Redox Cycling Of Copper Research Articles

Efficient activation of peracetic acid for abatement of tetracycline by W-doped CuS via regulating copper redox cycling

Recently, activation of peracetic acid (PAA) with the copper-based heterogeneous catalysts has been applied for organic pollutant abatement, yet the efficiency is limited by the sluggish Cu(II)/Cu(I) redox cycling. Regarding that reductive W(IV) can continuously effectively promote the regeneration of Cu(I) via the redox-reversible W(IV)/W(VI) couple, the W(IV) component is introduced to copper sulfide for efficient activation of PAA (simplified as the W-CuS/PAA process). Morphological and structural characterizations validate the incorporation of W(IV) component into the CuS lattice structure. Under neutral conditions, tetracycline (TC) can be completely removed within 30 min with the apparent first-order rate constant 5 times higher than that of the CuS/PAA process. CH3C(=O)OO is demonstrated as the main radical responsible for TC abatement by radical quenching experiment and electron paramagnetic resonance (EPR). The redox properties and electron transfer capacity of CuS are significantly improved by the W(IV) component. The reductive W(IV) and S(–II) components facilitate the Cu(II)/Cu(I) redox cycling with the concomitant generation of reactive radicals. In addition, the influence of various parameters and coexisting anions on TC abatement is systematically investigated. Four degradation pathways of TC are put forward and the toxicity of intermediates is evaluated. The W-CuS/PAA process shows superior reuse performance and excellent adaptability in different water matrices. This study highlights the capability of the W(IV) to modulate the Cu(II)/Cu(I) redox cycle and proposes a feasible activator amelioration strategy for the efficient activation of PAA.

Redox cycling of copper mediated by hydrazine: efficient catalyst systems for oxidative degradation of rhodamine B

Catalytic processes based on Fenton-like reactions on the degradation of organic pollutants have been improved by accelerating the redox cycling of metal ions. This work presents, at first, the results obtained for the heterogeneous degradation of rhodamine B (RhB) by copper ferrite (CuFe2O4) in presence of hydrogen peroxide (H2O2) and hydrazine (N2H4) as redox cycle accelerator. Atomic absorption spectroscopy showed small amounts of Cu2+ are leached from ferrite highlighting the influence of the homogeneous catalysis in the whole process. The data obtained for the homogeneous process using Cu2+ in solution containing both N2H4 and H2O2 indicated such system is highly efficient mineralizing 73% of RhB within only 10 min of reaction and having H2O and CO2 as major products. Using tert-butyl alcohol as radical scavenger, it was confirmed hydroxyl radical (HO•) is the active oxidant species regarding the RhB degradation. The homogeneous catalyst was applied to a real sample of textile effluent spiked with RhB and showed reasonable efficiency, although lower than that obtained for the standard solutions of RhB. This result was assigned to the interference of salts in the medium that react with HO• thus acting as radical scavenger.

Cancer Pro-oxidant Therapy Through Copper Redox Cycling: Repurposing Disulfiram and Tetrathiomolybdate.

Copper (Cu) is a transition metal active in Fenton redox cycling from reduced Cu+ and H2O2, to oxidized Cu2+ and the hydroxyl radical (·OH) highly reactive oxygen species (ROS). At homeostatic Cu levels, ROS promote cell proliferation, migration, angiogenesis, and wound repair. To limit ROS toxicity, cells use Cu-dependent chaperone proteins, Cu-binding ceruloplasmin, and Cu-modulated enzymes like superoxide dismutases (SOD) like SOD1 and SOD3 to scavenge excess superoxide anions which favour Cu+ reduction, and mitochondrial cytochrome c oxidase, important in aerobic energy production. Because Cu helps drive tumor cell proliferation by promoting growth factor-independent receptor tyrosine kinase signaling, and Cu-dependent MEK1 involved in oncogenic BRAF-V600E signaling, further augmenting bioavailable Cu may promote ROS overproduction, cancer progression and eventually tumor cell death. For these reasons, the following clinically approved copper chelators are being repurposed as anti-cancer agents: a) ammonium tetrathiomolybdate (TTM) used to treat Wilson's disease (copper overload) and Menkes disease (copper deficiency); b) Disulfiram (DSF), used against alcoholism, since it inhibits Aldehyde Dehydrogenase (ALDH1) enzyme, important in ethanol detoxification, and a key target against cancer stem cells. Moreover, TTM and DSF are also relevant in cancer clinical trials, because they increase the uptake of both Cu and Platinum (Pt)-containing anti-cancer drugs, since Pt and Cu share the same CTR1 copper transporter. The majority of reports on Cu chelators dealt separately with either TTM, DSF or others. Here, we compare in parallel, the anti-cancer efficacy of low doses of TTM and DSF, asking whether they can be synergistic or antagonistic. The relevance of their unequal ROS inducing abilities and their different behavior as ionophores is also addressed. The potential of Cu chelators as repurposed anti-cancer drugs, should be greater in patients with higher endogenous Cu levels. Since platinum and Cu share uptake receptors, the synergism by drugs containing these metals should not be under-estimated. The potential of disulfiram or its metabolically active Cu-containing form, to inhibit ALDH1-positive tumor cells is therapeutically very important.

Redox cycling of copper by coumarin-di(2-picolyl)amine hybrid molecule leads to ROS-mediated modulation of redox scavengers, DNA damage and cell death in diethylnitrosamine induced hepatocellular carcinoma.

Targeted therapy is a new strategy for cancer treatment that targets chemical entities specific to cancer cells than normal ones. One of the features associated with malignancy is the elevated copper which plays an integral role in angiogenesis. Work is in progress in our lab to identify new copper chelators to target elevated copper under targeted therapy for the killing of cancer cells. Recently, a coumarin-based copper chelator, di(2-picolyl)amine-3(bromoacetyl)coumarin hybrid molecule (ligand-L) has been synthesized by us, and also studied its copper-dependent macromolecular damage response in copper overloaded lymphocytes. The present study investigates the anticancer activity of ligand-L and its mode of action in rat model of diethylnitrosamine (DEN) induced hepatocellular carcinoma. It has been found that liver tissue has a marked increase in copper levels in DEN induced hepatocellular carcinoma. Ex vivo results showed that ligand-L inhibited cell viability, induced reactive oxygen species (ROS) generation, DNA damage, loss of mitochondrial membrane potential and caspase-3 activation in isolated hepatocellular carcinoma cells (HCC). All these effects induced by ligand-L were abrogated by neocuproine and N-acetylcysteine (ROS scavenger). Further, ligand-L treatment of animals bearing hepatocellular carcinoma results in an increment in the cellular redox scavengers, lipid peroxidation and DNA breakage in malignant hepatocytes. In vivo studies using ligand-L also showed that ligand-L possesses anticancer properties as evidenced by improvement in liver marker enzymes and liver surface morphology, and reduced alpha-fetoprotein in the treated group compared to untreated cancer-induced group. Overall, this study suggests that copper-ligand-L interaction leads to ROS generation which caused DNA damage and apoptosis in malignant cells. This study provides enough support to establish ligand-L as a clinically relevant lead molecule for the treatment of different malignancies.

Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper

Therapies for lung cancer patients initially elicit desirable responses, but the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer vs. normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50–150 nM) combined with physiological levels of Cu (15 μM CuSO4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O2, relative to 4 or 21% O2. This selective toxicity of DSF/Cu was associated with differential Cu ionophore capabilities. DSF/Cu treatment caused a >20-fold increase in cellular Cu in NSCLCs, with nearly two-fold higher Cu present in NSCLCs vs. HBECs and in cancer cells at 1% O2vs. 21% O2. DSF toxicity was shown to be dependent on the retention of Cu as well as oxidative stress mechanisms, including the production of superoxide, peroxide, lipid peroxidation, and mitochondrial damage. DSF was also shown to selectively (relative to HBECs) enhance radiation and chemotherapy-induced NSCLC killing and reduce radiation and chemotherapy resistance in hypoxia. Finally, DSF decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF could be a promising adjuvant to enhance cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.

Binding and Reactivity of Copper to R1 and R3 Fragments of tau Protein.

Tau protein is present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble neurofibrillary tangles of tau are associated with several neurological disorders known as tauopathies, among which is Alzheimer's disease. In neurons, tau binds tubulin through its microtubule binding domain which comprises four imperfect repeats (R1-R4). The histidine residues contained in these fragments are potential binding sites for metal ions and are located close to the regions that drive the formation of amyloid aggregates of tau. In this study, we present a detailed characterization through potentiometric and spectroscopic methods of the binding of copper in both oxidation states to R1 and R3 peptides, which contain one and two histidine residues, respectively. We also evaluate how the redox cycling of copper bound to tau peptides can mediate oxidation that can potentially target exogenous substrates such as neuronal catecholamines. The resulting quinone oxidation products undergo oligomerization and can competitively give post-translational peptide modifications yielding catechol adducts at amino acid residues. The presence of His-His tandem in the R3 peptide strongly influences both the binding of copper and the reactivity of the resulting copper complex. In particular, the presence of the two adjacent histidines makes the copper(I) binding to R3 much stronger than in R1. The copper-R3 complex is also much more active than the copper-R1 complex in promoting oxidative reactions, indicating that the two neighboring histidines activate copper as a catalyst in molecular oxygen activation reactions.

Disulfiram Causes Selective Hypoxic Cancer Cell Toxicity and Radio-Chemo-Sensitization via Redox Cycling of Copper

Disulfiram Causes Selective Hypoxic Cancer Cell Toxicity and Radio-Chemo-Sensitization via Redox Cycling of Copper

Multi-target-directed triazole derivatives as promising agents for the treatment of Alzheimer’s disease

A novel series of triazole-based compounds have been designed, synthesised and evaluated as multi-target-directed ligands (MTDLs) against Alzheimer disease (AD). The triazole-based compounds have been designed to target four major AD hallmarks that include Aβ aggregation, metal-induced Aβ aggregation, metal dys-homeostasis and oxidative stress. Among the synthesised compounds, 6n having o-CF3 group on the phenyl ring displayed most potent inhibitory activity (96.89% inhibition, IC50 = 8.065 ± 0.129 μM) against Aβ42 aggregation, compared to the reference compound curcumin (95.14% inhibition, IC50 = 6.385 ± 0.009 μM). Compound 6n disassembled preformed Aβ42 aggregates as effectively as curcumin. Furthermore, 6n displayed metal chelating ability and significantly inhibited Cu2+-induced Aβ42 aggregation and disassembled preformed Cu2+-induced Aβ42 aggregates. 6n successfully controlled the generation of the reactive oxygen species (ROS) by preventing the copper redox cycle. In addition, 6n did not display cytotoxicity and was able to inhibit toxicity induced by Aβ42 aggregates in SH-SY5Y cells. The preferred binding regions and key interactions of 6n with Aβ42 monomer and Aβ42 protofibril structure was evaluated with molecular docking. Compound 6n binds preferably to the C-terminal region of Aβ42 that play a critical role in Aβ42 aggregation. The results of the present study highlight a novel triazole-based compound, 6n, as a promising MTDL against AD.

Interaction of C20-substituted derivative of pregnenolone acetate with copper (II) leads to ROS generation, DNA cleavage and apoptosis in cervical cancer cells: Therapeutic potential of copper chelation for cancer treatment

Cervical cancer is a leading cause of cancer-related deaths among women in developing countries. Therefore, development of new chemotherapeutic agents is required. Unlike normal cells, cancer cells contain elevated copper levels which play an integral role in angiogenesis. Thus, targeting copper via copper-specific chelators in cancer cells can serve as effective anticancer strategy. In this work, a copper chelator pregnenolone acetate nucleus-based tetrazole derivative (ligand-L) was synthesized and characterized by elemental analysis, ESI-MS, 1H NMR and 13C NMR. DNA binding ability of ligand-L was studied using UV–Vis and fluorescence spectroscopy. Fluorescence spectroscopy studies reveal that quenching constant of ligand-l-DNA and ligand-L-Cu(II) were found to be 7.4 × 103 M−1 and 8.8 × 103 M−1, respectively. In vitro toxicity of ligand-L was studied on human cervical cancer C33A cancer cells. Results showed that ligand-L exhibit significant cytotoxic activity against cervical cancer C33A cells with IC50 value 5.0 ± 1.8 µM. Further, it was found that ligand-L cytotoxicity is due to redox cycling of copper to generate ROS which leads to DNA damage and apoptosis. In conclusion, this is the report where we synthesized pregnenolone acetate-based tetrazole derivative against C33A cells that targets cellular copper to induce pro-oxidant death in cancer cells. These findings will provide significant insights into the development of new chemical molecules with better copper chelating and pro-oxidant properties against cancer cells.

136 - Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper

136 - Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper

Copper-redox cycling by coumarin-di(2-picolyl)amine hybrid molecule leads to ROS-mediated DNA damage and apoptosis: A mechanism for cancer chemoprevention

Coumarin is an important bioactive pharmacophore. It is found in plants as a secondary metabolite and exhibits diverse pharmacological properties including anticancer effects against different malignancies. Therapeutic efficacy of coumarin derivatives depends on the pattern of substitution and conjugation with different moieties. Cancer cells contain elevated copper as compared to normal cells that plays a role in angiogenesis. Thus, targeting copper in malignant cells via copper chelators can serve as an attractive targeted anticancer strategy. Our previous efforts led to the synthesis of di(2-picolyl)amine-3(bromoacetyl)coumarin hybrid molecule (ligand-L) endowed with DNA/Cu(II) binding properties, and ROS generation ability in the presence of copper ions. In the present study, we aimed to validate copper-dependent cytotoxic action of ligand-L against malignant cells. For this, we used a cellular model system of copper (Cu) overloaded lymphocytes (CuOLs) to simulate malignancy-like condition. In CuOLs, lipid peroxidation/protein carbonylation, ROS generation, DNA fragmentation and apoptosis were investigated in the presence of ligand-L. Results showed that ligand-L-Cu(II) interaction leads to ROS generation, lipid peroxidation/protein carbonylation (oxidative stress parameters), DNA damage, up-regulation of p53 and mitochondrial-mediated apoptosis in treated lymphocytes. Further, pre-incubation with neocuproine (membrane permeable copper chelator) and ROS scavengers attenuated the DNA damage and apoptosis. These results suggest that cellular copper acts as molecular target for ligand-L to propagate redox cycling and generation of ROS via Fenton-like reaction leading to DNA damage and apoptosis. Further, we showed that ligand-L targets elevated copper in breast cancer MCF-7 and colon cancer HCT116 cells leading to a pro-oxidant inhibition of proliferation of cancer cells. In conclusion, we propose copper-dependent ROS-mediated mechanism for the cytotoxic action of ligand-L in malignant cells. Thus, targeting elevated copper represents an effective therapeutic strategy for selective cytotoxicity against malignant cells.

Mineralogy and geochemistry of the copper-dominated neutral mine drainage at the Cu deposit Ľubietová-Podlipa (Slovakia)

The neutral mine drainage system at Ľubietová in Slovakia is overwhelmingly dominated by copper (17 ppm in the most concentrated aqueous sample) and sulfate and almost devoid of iron (0.7 ppb in the most concentrated aqueous sample). This mine drainage is generated by oxidative dissolution of chalcopyrite (CuFeS2), as confirmed by sulfur isotopes, and its neutral nature is maintained by buffering via the primary carbonates. The rich oxidation zone, composed of abundant libethenite (Cu2(PO4)(OH)), pseudomalachite (Cu5(PO4)2(OH)4), and malachite (Cu2(CO3)(OH)2), appears to play only a passive and negligible role in the mine drainage generation. The copper- and sulfate-laden water is discharged through the lowermost adits in the ore field; inside the adits, blue gel covers the stream and converts slowly to crystalline langite (Cu4(SO4)(OH)6·2H2O). X-ray absorption spectroscopy on Cu K edge showed that the local structure inside the gel is similar to that in langite. Calculation of saturation indices showed supersaturation with respect to sulfates (langite, brochantite (Cu4(SO4)(OH)6)), malachite, and copper arsenates and phosphates. Arsenates and phosphates were not found and do not form, probably because of kinetic hindrance. The solubility product of langite was estimated as log Ksp = 17.73. Outside of the adits, transmission electron microscopy showed redox cycling of copper via the formation of nanocrystals of native copper on rock-forming sheet silicates. The copper concentrations are naturally attenuated, being reduced by a factor of 100 once the mine drainage reaches the Hutná river, about 1 km away from the discharge site. Sulfur isotopes document that no copper sulfides are precipitating; copper must be removed from the contaminated water in the form of sulfates or carbonates. The natural attenuation could be responsible for the enrichment of hyporheic sediments with copper whose mineralogy and geochemistry is unknown but its size is estimated to several metric tonnes of copper.

A new triazine bearing a pyrazolone group capable of copper, nickel, and zinc chelation.

Interest in inorganic applications of triazines is growing. In this report, metal complexes of copper(ii), nickel(ii), and zinc(ii) and a novel class of chelates composed of a triazine ring substituted with a hydrazine group and pyrazolone are evaluated using spectrophotometric methods, single crystal X-ray diffractometry, and electrochemistry. Complexes with copper(ii) include a single chelate and chloride ion(s)/water to satisfy a trigonal bipyramidal coordination sphere. The nickel(ii) and zinc(ii) complexes are composed of two chelating groups that adopt an octahedral geometry around the metal ion. Irreversible redox activity was observed with the copper(ii) complex but no redox activity was observed with the ligand alone or zinc(ii) and nickel(ii) complexes. Use of the coumarin carboxylic acid assay shows that the ligand motif is capable of preventing redox cycling of copper in biological conditions and could thus serve as an antioxidant preventative agent. Cellular toxicity studies show that the new triazine molecule could have therapeutic applications in the μM concentration range based on the measured EC50 = 1.183 ± 0.002 mM. Altogether this work shows that by merging triazine chemistry into inorganic compounds, there is potential to explore a range applications thanks to the new architecture.

115 - Disulfiram causes Selective Hypoxic Cancer Cell Toxicity and Radio-chemo-sensitization via Redox Cycling of Copper and O2•‒ mediated Oxidative Stress

Lung cancer is one of the most deadly cancers in the USA, and despite multiple modalities of treatment, the 5 year survival rate remains

Activation of oxygen via accelerating the copper-redox cycle by UV irradiation

This study proposes a new method showing that the addition of UV strongly enhanced the oxidation capacity of the Cu(II)/O 2 process, which subsequently produced reactive oxidants using copper as a catalyst via the Fenton-like reactions of in situ generated hydrogen peroxide (H 2 O 2 ). Acid orange 7 (AO7) was degraded more than 70% after 70 min at an initial pH of 3.5 in the Cu(II)/O 2 /UV process. The mechanism investigation showed that a remarkable amount of Cu(I) and H 2 O 2 was produced in the Cu(II)/O 2 /UV process, which was due to UV irradiation and activation of oxygen (O 2 ). The addition of a radical scavenger, tert-butyl alcohol, inhibited AO7 degradation, which verified that hydroxyl radical (·OH) was the primary radical for the degradation of AO7. Superoxide radical (O 2 · − ) was also generated in the Cu(II)/O 2 /UV process, indicating that (O 2 · − ) was considered as the intermediate of the production of ·OH. The results showed that the acidic condition was favorable for AO7 degradation, and the degradation of AO7 followed the pseudo-first-order kinetics. The high dosage of Cu(II) enhanced AO7 degradation, but exceeding the optimal dosage of Cu(II) would inhibit AO7 degradation.

Mammalian Metallothionein-3: New Functional and Structural Insights.

Metallothionein-3 (MT-3), a member of the mammalian metallothionein (MT) family, is mainly expressed in the central nervous system (CNS). MT-3 possesses a unique neuronal growth inhibitory activity, and the levels of this intra- and extracellularly occurring metalloprotein are markedly diminished in the brain of patients affected by a number of metal-linked neurodegenerative disorders, including Alzheimer’s disease (AD). In these pathologies, the redox cycling of copper, accompanied by the production of reactive oxygen species (ROS), plays a key role in the neuronal toxicity. Although MT-3 shares the metal-thiolate clusters with the well-characterized MT-1 and MT-2, it shows distinct biological, structural and chemical properties. Owing to its anti-oxidant properties and modulator function not only for Zn, but also for Cu in the extra- and intracellular space, MT-3, but not MT-1/MT-2, protects neuronal cells from the toxicity of various Cu(II)-bound amyloids. In recent years, the roles of zinc dynamics and MT-3 function in neurodegeneration are slowly emerging. This short review focuses on the recent developments regarding the chemistry and biology of MT-3.

High efficient catalytic degradation of PNP over Cu-bearing catalysts with microwave irradiation

The catalytic capabilities of copper doped attapulgite (Cu/ATP) and silicon carbide (Cu/SiC) nanocomposites for p-nitrophenol (PNP) were characterized under microwave (MW) irradiation. Higher catalytic performance of Cu/SiC was obtained than that of Cu/ATP due to higher production rate of reactive oxygen species (ROS) as well as higher MW absorptivity. The electron (e−) – hole (h+) pairs were related to the generation of ROS on Cu/SiC. The modification Cu with SiC had an enhancement on the band gap and large EVB absolute values, which promoted the catalytic activity of Cu/SiC. An ROS-producing copper-redox cycle between CuI and CuII of Cu/ATP was responsible for PNP degradation. The produced CuI via thermal dissociation of [Cu–O–Cu]n species in Cu/ATP activated molecular oxygen, followed by Fenton-like reactions to produce ROS. The reusability of the catalysts and negligible phase transformation of Cu species after five rounds of cycle verified the catalyst stability. Based on Density functional theory (DFT) calculation and catalytic experimental analyses, the denitro-hydroxylation of PNP was the initial oxidation reaction step followed by further oxidation to form short-chain carboxylic acids. OH oxidation was predominant whereas O2− was not involved in the OH addition on the meta-position due to lower oxidation energy than the corresponding activation energies. Other than reactive species attack, the “hot spots” on the surface of Cu/SiC was responsible for thermal stabilization of PNP/intermediates in soil. In this study, MW-assisted Cu/ATP and Cu/SiC catalytic degradation of PNP was proved to be an efficient technology for wastewater treatment and soil remediation.

Iron Complex Facilitated Copper Redox Cycling for Nitric Oxide Generation as Nontoxic Nitrifying Biofilm Inhibitor.

In this study, we developed poly(vinyl chloride) (PVC)-solvent casted mixed metal copper and iron complexes capable of catalytic generation of the antibiofilm nitric oxide (NO) from endogenous nitrite. In the absence of additional reducing agent, we demonstrated that the presence of iron complex facilitates a redox cycling, converting the copper(II) complex to active copper(I) species, which catalyzes the generation of NO from nitrite. Assessed by protein assay and surface coverage analyses, the presence of the mixed metal complexes in systems containing water industry-relevant nitrite-producing nitrifying biofilms was shown to result in a "nontoxic mode" of biofilm suppression, while confining the bacterial growth to the free-floating planktonic phase. Addition of an NO scavenger into the mixed metal system eliminated the antibiofilm effects, therefore validating first, the capability of the mixed metal complexes to catalytically generate NO from the endogenously produced nitrite and second, the antibiofilm effects of the generated NO. The work highlights the development of self-sustained antibiofilm materials that features potential for industrial applications. The novel NO-generating antibiofilm technology diverts from the unfavorable requirement of adding a reducing agent and importantly, the less tendency for development of bacterial resistance.

Copper-Aβ Peptides and Oxidation of Catecholic Substrates: Reactivity and Endogenous Peptide Damage.

The oxidative reactivity of copper complexes with Aβ peptides 1-16 and 1-28 (Aβ16 and Aβ28) against dopamine and related catechols under physiological conditions has been investigated in parallel with the competitive oxidative modification undergone by the peptides. It was found that both Aβ16 and Aβ28 markedly increase the oxidative reactivity of copper(II) towards the catechol compounds, up to a molar ratio of about 4:1 of peptide/copper(II). Copper redox cycling during the catalytic activity induces the competitive modification of the peptide at selected amino acid residues. The main modifications consist of oxidation of His13/14 to 2-oxohistidine and Phe19/20 to ortho-tyrosine, and the formation of a covalent His6-catechol adduct. Competition by the endogenous peptide is rather efficient, as approximately one peptide molecule is oxidized every 10 molecules of 4-methylcatechol.

Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells

ABSTRACTRecently, we showed that the metal chelator TPEN targets colon cancer cells through redox cycling of copper. Here, we studied the DNA damage potential of TPEN and deciphered the role of Chk1, ATM and DNA-PK in TPEN-induced toxicity in 3 human colon cancer cell lines, HCT116, SW480 and HT29. We also investigated the role of reactive oxygen species (ROS) in TPEN-induced DNA damage. TPEN reduced cell viability in a dose- and time-dependent manner. Cytotoxicity was associated with significant DNA damage and higher expression of γ-H2AX protein and activation of ATM/ATR signaling pathway. Cell death by TPEN was dependent on ROS generation as evidenced by the reversal of cell viability, and DNA damage and the abrogation of γ-H2AX levels in the presence of antioxidants. Treatment with antioxidants, however, failed to reverse cytotoxicity at high TPEN concentrations (10µM). TPEN-induced cell death was also dependent on the redox cycling of copper since the copper chelator neocuproine inhibited DNA damage and reduced pChk1, γ-H2AX, and ATM protein expression. Cell death by low TPEN concentrations, involved ATM/ATR signaling in all 3 cell lines, since pre-incubation with specific inhibitors of ATM and DNA-PK led to the recovery of cells from TPEN-induced DNA damage. In addition, siRNA silencing of Chk1, DNA-PK and ATM abrogated the expression of γ-H2AX and reversed cell death, suggesting that Chk1 and DNA-PK mediate TPEN-induced cytotoxicity in colon cancer cells. This study shows for the first time the involvement of Chk1, DNA-PK and ATM in TPEN-induced DNA damage and confirms our previous findings that ROS generation and the redox cycling of copper in response to TPEN are the main mechanisms by which this compound induces cell death in human colon cancer cells. Inhibition of ATM or DNA-PK did not reverse cytotoxicity at high TPEN concentrations that cause excessive levels of ROS and irreversible cellular damage.