Development of drug resistance to gallium nitrate through modulation of cellular iron uptake.

We have shown that transferrin-gallium (Tf-Ga) blocks DNA synthesis through inhibition of cellular iron incorporation and a diminution in the activity of the iron-dependent M2 subunit of ribonucleotide reductase. To examine the mechanisms of drug resistance to gallium, we developed a subline of HL60 cells (R cells) which is 29-fold more resistant to growth inhibition by gallium nitrate than the parent line (S cells). R cells displayed a 2.5-fold increase in transferrin (Tf) receptor expression, without a change in receptor affinity for Tf. The uptake and release of 67Ga were similar for both S and R cells. The uptake of 59Fe-Tf by S cells was inhibited by gallium nitrate over 24-48 h of incubation. In contrast, 59Fe-Tf uptake by R cells, although initially inhibited by gallium nitrate at 24 h, was no longer inhibited at 48 h of incubation. 59FeCl3 uptake by R cells was significantly greater than that of S cells, regardless of the time in culture. Despite the increase in 59Fe uptake by R cells, the ferritin content of these cells was lower than that of S cells. The ribonucleotide reductase electron spin resonance signal of R cells was comparable to that of S cells. R cells were not cross-resistant to Adriamycin, vincristine, cis-platinum or hydroxyurea. Resistance to gallium nitrate in this subline of HL60 cells results primarily from the ability of cells to overcome the gallium-induced block in iron incorporation. In addition, intracellular iron in R cells appears to traffic preferentially to a non-ferritin compartment.

Oxidation of low-density lipoprotein by Cu2+ and lipoxygenase: an electron spin resonance study.

The aim of this work was to obtain spectroscopic evidence for free radicals formed during copper ion- and lipoxygenase-catalyzed oxidation of the low-density lipoprotein. During the initial oxidation phase, a free-radical metabolite derived from the endogenous alpha-tocopherol present in the low-density lipoprotein was detected by the electron spin resonance technique. The divalent copper ions were bound to the residual EDTA present in the low-density lipoprotein and to the protein. Production of the alpha-tocopherol radical was suppressed in the presence of spin traps. Evidence for the low-density lipoprotein-lipid derived radicals was obtained by ESR-spin trapping methods. Implications of these findings in the oxidative modification of the low-density lipoprotein are discussed.

Inhibition of malignant cell growth by 311, a novel iron chelator of the pyridoxal isonicotinoyl hydrazone class: effect on the R2 subunit of ribonucleotide reductase.

The key roles of iron and iron proteins in cell proliferation make them potential targets for cancer therapy. However, clinical trials directed toward perturbation of tumor iron homeostasis by iron chelation have been limited to the use of deferoxamine (DFO). There is thus a need to develop agents with greater efficacy. In the present study, we investigated the mechanism of cytotoxicity of 311 (2-hydroxy-1-naphthylaldehyde benzoyl hydrazone), a novel iron chelator of the pyridoxal isonicotinoyl class. We found that 311 inhibited the growth of CCRF-CEM cells in a time- and concentration-dependent fashion with an IC(50) that was approximately 20-fold lower than that of DFO. 311 also inhibited the growth of breast, bladder, and head and neck cancer cell lines. Using electron spin resonance (ESR) spectroscopy analysis, we found that a 12-h exposure of CCRF-CEM cells to 311 inhibited the tyrosyl radical ESR signal of the R2 subunit of ribonucleotide reductase. However, overproduction of the R2 subunit in hydroxyurea-resistant CCRF-CEM cells was associated with a decrease in sensitivity of cells to 311 but not to DFO. Our studies show that 311 is a more potent cytotoxic agent than DFO, with activity against both hematopoietic and nonhematopoietic cell lines regardless of their p53 status. Furthermore, the ESR studies suggest that inhibition of the R2 subunit of ribonucleotide reductase is at least one mechanism by which 311 blocks cell proliferation.

The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells.

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine.

The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.

Spin-trapping studies of the oxidation-reduction reactions of iron bleomycin in the presence of thiols and buffer.

The reaction of ferrous bleomycin with dioxygen is reexamined to clarify whether radical species derived from molecular oxygen are generated. Detection of low levels of spin-trapped oxyradicals confirm the production of OH during this reaction when bleomycin is present in excess, but not when iron and drug concentrations are equal. In phosphate buffer, hydroxyl radicals continue to be spin trapped for at least 15 min after Fe(II)bleomycin has been oxidized to Fe(III)bleomycin. In HEPES buffer, detection of a HEPES radical in the absence of spin trap over the same period independently supports the conclusion that reactive radicals are present after the initial oxidation of Fe(II)bleomycin is complete. When glutathione is included in the aerobic reaction mixture, thiyl radical species are spin trapped. The reaction of Fe(III)bleomycin with cysteine produces thiyl radical without spin-trapped hydroxyl radical.

Characterization of the adduct formed from the reaction between homocysteine thiolactone and low-density lipoprotein: antioxidant implications.

Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol/mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactive nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spin labeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2(+)-mediated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase.

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu-Cu interaction in both enzymes. C-band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well-resolved 7-line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g-values are calculated for gz and gx at four frequencies. No consistent g-values are obtained with the assumption of a 4-line pattern indicative for a mononuclear Cu site.

Inhibition of tumor cell transplantability by iron and copper complexes of 5-substituted 2-formylpyridine thiosemicarbazones.

The cytotoxicity of copper and iron complexes of 5-substituted 2-formylpyridine thiosemicarbazones against Ehrlich ascites tumor cells has been measured. Brief in vitro incubation of cells and drugs is followed by implantation into host mice. Subsequent degree of tumor development is a measure of cytotoxicity. A spectrum of activities for the iron complexes is observed, starting with the least active as designated by its 5-substitution: OH less than OCOCH3 approximately N(CH3)2 less than H less than CH3 approximately Cl approximately CF3. The last three complexes can prevent completely tumor growth in the new host. Copper complexes of 5-H and 5-CH3 also prevent successful tumor cell transplantation.

Cytotoxic and antitumor properties of bleomycin and several of its metal complexes.

This study examines the concentration-dependent cytotoxicity and antitumor activity of bleomycin (Blm) and Cu-, Zn-, Fe(III)-, and CoBlm using Ehrlich cells in culture and the Ehrlich ascites tumor. The order of activity in culture under several conditions is CuBlm approximately equal to Blm approximately equal to ZnBlm > Fe(III)Blm > CoBlm approximately equal to control. Short exposures of cells to drugs in the presence or absence of serum produced effects on cell proliferation similar to 48-h incubations. With Blm and CuBlm there was no obvious relationship between cytotoxicity and the modest short-term inhibition of DNA synthesis by the drugs. The antitumor experiments produced qualitatively similar results with the order of antitumor potency being CuBlm > Blm > ZnBlm approximately equal to FeBlm > CoBlm approximately equal to control tumor. The host toxicity produced by these drugs as measured by weight loss had the opposite ordering: CoBlm < FeBlm < ZnBlm < Blm < CuBlm. At therapeutically effective concentrations, FeFlm was significantly less toxic relative to ther other active agents.

Effect of gallium on the tyrosyl radical of the iron-dependent M2 subunit of ribonucleotide reductase.

Gallium, a pharmacologically important metal which resembles iron, was shown in previous studies to inhibit ribonucleotide reductase. To better understand its mechanism of action, we have examined the interaction of gallium with the iron-dependent M2 subunit of ribonucleotide reductase. In its active form, M2 contains an iron center and a tyrosyl free radical which is detectable by ESR spectroscopy. In the present study, cytoplasmic extracts prepared from murine leukemic L1210 cells after an 18-hr incubation with 960 microM gallium nitrate displayed a > 60% inhibition in their M2 tyrosyl radical ESR signal. However, this signal was restored within 15 min to levels greater than that of controls by the addition of increasing concentrations of ferrous ammonium sulfate. Gallium citrate added directly to cytoplasmic extracts from control cells also decreased the tyrosyl radical signal, an effect which could be reversed by iron. Immunoblot analysis revealed that incubation with gallium did not diminish the amount of M2 protein in cells, thus indicating that the decrease in the tyrosyl radical signal was not due to a decrease in cellular M2 content. In immunoprecipitation studies of 59Fe-labeled M2, gallium displaced 55-60% of the 59Fe incorporated into M2. Our studies suggest that gallium displaces iron from the M2 subunit of ribonucleotide reductase, resulting in a loss of the tyrosyl radical and an accumulation of inactive M2 within the cell.

Activity of bleomycin in iron- and copper-deficient cells.

Three models were used to examine the requirement of bleomycin (Blm) for iron (Fe) to carry out its antitumor or cytotoxic activity. Mice were made iron deficient by dietary means. Animals with depressed iron stores in liver and low plasma and ascites fluid iron supported Ehrlich tumor growth as well as mice maintained on a control diet. Bleomycin was equally effective against this tumor in iron-deficient mice as it was against the tumor in iron-sufficient controls. Likewise, nutrient copper deficiency did not change the efficacy of the drug. Ehrlich cells in culture were treated with a non-growth inhibiting concentration of the chelating agent, 1,10-phenanthroline before or during their exposure to bleomycin. Again, the treated cells were as sensitive to drug as controls, despite the fact that this ligand reduces cellular iron and zinc and can extract iron from Fe(II)Blm. Lastly, it was demonstrated that iron-depleted Euglena gracilis cells growing at reduced rates were as sensitive to growth inhibition by bleomycin as control cells.

Properties of redox-inactivated bleomycins. In vitro DNA damage and inhibition of Ehrlich cell proliferation.

Blenoxane, bleomycin A2, bleomycin B2, and demethyl bleomycin A2 and products of their reactions with Fe2+ and oxygen were used to explore the relationship between their capacity to carry out in vitro DNA strand scission and their growth inhibitory activity against Ehrlich cells. Reaction of Fe2+, bleomycin and O2 in the absence of DNA decreased the subsequent effectiveness of various bleomycin congeners to degrade DNA in the presence of Fe2+ and oxygen. In comparison with controls, this loss of strand scission activity was not paralleled by equivalent decreases in growth inhibition. Demethyl bleomycin A2 retained full biological activity relative to bleomycin A2, despite being only 30% as effective as bleomycin A2 in its ability to cleave DNA in vitro. Prior reaction of bleomycins with Fe2+ did not alter their capacity to reduce oxygen or affect their ability to generate the activated intermediate which, for native bleomycin structures, is competent to cleave DNA in vitro.

ESR of copper and iron complexes with antitumor and cytotoxic properties.

The relatively few iron and copper metal complexes which have been examined in cells and tissues for their redox properties, radical generation properties, and antitumor activity are discussed for studies which utilized electron spin resonance spectroscopy (ESR). A common property of a number of metal complexes, which include bleomycin, adriamycin, and thiosemicarbazones described in this review, is that they are readily reduced by thiol compounds and oxidized by oxygen or reduced species of oxygen to produce radicals. Structural features of these reactions are identified by ESR spectroscopy in model systems and often in cells. Furthermore, ESR spectroscopy has been most useful to probe the environment of the complexes in cells and to measure the rate of reduction of their oxidized forms. As a result of these studies, it is anticipated that more attention will be given to the exploration of redox-active metal complexes as drugs.

The influence of gender on developing copper deficiency and on free radical generation of rats fed a fructose diet.

The present investigation was conducted to determine whether differences in copper and iron status between male and female rats can be detected during the development of copper deficiency. These differences may explain the protection of the female against the severity of copper deficiency. In addition, the livers of all rats were exposed to electron-spin resonance (ESR) spectroscopy for the presence of free radicals. Male and female rats were fed from weaning either copper-deficient or -adequate diets containing fructose for 31 days. Rats were killed at day 0, 8, 16, 24, and 31 of the study. Throughout the study, copper-deficient males exhibited the same organ copper concentrations as copper-deficient female rats. However, only in the male did copper deficiency cause a reduction in body weight and an increase in liver and heart sizes but a decrease in pancreas size. In contrast, organ iron concentrations were different between males and females. Only copper-deficient males were anemic. Only the livers of copper-deficient males showed the presence of free radicals. Although the livers of copper-deficient female rats exhibited higher concentrations of hepatic iron than their male counterparts, their livers did not show the presence of free radicals. The data of the present study suggest that changes in organ sizes and the severity of copper deficiency are not solely due to the total concentrations of iron and/or copper. The type of iron compound and the presence of free radicals may be involved in the pathology of copper deficiency of the male.

The severity of copper deficiency can be ameliorated by deferoxamine.

The present study was undertaken in order to determine whether hepatic iron overload plays a role in the exacerbation of copper deficiency. Weanling male Sprague-Dawley rats were fed a copper-deficient (0.6 microgram Cu/g) diet containing 62% fructose for 5 weeks. Some of the copper-deficient rats were injected daily with deferoxamine (DFX), an iron chelator that has been widely used to reduce iron overload. DFX reduced hepatic iron concentrations, which in turn ameliorated the pathology of copper deficiency when compared with nontreated copper-deficient animals. It is suggested that hepatic iron overload in a reduced environment plays a major role in the exacerbation of copper deficiency. Once the concentration of hepatic iron is reduced, the severity of the deficiency should be improved.

Cytochrome b(5) plays a key role in human microsomal chromium(VI) reduction.

The reduction of chromium(VI) to Cr(III) results in the formation of reactive intermediates that contribute to the cytotoxicity, genotoxicity, and carcinogenicity of Cr(VI)-containing compounds. Previous studies suggest that human microsomal Cr(VI) reduction likely proceeds through cytochrome b(5). In order to better understand Cr(VI) toxicity in humans, the role of cytochrome b(5) in combination with P450 reductase was examined in the reductive transformation of Cr(VI). Proteoliposomes containing human recombinant cytochrome b(5) and P450 reductase were constructed. The ability of P450 reductase to mediate efficient electron transfer from NADPH to cytochrome b(5) was confirmed by spectral analysis. The NADPH-dependent Cr(VI) reduction rate mediated by proteoliposomes was then compared to that of human microsomes. When these rates were normalized to equivalent cytochrome b(5) concentrations, the NADPH-dependent Cr(VI) reduction rates mediated by human microsomes were essentially identical to those for proteoliposomes containing cytochrome b(5) plus P450 reductase. Proteoliposomes containing only P450 reductase or cytochrome b(5) exhibited poor Cr(VI) reducing capabilities. Since it had been previously shown that trace amounts of iron (Fe) could dramatically stimulate microsomal Cr(VI) reduction, the ability of Fe to stimulate Cr(VI) reduction by proteoliposomes was examined. Both ferric chloride (FeCl(3)) and ferric adenosine-5'-diphosphate (FeADP) were shown to stimulate Cr(VI) reduction; this stimulation could be abolished by the addition of deferoxamine, a specific Fe(III) chelator. The NADPH-dependent reduction rates of various ferric complexes by proteoliposomes were sufficient to account for the increased Cr(VI) reduction rates seen with the addition of FeCl(3) or FeADP. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy as a transient intermediate formed during NADPH-dependent Cr(VI) reduction mediated by proteoliposomes containing cytochrome b(5) and P450 reductase. Overall, cytochrome b(5) in combination with P450 reductase can account for the majority of the NADPH-dependent Cr(VI) reduction seen with human microsomes.

ESR parameters for cupric bleomycin in the mobilized state.

The configuration of the copper complex of the glycopeptide bleomycin, CuBlm, is presumed to be pyramidal square planar from a previous X-ray structural determination of a fragment of cupric bleomycin. This study presents evidence for a difference in the ESR parameters for cupric bleomycin in the liquid as opposed to the solid state. A decrease in Aiso for CuBlm in the liquid state can be directly surmised from the low frequency S-band spectrum for which three of the four cupric hyperfine lines are partially resolved. Computer simulated spectra infer that the absolute value of All increases about 100 MHz and the value of Al may change sign for CuBlm in the liquid state. Simulations using a rotational correlation time of about 250 psec. indicate that CuBLM may not be spherical in the liquid phase. The fastest component for anisotropic motion could dominate and account for the well resolved cupric hyperfine structure. Furthermore, it is argued from an analysis of the cupric hyperfine coupling constants that the CuBlm structure opens up at room temperature and that the cupric ion is displaced from the square plane.

The oxidation of cat, human, and the cat-human hybrid hemoglobins alpha 2 human beta 2 cat and alpha 2 cat beta 2 human by copper(II).

The heme iron of the beta chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the beta globin chain. The presence of freely reactive -SH groups on the alpha chains of cat and sheep hemoglobins does not alter the course of this reaction; only the beta hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two beta chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, alpha 2 Human beta 2 Cat and alpha 2 Cat beta 2 Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the beta subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid alpha 2 Cat beta 2 Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or alpha 2 Human beta 2 Cat. Cat beta globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2 beta. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the beta chain and is associated with histidine 2 beta.