Thioredoxin reductase (TRX) is a selenoprotein that reduces oxidized protein substrates in an NADPH-dependent process (cf. Fig. 1). The thioredoxins (TX) are a family of small redox active proteins that undergo reversible oxidation/reduction and help to maintain the redox state of cells. TX serves as a cofactor in many TRX-catalyzed reductions in a manner similar to glutathione (GSH) in thioltransferase reactions. For example, TX is a cofactor in protein disulfide reduction and DNA synthesis, but independently, it inhibits apoptosis, stimulates cell proliferation and angiogenesis, and increases transcription factor activity. The role of the TRX/TX system is limited by its reducing capacity as well as the additional supply of electrons in the form of NADPH provided by hexose monophosphate shunt (HMPS). TX is limited by the reduction capacity of its vicinal sulfhydryls and needs a source of electrons from the HMPS and TRX- coupled system to reduce disulfides. Oxidized TX is reduced by TRX and NADPH. Several lines of evidence suggest that the coupled HMPS/TRX/TX system represents an important target for cancer therapy. TX overexpression has been reported in several malignancies and may be associated with aggressive tumor growth and poor survival. In some cells, TX is an important factor in conferring resistance to chemotherapy and in stimulating production of hypoxia-inducible factor (HIF-1). Several inhibitors of the TRX/TX system have been evaluated in experimental cancer models: these include HMPS inhibitors, carbohydrate analogues, NADP synthesis blockers, vicinal thiol reactants, cisplatin, and TRX inhibitors. More recently, the targeted anti-cancer agent motexafin gadolinium has been identified. Motexafin gadolinium is a redox mediator that selectively localizes to cancer cells, and reacts with reducing metabolites and vicinal thiols to generate reactive oxygen species that ultimately block the TRX enzyme as well as the analogous glutaredoxin activity. In cell and animal models, motexafin gadolinium is directly cytotoxic to various tumor cells and enhances the activity of radiation therapy and chemotherapy. This drug is now in a broad range of clinical trials investigating its therapeutic potential when used as a single agent or in combination with either chemotherapy or radiation therapy. Promising clinical activity has been reported in a clinical trial with motexafin gadolinium and whole brain radiation therapy for treatment of brain metastases from solid tumors. These findings suggest that the TRX/TX system may represent an attractive target for development of new cancer therapeutics.

Coumarin-3-carboxylic acid (3-CCA) was used as a detector for hydroxyl radicals (.OH) in aqueous solution. The .OH was generated by gamma irradiation or chemically by the Cu2+-mediated oxidation of ascorbic acid (ASC). The excitation and emission spectra of 3-CCA, hydroxylated either chemically or by gamma irradiation, were nearly identical to those of an authentic 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). The pH-titration curves for the fluorescence at 450 nm (excitation at 395 nm) of 3-CCA, hydroxylated either chemically or by gamma radiation, were also identical to those of authentic 7-OHCCA (pK = 7.4). Time-resolved measurements of the fluorescence decays of radiation- or chemically hydroxylated 3-CCA, as well as those of 7-OHCCA, indicate a monoexponential fit. The fluorescence lifetime for the product of 3-CCA hydroxylation was identical to that of 7-OHCCA (approximately 4 ns). These data, together with analysis of end products by high-performance liquid chromatography, show that the major fluorescent product formed by radiation-induced or chemical hydroxylation of 3-CCA is 7-OHCCA. Fluorescence detection of 3-CCA hydroxylation allows real-time measurement of the kinetics of .OH generation. The kinetics of 3-CCA hydroxylation by gamma radiation is linear, although the kinetics of 3-CCA hydroxylation by the Cu2+-ASC reaction shows a sigmoid shape. The initial (slow) step of 3-CCA hydroxylation is sensitive to Cu2+, but the steeper (fast) step is sensitive to ASC. Analysis of the kinetics of 3-CCA hydroxylation shows a diffusion-controlled reaction with a rate constant 5.0 +/- 1.0 x 10(9) M(-1) s(-1). The scavenging of .OH by 3-CCA was approximately 14% for chemical generation with Cu2+-ASC and approximately 50% for gamma-radiation-produced .OH. The yield of 7-OHCCA under the same radiation conditions was approximately 4.4% and increased linearly with radiation dose. The 3-CCA method of detection of .OH is quantitative, sensitive, specific and therefore accurate. It has an excellent potential for use in biological systems.

The kinetics of copper-catalyzed autoxidation of cysteine and its derivatives were investigated using oxygen consumption, spectroscopy and hydroxyl radical detection by fluorescence of a coumarin probe. The process has complex two-phase kinetics. During the first phase a stoichiometric amount of oxygen (0.25 moles per mole of thiol) is consumed without production of hydroxyl radicals. In the second reaction phase excess oxygen is consumed in a hydrogen peroxide-mediated process with significant *OH production. The reaction rate in the second phase is decreased for cysteine derivatives with a free aminogroup and increased for compounds with a modified aminogroup. The kinetic data suggest the catalytic action of copper in the form of a cysteine complex. The reaction mechanism consists of two simultaneous reactions (superoxide-dependent and peroxide-dependent) in the first phase, and peroxide-dependent in the second phase. The second reaction phase begins after oxidation of free thiol. This consists of a Fenton-type reaction between cuprous-cysteinyl complex and following oxidation of cysteinyl radical to sulfonate with the consumption of excessive oxygen and significant production of hydroxyl radicals.

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The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal .OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and .OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.

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Under certain conditions, many radioprotective thiols can be toxic, causing loss of colony-forming ability in cultured mammalian cells in a biphasic fashion whereby the thiols are not toxic at high or low concentrations of the drug, but cause decreased clonogenicity at intermediate (0.2-1.0 mM) drug levels. This symposium paper summarizes our studies using dithiothreitol (DTT) as a model thiol to demonstrate the role of Fenton chemistry in thiol toxicity. The toxicity of DTT in V79 cells has several characteristics: it is dependent on the medium used during exposure of cells to the drug; the toxicity is decreased or prevented by addition of catalase exogenously, but superoxide dismutase has no effect; the toxicity is increased by addition of copper, either free or derived from ceruloplasmin in serum; and the toxicity can be modified intracellularly by altering glucose availability or pentose cycle activity. Thus the data are consistent with a mechanism whereby DTT oxidation produces H2O2 in a reaction catalyzed by metals, predominantly copper, followed by reaction of H2O2 in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, .OH. Studies comparing 12 thiols have shown that the magnitude of cell killing and pattern of dependence on thiol concentration vary among the different agents, with the toxicity depending on the interplay between the rates of two reactions: thiol oxidation and the reaction between the thiol and the H2O2 produced during the thiol oxidation. The addition of other metals, e.g. Zn2+, and metal chelators, e.g. EDTA, can also alter DTT toxicity by altering the rates of thiol oxidation or the Fenton reaction. Recent studies have shown that in certain cell lines thiols can also cause apoptosis in a biphasic pattern, with little apoptosis at low or high drug concentrations but greatly increased apoptosis levels at intermediate (approximately 3 mM) thiol concentrations. There appears to be a good correlation between those thiols that cause loss of clonogenicity and those that induce apoptosis, suggesting similar mechanisms may be involved in both end points. However, thiol-induced apoptosis is not prevented by addition of exogenous catalase. These observations are discussed in relation to the possible role of Fenton chemistry in induction of apoptosis by thiols.

To gain a better understanding of the mechanism of action of the metal cation-containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (ZnT1) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer. (Cancer Res 2005; 65(9): 3837-45)