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The possible nuclear compartmentalization of glutathione S-transferase (GST) isoenzymes has been the subject of contradictory reports. The discovery that the dinitrosyl-diglutathionyl- iron complex binds tightly to Alpha class GSTs in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M., and Ricci, G. (2007) J. Biol. Chem. 282, 6364–6371) prompted us to reconsider the nuclear localization of GSTs in these cells. Surprisingly, we found that a considerable amount of GSTs corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2, and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy, immunofluorescence experiments, and molecular modeling have been used to detail the electrostatic association in hepatocytes and liposomes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent an amazing novelty in cell physiology. The interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intranuclear localization of Alpha GSTs.

It is now well established that exposure of cells and tissues to nitric oxide leads to the formation of a dinitrosyl-iron complex bound to intracellular proteins, but little is known about how the complex is formed, the identity of the proteins, and the physiological role of this process. By using EPR spectroscopy and enzyme activity measurements to study the mechanism in hepatocytes, we here identify the complex as a dinitrosyl-diglutathionyl-iron complex (DNDGIC) bound to Alpha class glutathione S-transferases (GSTs) with extraordinary high affinity (K(D) = 10(-10) m). This complex is formed spontaneously through NO-mediated extraction of iron from ferritin and transferrin, in a reaction that requires only glutathione. In hepatocytes, DNDGIC may reach concentrations of 0.19 mm, apparently entirely bound to Alpha class GSTs, present in the cytosol at a concentration of about 0.3 mm. Surprisingly, about 20% of the dinitrosyl-glutathionyl-iron complex-GST is found to be associated with subcellular components, mainly the nucleus, as demonstrated in the accompanying paper (Stella, L., Pallottini, V., Moreno, S., Leoni, S., De Maria, F., Turella, P., Federici, G., Fabrini, R., Dawood, K. F., Lo Bello, M., Pedersen, J. Z., and Ricci, G. (2007) J. Biol. Chem. 282, 6372-6379). DNDGIC is a potent irreversible inhibitor of glutathione reductase, but the strong complex-GST interaction ensures full protection of glutathione reductase activity in the cells, and in vitro experiments show that damage to the reductase only occurs when the DNDGIC concentration exceeds the binding capacity of the intracellular GST pool. Because Pi class GSTs may exert a similar role in other cell types, we suggest that specific sequestering of DNDGIC by GSTs is a physiological protective mechanism operating in conditions of excessive levels of nitric oxide.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i)10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

Colorectal cancer is a serious medical problem in Taiwan. New, effective therapeutic approaches are needed. The selection of promising anticancer drugs and the transition from pre-clinical investigations to clinical trials are often challenging. The deaminated thyroid hormone analog (tetraiodothyroacetic acid, tetrac) and its nanoparticulate analog (NDAT) have been shown to have anti-proliferative activity in vitro and in xenograft model of different neoplasms, including colorectal cancers. However, mechanisms involved in tetrac- and NDAT-induced anti-proliferation in colorectal cancers are incompletely understood. We have investigated possible mechanisms of tetrac and NDAT action in colorectal cancer cells, using a perfusion bellows cell culture system that allows efficient, large-scale screening for mechanisms of drug actions on tumor cells. Although integrin αvβ3 in K-RAS wild type colorectal cancer HT-29 cells was far less than that in K-RAS mutant HCT116 cells, HT-29 was more sensitive to both tetrac and NDAT. Results also indicate that both tetrac and NDAT bind to tumor cell surface integrin αvβ3, and the agents may have different mechanisms of anti-proliferation in colorectal cancer cells. K-RAS status appears to play an important role in drug resistance that may be encountered in treatment with this drug combination.

In order to better understand the antioxidant behavior of a series of polyphenolic 2'-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV-vis spectroscopic method indicate that a hydroxyl group in position 5' induces the highest antioxidant activity. Consequently, 2,2',5'-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2',5'-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa(-)/fa(-)) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2',5'-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.

Developing a rational strategy to control intracellular reactive oxygen species (ROS) requires understanding the mechanism of antioxidant activity. In this investigation the properties of a novel synthetic analog of vitamin E (IRFI005) with potent antioxidant activity are described. A mechanism is proposed for its efficient radical-scavenging effects. Cellular antioxidant and antitoxicity assays showed IRFI005 to freely permeate across cellular membranes, enabling it to be an effective suppressor of intracellular ROS and to protect cells against toxicity induced by free radical generating compounds. The free radical-scavenging activity of IRFI005 examined by UV-Vis and electron spin resonance (ESR) techniques clearly confirmed a "two electrons and/or H-atom" donation mechanism for each molecule of IRFI005. Reducing power assay as well as semi-empirical calculations revealed that under physiological conditions (pH∼7) almost all IRFI005 molecules are in the anionic state (IRFI005(-)). Data indicated that the electron donating ability of IRFI005(-) was dominant at physiological pH because of higher stability of quinine-IRFI005(-) and less barrier energy of IRFI005(-) than neutral IRFI005. Consequently, the efficient cellular protection of IRFI005 against toxic free radicals can be explained by a two electron-transfer process, because of reduced inter-frontier molecular orbital energy gap barrier at physiological pH. Our findings suggest that hydrophilic vitamin E-like antioxidants are good candidates in designing novel therapeutic strategies for inhibition of oxidative stress associated with different human diseases.

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 mu M cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.

Extranuclear or nongenomic effects of thyroid hormones are mediated by receptors located at the plasma membrane or inside cells, and are independent of protein synthesis. Recently the alphaVbeta3 integrin was identified as a cell membrane receptor for thyroid hormones, and a wide variety of nongenomic effects have now been shown to be induced through binding of thyroid hormones to this receptor. However, also other thyroid hormone receptors can produce nongenomic effects, including the cytoplasmic TRalpha and TRbeta receptors and probably also a G protein-coupled membrane receptor, and increasing importance is now given to thyroid hormone metabolites like 3,5-diiodothyronine and reverse T(3) that can mimick some nongenomic effects of T(3) and T(4). Signal transduction from the alphaVbeta3 integrin may proceed through at least three independent pathways (protein kinase C, Src or mitogen-activated kinases) but the details are still unknown. Thyroid hormones induce nongenomic effects on at least three important Na(+)-dependent transport systems, the Na(+)/K(+)-ATPase, the Na(+)/H(+) exchanger, and amino acid transport System A, leading to a mitogenic response in embryo cells; but modulation of the same transport systems may have different roles in other cells and at different developmental stages. It seems that thyroid hormones in many cases can modulate nongenomically the same targets affected by the nuclear receptors through long-term mechanisms. Recent results on nongenomic effects confirm the old theory that the primary role of thyroid hormones is to keep the steady-state level of functioning of the cell, but more and more mechanisms are discovered by which this goal can be achieved.

Reactive oxygen species (ROS) is the common name used to describe the partially reduced forms of molecular oxygen that may be generated in cells during oxidative metabolism. They are normally considered to be toxic, and cells possess various defence systems to protect themselves including antioxidant enzymes and low molecular weight antioxidants like vitamin C and vitamin E. However, it is now clear that small amounts of ROS also act as messenger molecules in cell signal transduction pathways; the plasma membrane of eukaryotic cells in particular contains a variety of different ROS-producing oxidases and reductases, of which the best characterized are the superoxide-producing NADPH oxidases. It has been known for many years that membrane redox activity can be changed rapidly by various hormones and growth factors, but the molecular mechanisms involved and the physiological importance of this phenomenon have only recently begun to be unveiled. This review summarizes the state of the art on plasma membrane-based ROS signalling in the pathways of insulin, steroid and thyroid hormones and growth factors. The apparent paradox of ROS being essential biomolecules in the regulation of cellular functions, but also toxic by-products of metabolism, may be important for the pharmacological application of natural and synthetic antioxidants.

Extranuclear or nongenomic effects of thyroid hormones are unaffected by inhibitors of protein synthesis, and their rapid time course cannot be explained by interaction of the hormone molecule with nuclear receptors. Their origin has been localized at the plasma membrane, but also at organelles such as the endoplasmatic reticulum and mitochondria. Thyroid hormone has been reported to activate, by both genomic and non genomic mechanisms, the Ca2+-ATPase that stores calcium from the cytosol in the sarcoplasmic reticulum; the decrease in intracellular Ca2+ leads to muscle relaxation. Considering the important effects on the cardiovascular system, T3 can actually be envisaged as a potent inotropic drug. T3 is also a major regulator of the plasma membrane Na+/K+-ATPase activity; T3 and its analog 3,5-diiodothyronine rapidly inhibits Na+/K+-ATPase in chick embryo hepatocytes, whereas the activity is up-regulated in alveolar epithelial cells. Also the ubiquitous plasma membrane Na+/H+ exchanger, that regulates cell volume and pH by exchanging extracellular Na+ with cytoplasmic H+ according to the concentration gradient, is activated by T3 via both genomic and nongenomic mechanisms. A growing number of natural and synthetic thyroid hormone analogs are available to study the physiological importance of extranuclear effects; this may lead to compounds that selectively target either genomic or nongenomic receptors. Such drugs may make it possible to activate separately only a part of the complex effects normally induced by thyroid hormones, this could be of clinical relevance for the cardiovascular system, bone tissue and the Central Nervous System.

Several recent studies suggest that thyroid hormones role is not completely understood in insulin resistance as well as in the development of type 2 diabetes mellitus. Through the perturbation of gene expression linked to glucose metabolism both hyper- and hypothyroidism may cause impaired glucose utilization in skeletal muscle or overproduction of hepatic glucose, thus contributing to the induction of insulin resistance. The complex crosstalk between immune cells and skeletal muscle cells and adipose tissue, the ability of macrophages to release thyroid hormones, the ability of T3 to induce M2 macrophage polarization, the proinflammatory role of thyroid hormones and the antinflammatory effects of insulin all represent important events where thyroid hormone interference may lead to insulin resistance. The crosstalk between thyroid hormones and insulin in the modulation of oxidative status, and also to some extent in the antagonistic effects on several aspects of mitochondrial activities, could represent novel downstream targets for future therapeutic strategies in the treatment of insulin resistance and type 2 diabetes.