Thiazolides inhibit growth and induce glutathione-S-transferase Pi (GSTP1)-dependent cell death in human colon cancer cells

Thiazolides are a novel class of broad-spectrum anti-infective drugs with promising in vitro and in vivo activities against intracellular and extracellular protozoan parasites. The nitrothiazole-analogue nitazoxanide (NTZ; 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide) represents the thiazolide parent compound, and a number of bromo- and carboxy-derivatives with differing activities have been synthesized. Here we report that NTZ and the bromo-thiazolide RM4819, but not the carboxy-thiazolide RM4825, inhibited proliferation of the colon cancer cell line Caco2 and nontransformed human foreskin fibroblasts (HFF) at or below concentrations the compounds normally exhibit anti-parasitic activity. Thiazolides induced typical signs of apoptosis, such as nuclear condensation, DNA fragmentation and phosphatidylserine exposure. Interestingly, the apoptosis-inducing effect of thiazolides appeared to be cell cycle-dependent and induction of cell cycle arrest substantially inhibited the cell death-inducing activity of these compounds. Using affinity chromatography and mass spectrometry glutathione-S-transferase P1 (GSTP1) from the GST class Pi was identified as a major thiazolide-binding protein. GSTP1 expression was more than 10 times higher in the thiazolide-sensitive Caco2 cells than in the less sensitive HFF cells. The enzymatic activity of recombinant GSTP1 was strongly inhibited by thiazolides. Silencing of GSTP1 using siRNA rendered cells insensitive to RM4819, while overexpression of GSTP1 increased sensitivity to RM4819-induced cell death. Thiazolides may thus represent an interesting novel class of future cancer therapeutics.

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

In situ fiber-optical monitoring of cytosolic calcium in tissue explant cultures

We present a fluorescence-lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber-optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green-5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s.

The role of BCL-2 in regulation of apoptosis susceptibility by modulation of glutathione

The role of oxidative stress and apoptosis has recently been recognized as an important determinant in the development of a variety of diseases known to man. The oncogene BCL-2 is known to regulate sensitivity to induction of apoptosis and appears to function in an antioxidant pathway by regulating glutathione. We have investigated various steps in the oxidative stress cascade to determine possible sites of action for BCL-2. The fluorescent probes H2DCFDA, dihydroethidium and cis-parinaric acid were used to quantitate generation of peroxides, superoxide and lipid peroxidation, respectively. While each of these agents was able to detect substantial increases in oxidative stress following exposure of cells to ionizing radiation, there was no significant difference between cells expressing high or low levels of BCL-2. Investigation of mitochondrial dysfunction during apoptosis revealed a possible site of bcl-2 intervention, but, analysis of kinetic events occurring during apoptosis suggested that the observed effect is not in the direct apoptotic effector pathway. When glutathione was studied, localization to the nucleus was observed in cells overexpressing BCL-2 that did not occur in cells lacking BCL-2. Additionally, nuclear accumulation of glutathione was sufficient to block granzyme b-mediated nuclear DNA fragmentation, poly (ADP-ribose) polymerase cleavage and caspase activity suggesting that nuclear accumulation of glutathione via a bcl-2 dependent process is functionally relevant to suppression of apoptosis. Thus, a model system emerges where BCL-2 is able to regulate a cell's ability to prevent apoptosis by modifying the cell's antioxidant systems at the organelle level to compensate for oxidative stresses placed upon it. ^

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

A novel family of transporters mediating the transport of glutathione derivatives in plants

Uptake and compartmentation of reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione conjugates are important for many functions including sulfur transport, resistance against biotic and abiotic stresses, and developmental processes. Complementation of a yeast (Saccharomyces cerevisiae) mutant (hgt1) deficient in glutathione transport was used to characterize a glutathione transporter cDNA (OsGT1) from rice (Oryza sativa). The 2.58-kb full-length cDNA (AF393848, gi 27497095), which was obtained by screening of a cDNA library and 5'-rapid amplification of cDNA ends-polymerase chain reaction, contains an open reading frame encoding a 766-amino acid protein. Complementation of the hgt1 yeast mutant strain with the OsGT1 cDNA restored growth on a medium containing GSH as the sole sulfur source. The strain expressing OsGT1 mediated H-3]GSH uptake, and this uptake was significantly competed not only by unlabeled GSSG and GS conjugates but also by some amino acids and peptides, suggesting a wide substrate specificity. OsGT1 may be involved in the retrieval of GSSG, GS conjugates, and nitrogen-containing peptides from the cell wall.

The role of cytochromes P450 and peroxidases in the detoxification of sulphonated anthraquinones by rhubarb and common sorrel plants cultivated under hydroponic conditions

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation and thus not eliminated by classical wastewater treatments. In the development of a phytotreatment to remove sulphonated aromatic compounds from dye and textile industrial effluents, it has been shown that rhubarb (Rheum rabarbarum) and common sorrel (Rumex acetosa) are the most efficient plants. Both species, producing natural anthraquinones, not only accumulate, but also transform these xenobiotic chemicals. Even if the precise biochemical mechanisms involved in the detoxification of sulphonated anthraquinones are not yet understood, they probably have cross talks with secondary metabolism, redox processes and plant energy metabolism. The aim of the present study was to investigate the possible roles of cytochrome P450 monooxygenases and peroxidases in the detoxification of several sulphonated anthraquinones. Both plant species were cultivated in a greenhouse under hydroponic conditions, with or without sulphonated anthraquinones. Plants were harvested at different times and either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 toward several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. A significant activity of cytochromes P450 was detected in rhubarb leaves, while no (rhizome) or low (petioles and roots) activity was found in other parts of the plants. An induction of this enzyme was observed at the beginning of the exposition to sulphonated anthraquinones. The results also indicated that cytochromes P450 were able to accept as substrate the five sulphonated anthraquinones, with a higher activity toward AQ-2,6-SS (0.706 nkat/mg protein) and AQ-2-S (0.720 nkat/mg protein). An activity of the cytochromes P450 was also found in the leaves of common sorrel (1.212 nkat/mg protein (AQ-2,6-SS)), but no induction of the activity occurred after the exposition to the pollutant. The activity of peroxidases increased when rhubarb was cultivated in the presence of the five sulphonated anthraquinones (0.857 nkat/mg protein). Peroxidase activity was also detected in the leaves of the common sorrel (0.055 nkat/mg protein), but in this plant, no significant difference was found between plants cultivated with and without sulphonated anthraquinones. Results indicated that the activity of cytochromes P450 and peroxidases increased in rhubarb in the presence of sulphonated anthraquinones and were involved in their detoxification mechanisms. These results suggest the existence in rhubarb and common sorrel of specific mechanisms involved in the metabolism of sulphonated anthraquinones. Further investigation should be performed to find the next steps of this detoxification pathway. Besides these promising results for the phytotreatment of sulphonated anthraquinones, it will be of high interest to develop and test, at small scale, an experimental wastewater treatment system to determine its efficiency. On the other hand, these results reinforce the idea that natural biodiversity should be better studied to use the most appropriate species for the phytotreatment of a specific pollutant.

Metabolism of sulphonated anthraquinones in rhubarb, maize and celery: the role of cytochromes P450 and peroxidases

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation, and thus contaminating many industrial effluents and rivers. In the development of a phytotreatment to remove sulphonated aromatic compounds, rhubarb (Rheum rhaponticum), a plant producing natural anthraquinones, as well as maize (Zea mays) and celery (Apium graveolens), plants not producing anthraquinones, were tested for their ability to metabolise these xenobiotics. Plants were cultivated under hydroponic conditions, with or without sulphonated anthraquinones, and were harvested at different times. Either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 towards several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. Results indicated that the activity of cytochromes P450 and peroxidases significantly increased in rhubarb plants cultivated in the presence of sulphonated anthraquinones. A higher activity of cytochromes P450 was also detected in maize and celery exposed to the pollutants. In these two plants, a peroxidase activity was also detected, but without a clear difference between the control plants and the plants exposed to the organic contaminants. This research demonstrated the existence in rhubarb, maize and celery of biochemical mechanisms involved in the metabolism and detoxification of sulphonated anthraquinones. Taken together, results confirmed that rhubarb might be the most appropriate plant for the phytotreatment of these organic pollutants.

Prefrontal GABA and glutathione imbalance in posttraumatic stress disorder: Preliminary findings.

Although posttraumatic stress disorder (PTSD) is associated with a variety of structural and functional brain changes, the molecular pathophysiological mechanisms underlying these macroscopic alterations are unknown. Recent studies support the existence of an altered excitation-inhibition balance in PTSD. Further, there is preliminary evidence from blood-sample studies suggesting heightened oxidative stress in PTSD, potentially leading to neural damage through excessive brain levels of free radicals. In this study we investigated PTSD (n=12) and non-PTSD participants (n=17) using single-voxel proton magnetic resonance spectroscopy (MRS) in dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC). We found significantly higher levels of γ-amino butyric acid (GABA) (a primary inhibitory neurotransmitter) and glutathione (a marker for neuronal oxidative stress) in PTSD participants. Atypically high prefrontal inhibition as well as oxidative stress may be involved in the pathogenesis of PTSD.

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.