide critical insights into this deadly disease.

The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols.

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma.

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.

New perspectives on melanoma pathogenesis and chemoprevention.

Epidemiologic studies implicate ultraviolet radiation (sunlight) as an etiologic agent for the pathogenesis of melanoma. However, the experimental evidence is less convincing. We present information from recent experimental findings that elevation of reactive oxygen species follows from melanin serving as a redox generator, and that this may play an important role in the etiology and pathogenesis of cutaneous melanoma. These observations offer a new paradigm for the development of preventive (and therapeutic) approaches to this disease.

Disulfiram facilitates intracellular Cu uptake and induces apoptosis in human melanoma cells.

The alcohol-abuse deterrent disulfiram (DSF) is shown to have a highly selective toxicity against melanoma in culture, inducing a largely apoptotic response, with much lower toxicity against several other cell lines. Melanoma cell lines derived from different stages (radial, vertical, and metastatic phase) were all sensitive to DSF treatment in vitro; melanocytes were only slightly affected. A required role of extracellular Cu is demonstrated for DSF toxicity. Low concentrations of DSF alone decreased the number of viable cells, and the addition of CuCl(2) significantly enhanced the DSF-induced cell death to less than 10% of control. Significantly, the intracellular Cu concentration of melanoma cells increased rapidly upon DSF treatment. Both the intracellular Cu uptake and the toxicity induced by DSF were blocked by co-incubation with bathocuproine disulfonic acid (BCPD, 100 muM), a non-membrane-permeable Cu chelator. Chemical studies demonstrated a complicated, extracellular redox reaction between Cu(II) and DSF, which forms the complex Cu(deDTC)(2) in high yield, accompanied by oxidative decomposition of small amounts of disulfiram. The Cu complex has somewhat higher activity against melanoma and is suggested to be the active agent in DSF-induced toxicity. The redox conversion of DSF was unique to Cu(II) and not engendered by the other common biological metal ions Fe(II or III), Mn(III), and Zn(II). The implications of this work are significant both in the possible treatment of melanoma as well as in limiting the known side-effects of DSF, which we propose may be diminished by cotreatment to decrease adventitious Cu.

Melanin as a target for melanoma chemotherapy: pro-oxidant effect of oxygen and metals on melanoma viability.

Melanoma cells have a poor ability to mediate oxidative stress, which may be attributed to constitutive abnormalities in their melanosomes. We hypothesize that disorganization of the melanosomes will allow chemical targeting of the melanin within. Chemical studies show that under oxidative conditions, synthetic melanins demonstrate increased metal affinity and a susceptibility to redox cycling with oxygen to form reactive oxygen species. The electron paramagnetic resonance (EPR)-active 5,5'-dimethyl-pyrollidine N-oxide spin adduct was used to show that binding of divalent Zn or Cu to melanin induces a pro-oxidant response under oxygen, generating superoxide and hydroxyl radicals. A similar pro-oxidant behaviour is seen in melanoma cell lines under external peroxide stress. Melanoma cultures grown under 95% O2/5% CO2 atmospheres show markedly reduced viability as compared with normal melanocytes. Cu- and Zn-dithiocarbamate complexes, which induce passive uptake of the metal ions into cells, show significant antimelanoma activity. The antimelanoma effect of metal- and oxygen-induced stress appears additive rather than synergistic; both treatments are shown to be significantly less toxic to melanocytes.

A Coordination Polymer of Nickel(II) Based on a Pentadentate N, S, and O Donor Ligand.

The synthesis, structural characterization, and electrochemical properties of a Ni(II) complex derived from the template reaction of N,N'-bis(2-mercaptoethyl)-1,5-diazacyclooctane nickel(II), Ni-1, with ICH(2)CO(2)Na are described. Blue N-(3-thiabutyl)-N'-(3-thiapentanoate)-1,5-diazacyclooctanenickel(II)iodide, [(tbtp-daco)Ni][I], [5]I, contains Ni(II) in an octahedral environment with N(2)S(2)O(2) donor atoms; one oxygen is from an adjacent [(tbtp-daco)Ni] ion and has the same distance to Ni(II) as the intramolecular oxygen, resulting in a coordination polymer. Complex [5]I.H(2)O, C(13)H(27)N(2)O(3)S(2)NiI, crystallizes in the orthorhombic space group Pbca with a = 10.898(3), b = 18.103(5), c = 19.020(5), and Z = 8. The extent to which the polymer is retained in solution is counterion dependent, which influences redox properties (accessibility of Ni(I) and Ni(III)).

Polydentate N(2)S(2)O and N(2)S(2)O(2) Ligands as Alcoholic Derivatives of (N,N'-Bis(2-mercaptoethyl)-1,5-diazacyclooctane)nickel(II) and (N,N'-Bis(2-mercapto-2-methylpropane)-1,5-diazacyclooctane)nickel(II).

The synthesis, structural characterization, spectroscopic, and electrochemical properties of N(2)S(2)-ligated Ni(II) complexes, (N,N'-bis(2-mercaptoethyl)-1,5-diazacyclooctane)nickel(II), (bme-daco)Ni(II), and (N,N'-bis(2-mercapto-2-methylpropane)1,5-diazacyclooctane)nickel(II), (bme-daco)Ni(II), derivatized at S with alcohol-containing alkyl functionalities, are described. Reaction of (bme-daco)Ni(II) with 2-iodoethanol afforded isomers, (N,N'-bis(5-hydroxy-3-thiapentyl)-1,5-diazacyclooctane-O,N,N',S,S')halonickel(II) iodide (halo = chloro or iodo), 1, and (N,N'-bis(5-hydroxy-3-thiapentyl)-1,5-diazacyclooctane-N,N',S,S')nickel(II) iodide, 2, which differ in the utilization of binding sites in a potentially hexadentate N(2)S(2)O(2) ligand. Blue complex 1 contains nickel in an octahedral environment of N(2)S(2)OX donors; X is best modeled as Cl. It crystallizes in the monoclinic space group P2(1)/n with a = 12.580(6) Å, b = 12.291(6) Å, c = 13.090(7) Å, beta = 97.36(4) degrees, and Z = 4. In contrast, red complex 2 binds only the N(2)S(2) donor set forming a square planar nickel complex, leaving both -CH(2)CH(2)OH arms dangling; the iodide ions serve strictly as counterions. 2 crystallizes in the orthorhombic space group Pca2(1) with a = 15.822(2) Å, b = 13.171(2) Å, c = 10.0390(10) Å, and Z = 4. Reaction of (bme-daco)Ni(II) with 1,3-dibromo-2-propanol affords another octahedral Ni species with a N(2)S(2)OBr donor set, ((5-hydroxy-3,7-dithianonadiyl)-1,5-diazacyclooctane-O,N,N',S,S')bromonickel(II) bromide, 3. Complex 3 crystallizes in the orthorhombic space group Pca2(1) with a = 15.202(5) Å, b = 7.735(2) Å, c = 15.443(4) Å, and Z = 4. Complex 4.2CH(3)CN was synthesized from the reaction of (bme-daco)Ni(II) with 1,3-dibromo-2-propanol. It crystallizes in the monoclinic space group P2/c with a = 20.348(5) Å, b = 6.5120(1) Å, c = 20.548(5) Å, and Z = 4.

Influence of Sulfur Metalation on the Accessibility of the Ni(II/I) Couple in [N,N'-Bis(2-mercaptoethyl)-1,5-diazacyclooctanato]nickel(II): Insight into the Redox Properties of [NiFe]-Hydrogenase.

A redox model study of [NiFe] hydrogenase has examined a series of five polymetallics based on the metalation of the dithiolate complex [1,5-bis(mercaptoethyl)-1,5-diazacyclooctane]Ni(II), Ni-1. Crystal structures of three polymetallics of the series have been reported earlier: [(Ni-1)(2)()Ni]Cl(2)(), [(Ni-1)(2)()FeCl(2)()](2)(), and [(Ni-1)(3)()(ZnCl)(2)()]Cl(2)(). Two are described here: [(Ni-1)(2)()Pd]Cl(2)().2H(2)()Ocrystallizes in the monoclinic system, space group P2(1)/c with cell constants a = 12.212(4) Å, b = 7.642(2) Å, c = 16.625(3) Å, beta = 107.69(2) degrees, V = 1443.230(0) Å(3), Z = 2, R = 0.051, and R(w) = 0.056. [(Ni-1)(2)()CoCl]PF(6)() crystallizes in the triclinic system, space group P&onemacr;, with cell constants a = 8.14(2) Å, b = 13.85(2) Å, c = 15.67(2) Å, alpha = 113.59(10) degrees, beta = 101.84(14) degrees, gamma = 94.0(2) degrees, V = 1561.620(0)Å(3), Z = 2, R = 0.072, and R(w) = 0.077. In all Ni-1 serves as a bidentate metallothiolate ligand with a "hinge" angle in the range 105-118 degrees and Ni-M distances of 2.7- 3.7 Å. The most accessible redox event is shown by EPR and electrochemistry to reside in the N(2)S(2)Ni unit and is the Ni(II/I) couple. Charge neutralization of the thiolate sulfurs by metalation can (dependent on the interacting metal) stabilize the Ni(I) state as efficiently as methylation forming a thioether. The implication of these results for the heterometallic active site of [NiFe]-hydrogenase as structured from Desulfovibrio gigas (Volbeda, A., et al. Nature, 1995, 373, 580), the generality of the Ni(&mgr;-SR)(2)M hinge structure, and a possible explanation for the unusual redox potentials are discussed.