The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase

The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a beta-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.     

Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine

Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase). 14C chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding 14C chloroethyl-thioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated beta-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with NADPH as electron donor and DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intracellularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase or glutathione reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.     

The role of calcium in the regulation of free radical reduction by thioredoxin reductase at the surface of the skin

Membrane associated thioredoxin reductase has been previously shown to reduce free radicals on the outer plasma membranes of human keratinocytes and melanocytes to provide a possible first line of defense against free radical damage at the surface of the skin. Preliminary experiments with cell cultures of human keratinocytes and melanocytes grown in serum-free medium showed that the enzyme activity depends on extracellular calcium concentration in the medium. Thioredoxin reductase activity at the surface of the skin, at the surface of human keratinocytes and melanocytes, and purified thioredoxin reductase from E. coli and adult human keratinocytes all exhibited calcium-dependent allosteric control. Since thioredoxin reductase contains two extremely reactive thiolate groups at the active site with pK values close to neutrality, both of these anions can form covalent complexes with N-ethylmaleimide by nucleophilic attack on the double bond. In our experiments we used spin-labeled maleimide [4-maleimido-tempo] to examine the local environment in the active site of thioredoxin reductase in the presence and absence of calcium. Both spin-labeled thioethers are distinguishable by EPR spectroscopy, with one site being significantly more immobilized than the other. Hence, it has been possible to observe direct evidence for active site closure by calcium. These results suggest that extracellular calcium may play an important role in regulation of thioredoxin reductase activity for the defense mechanism against UV-mediated free radical damage at the surface of human skin.     

EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes

Thioredoxin reductases purified from Escherichia coli from human metastatic melanoma tissue and from human keratinocytes are subject to allosteric inhibition by calcium. 45Calcium has been used to show that this enzyme contains a single binding site. Bound calcium does not exchange from thioredoxin reductase upon dialysis for 48 hours or upon exposure to 10(-3) M EGTA. An intelligenetics computer analysis yielded a single EF-hands calcium binding site on E. coli thioredoxin reductase with homology to the first EF-hands site on calmodulin. Calcium exchange from the enzyme requires the addition of the natural electron acceptor oxidized thioredoxin which causes a concentration dependent slow exchange. Due to the large conformational change caused by calcium binding to thioredoxin reductase it has been possible to separate Calcium-free and Calcium-bound enzyme by FPLC chromatography. Human keratinocytes contain 5% thioredoxin reductase in their acidic protein cytosol fraction. The influence of extracellular calcium concentration on the intracellular equilibrium between calcium bound versus calcium free thioredoxin reductase has been assessed. This equilibrium was shown to determine the redox status of keratinocytes via the reduction of thioredoxin. Our results provide the first evidence for calcium dependent regulation of redox conditions in the human epidermis.     

Calcium regulates thioredoxin reductase in human metastatic melanoma

Thioredoxin reductase has been purified from human metastatic melanotic melanoma and amelanotic melanoma tissues. Enzyme from the melanotic melanoma tissue contains bound calcium showing classical sigmoidal allosteric kinetics, whereas enzyme from the amelanotic melanoma yielded normal Michaelis-Menten saturation with substrate. Calcium inhibition can be partially reversed by oxidized thioredoxin. 45Ca has been used to label the amelanotic melanoma enzyme in order to determine the number of calcium-binding sites. These isotope experiments yielded only one calcium-binding site per enzyme molecule. Enzyme labeled with 45Ca was dialyzed for 24 h without loss of radioactivity, but the addition of oxidized thioredoxin to this labeled enzyme caused 60% calcium exchange in 24 h. Comparative studies with Escherichia coli thioredoxin reductase showed similar calcium inhibition as well as partial reactivation with oxidized thioredoxin. The enzyme from E. coli previously sequenced by others, showed considerable homology with the first EF-hands calcium-binding site of calmodulin. Detailed calcium-binding studies indicated that 10(-5) M of this fast exchange ion was sufficient to cause allosteric regulation in 10 min. This strong calcium-binding property could explain the allosteric nature of the thioredoxin reductase purified from human metastatic melanotic melanoma and its role in the regulation of melanin biosynthesis.     

Characterization of Escherichia coli thioredoxins with altered active site residues

Escherichia coli thioredoxin is a small disulfide-containing redox protein with the active site sequence Cys-Gly-Pro-Cys-Lys. Mutations were made in this region of the thioredoxin gene and the mutant proteins expressed in E. coli strains lacking thioredoxin. Mutant proteins with a 17-membered or 11-membered disulfide ring were inactive in vivo. However, purified thioredoxin with the active site sequence Cys-Gly-Arg-Pro-Cys-Lys is still able to serve as a substrate for thioredoxin reductase and a reducing agent in the ribonucleotide reductase reaction, although with greatly reduced catalytic efficiency. A smaller disulfide ring, with the active site sequence Cys-Ala-Cys, does not turn over at a sufficient rate to be an effective reducing agent. Strain in the small ring favors the formation of intermolecular disulfide bonds. Alteration of the invariant proline to a serine has little effect on redox activity. The function of this residue may be in maintaining the stability of the active site region rather than participation in redox activity or protein-protein interactions. Mutation of the positively charged lysine in the active site to a glutamate residue raises the Km values with interacting enzymes. Although it has been proposed that the positive residue at position 36 is conserved to maintain the thiolate anion on Cys-32 (Kallis & Holmgren, 1985), the presence of the negative charge at this position does not alter the pH dependence of activity or fluorescence behavior. The lysine is most likely conserved to facilitate thioredoxin-protein interactions.     

The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.     

The role of thioredoxin reductase in the reduction of free radicals at the surface of the epidermis

A study of guinea pig and human skin in vivo has revealed that keratinocytes contain a thioenzyme which reduces radicals. This enzyme has been purified by affinity column chromatography and identified as thioredoxin reductase. In vivo and in vitro bioassays were performed by using a spin-labelled surfactant as the radical substrate, because it can diffuse through the stratum corneum and react by surface complexation with the epidermis and also on the outer plasma membrane of keratinocytes from cell cultures. Thioredoxin, the native substrate for thioredoxin reductase effectively competes for electrons with radical substrates. Nicotinamide adenine dinucleotide phosphate (NADPH) is the electron donating coenzyme in both the reduction of radicals and thioredoxin. Reduced thioredoxin has been shown to be an inhibitor of tyrosinase, whereas oxidized thioredoxin has no effect on this enzyme activity. Taken together these results indicate that the thioredoxin/thioredoxin reductase system plays an important role in preventing cell damage from UV-generated free radicals on the skin.     

The activity and purification of membrane-associated thioredoxin reductase from human metastatic melanotic melanoma

Electron spin resonance spectroscopy has been used to measure the reduction of nitroxide radicals on a spin-labeled quaternary ammonium substrate by plasma membrane-associated thioredoxin reductase (EC 1.6.4.5) at the surface of cutaneous and subcutaneous melanoma metastases from one patient (B.M.). Enzyme activity in these metastases was shown to be hyperactive compared to normal skin and was subject to inhibition by calcium. From the remainder of the tissue (50.6 g), plasma membrane-associated thioredoxin reductase has been isolated and its molecular properties were compared with the same enzyme purified from the cytosol of rat liver and Escherichia coli. The enzyme from melanoma possessed an identical molecular weight to that from rat liver as determined by SDS-polyacrylamide gel electrophoresis (Mr 58,000). Upon fluorescence spectroscopic examination, the enzyme from melanoma was shown to contain flavin adenine dinucleotide as previously shown in the enzymes from E. coli and rat liver. The increased activities in plasma membrane-associated thioredoxin reductase in metastases of malignant melanotic melanoma are discussed in terms of the cellular functions of this important enzyme.     

Protein and ligand adaptation in a retinoic acid binding protein.

A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any structural differences in the protein when bound to the all-trans and 9-cis isomers, the structures of all-trans retinoic acid-ERABP and 9-cis retinoic acid ERABP were determined. Our results indicate that the all-trans isomer of retinoic acid adopts an 8-cis structure in the binding cavity with no concomitant conformational change in the protein. The structure of TTNPB-ERABP is also reported herein. To accommodate this all-trans analog, which cannot readily adopt a cis-like structure, alternative positioning of critical binding site side chains is required. Consequently, both protein and ligand adaption are observed in the formation of the various holo-proteins.     

The biological activity of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid in the rat vaginal smear assay

The biological activity of a series of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid has been evaluated in the vaginal smear assay carried out in vitamin A-deficient rats. These analogs were designed to probe the role of the 13-cis isomer in the actions of the parent all-trans-retinoic acid by blocking the interconversion of these two compounds. Although relatively less active, the potency of some of the cyclopropyl analogs suggests that 13-cis-retinoic acid is a fully active metabolite of all-trans-retinoic acid. Since 13-cis-retinoic acid represents a small percentage of the retinoic acid metabolites, the physiological significance of this activity is still unclear. Possible reasons for the reduced activity of the cyclopropyl analogs, as well as an aromatic analog of retinoic acid, are discussed.     

Ethaselen: a potent mammalian thioredoxin reductase 1 inhibitor and novel organoselenium anticancer agent

Mammalian thioredoxin reductase 1 (TrxR1) is considered to be an important anticancer drug target and to be involved in both carcinogenesis and cancer progression. Here, we report that ethaselen, a novel organoselenium compound with anticancer activity, specifically binds to the unique selenocysteine-cysteine redox pair in the C-terminal active site of mammalian TrxR1. Ethaselen was found to be a potent inhibitor rather than an efficient substrate of mammalian TrxR1. It effectively inhibits wild-type mammalian TrxR1 at submicromolar concentrations with an initial mixed-type inhibition pattern. By using recombinant human TrxR1 variants and human glutathione reductase, we prove that ethaselen specifically targets the C-terminal but not the N-terminal active site of mammalian TrxR1. In A549 human lung cancer cells, ethaselen significantly suppresses cell viability in parallel with direct inhibition of TrxR1 activity. It does not, however, alter either the disulfide-reduction capability of thioredoxin or the activity of glutathione reductase. As a downstream effect of TrxR1 inactivation, ethaselen causes a dose-dependent thioredoxin oxidation and enhances the levels of cellular reactive oxygen species in A549 cells. Thus, we propose ethaselen as the first selenium-containing inhibitor of mammalian TrxR1 and provide evidence that selenium compounds can act as anticancer agents based on mammalian TrxR1 inhibition.     

Median effect and long term recovery analysis of biological modifier interactions with difluoromethylornithine on the proliferation of human melanoma cells

The ability of difluoromethylornithine (DFMO), a specific polyamine synthesis inhibitor, to interact with various biological modifiers to inhibit the colony-forming growth of human melanoma cells was determined by using the median effect principle to computer model the strength of two agent interactions. Either alpha- or gamma-IFN (interferon) in combination with DFMO resulted in a synergistic inhibition on human melanoma colony formation. For human melanoma cells which were not resistant to 13-cis RA (retinoic acid), an additive suppression on colony formation was obtained with the retinoid-DFMO combination. Dexamethasone (DEX) interacted with DFMO to yield a synergistic reduction in melanoma colony number on glucocorticoid sensitive cells and no growth enhancement with DFMO on glucocorticoid resistant melanoma lines. Human melanoma cells displayed differential long-term growth sensitivity to DFMO treatment. C8146C human melanoma cells were terminally growth-inhibited by a 96 h exposure to DFMO, in a manner which was concentration and time dependent. The proliferation of C82-7A melanoma cells was inhibited by 95% in presence of DFMO, but upon removal of DFMO the cells regained their ability to proliferate and form colonies. The simultaneous addition of DEX plus alpha-IFN plus 13-cis-RA with DFMO caused most of the human melanoma cells in these lines to become permanently growth arrested. Pre-treatment with DEX plus alpha-IFN plus 13-cis RA, but without DFMO, did not have any long term effect on the ability of melanoma cells to recover and proliferate in soft agar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications of the active center of T4 thioredoxin by site-directed mutagenesis

The active site sequence of T4 thioredoxin, Cys-Val-Tyr-Cys, has been modified in two positions to Cys-Gly-Pro-Cys to mimic that of Escherichia coli thioredoxin. The two point mutants Cys-Gly-Tyr-Cys and Cys-Val-Pro-Cys have also been constructed. The mutant proteins have similar reaction rates with T4 ribonucleotide reductase as has the wild-type T4 thioredoxin. Mutant T4 thioredoxins with Pro instead of Tyr at position 16 in the active site sequence have three to four times lower apparent KM with E. coli ribonucleotide reductase than wild-type T4 thioredoxin. The KM values for these mutant proteins which do not have Tyr in position 16 are thus closer to E. coli thioredoxin than to the wild-type T4 thioredoxin. The bulky tyrosine side chain probably prevents proper interactions to E. coli ribonucleotide reductase. Also the redox potentials of these two mutant thioredoxins are lower than that of the wild-type T4 thioredoxin and are thereby more similar to the redox potential of E. coli thioredoxin. Mutations in position 15 behave more or less like the wild-type protein. The kinetic parameters with E. coli thioredoxin reductase are similar for wild-type and mutant T4 thioredoxins except that the apparent kcat is lower for the mutant protein with Pro instead of Tyr in position 16. The active site sequence of T4 thioredoxin has also been changed to Cys-Pro-Tyr-Cys to mimic that of glutaredoxins. This change does not markedly alter the reaction rate of the mutant protein with T4 ribonucleotide reductase or E. coli thioredoxin reductase, but the redox potential is lower for this mutant protein than for wild-type T4 thioredoxin.     

Retinoic acids regulate apoptosis of T lymphocytes through an interplay between RAR and RXR receptors

Vitamin A deficiency has been known for a long time to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Increasing evidence suggests that retinoic acids derived from vitamin A are involved in the functional regulation of the immune system. Of the two groups of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) all-trans and 9-cis retinoic acids are high affinity ligands for RARs and 9-cis retinoic acid additionally binds to RXRs. In cells, at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid by unknown mechanisms. Apoptosis plays a major role in shaping the T cell repertoire and one way in which retinoids may affect immune functions is to influence the various apoptosis pathways. Indeed, it has been shown that retinoic acids can induce apoptosis, increase the rate of dexamethasone-induced death and inhibit activation-induced death of thymocytes and T lymphocytes. Therefore, retinoids together with glucocorticoids may be involved in regulating positive and negative selection of T lymphocytes. Here we demonstrate that retinoids can induce apoptosis of T cells through the stimulation of RARgamma. Specific stimulation of RARalpha, on the other hand, prevents both RARgamma-dependent and TCR-mediated cell death. In all these functions 9-cis retinoic acid proved to be more effective than all-trans retinoic acid suggesting the involvement of RXRs. Based on these results a possible mechanism through which costimulation of RARs and RXRs might affect spontaneous and activation-induced death of T lymphocytes is proposed.     

Studies on the reactions between human tyrosinase, superoxide anion, hydrogen peroxide and thiols.

Human tyrosinase (5.5 mg) has been purified from a single human melanotic melanoma metastasis (50.5 g). In the presence of dioxygen, L-tyrosine proved to be a very poor substrate for this enzyme with barely detectable activity compared to L-dopa. However, saturating superoxide anion (i.e., greater than 5 x 10(-3) M) enhanced the oxidation rate of L-tyrosine to dopachrome 40-fold. Hydrogen peroxide was shown to be a competitive inhibitor of tyrosinase when L-tyrosine was the substrate. This reversible inhibition is based on a slow pseudocatalase activity for tyrosinase. Monothiols and dithiols inhibit tyrosinase by different mechanisms. Reduced human thioredoxin and 2,3-dithiopropanol are allosteric inhibitors of tyrosinase yielding bis-cysteinate complexes with one of the copper atoms in the enzyme active site. Bis-cysteinate tyrosinase activity is down-regulated to 30% of native enzyme activity in the L-dopa assay; suggesting a true regulatory role for dithiols. Monothiols such as reduced glutathione and beta-mercaptoethanol are much less reactive with tyrosinase although 10(-3) M monothiol totally inhibits enzyme activity. Reduced thioredoxin inhibits tyrosinase 23-fold more than reduced glutathione under the same experimental conditions.