Glutathione (GSH) is the most abundant non-protein thiol present at millimolar concentrations in mammalian tissues. As an important intracellular antioxidant, it acts as a regulator of cellular redox state protecting cells from damage caused by lipid peroxides, reactive oxygen and nitrogen species, and xenobiotics. Recent studies have highlighted the importance of GSH in key signal transduction reactions as a controller of cell differentiation, proliferation, apoptosis, ferroptosis and immune function. Molecular changes in the GSH antioxidant system and disturbances in GSH homeostasis have been implicated in tumor initiation, progression, and treatment response. Hence, GSH has both protective and pathogenic roles. Although in healthy cells it is crucial for the removal and detoxification of carcinogens, elevated GSH levels in tumor cells are associated with tumor progression and increased resistance to chemotherapeutic drugs. Recently, several novel therapies have been developed to target the GSH antioxidant system in tumors as a means for increased response and decreased drug resistance. In this comprehensive review we explore mechanisms of GSH functionalities and different therapeutic approaches that either target GSH directly, indirectly or use GSH-based prodrugs. Consideration is also given to the computational methods used to describe GSH related processes for in silico testing of treatment effects.

The mammalian neocortex is established from neural stem and progenitor cells that utilize specific transcriptional and environmental factors to create functional neurons and astrocytes. Here, we examined the mechanism of Sox2 action during neocortical neurogenesis and gliogenesis. We established a robust Sox2 expression in neural stem and progenitor cells within the ventricular zone, which persisted until the cells exited the cell cycle. Overexpression of constitutively active Sox2 in neural progenitors resulted in upregulation of Notch1, recombination signal-sequence binding protein-J (RBP-J) and hairy enhancer of split 5 (Hes5) transcripts and the Sox2 high mobility group (HMG) domain seemed sufficient to confer these effects. While Sox2 overexpression permitted the differentiation of progenitors into astroglia, it inhibited neurogenesis, unless the Notch pathway was blocked. Moreover, neuronal precursors engaged a serine protease(s) to eliminate the overexpressed Sox2 protein and relieve the repression of neurogenesis. Glial precursors and differentiated astrocytes, on the other hand, maintained Sox2 expression until they reached a quiescent state. Sox2 expression was re-activated by signals that triggered astrocytic proliferation (i.e., injury, mitogenic and gliogenic factors). Taken together, Sox2 appears to act upstream of the Notch signaling pathway to maintain the cell proliferative potential and to ensure the generation of sufficient cell numbers and phenotypes in the developing neocortex.

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BackgroundIn addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit.MethodsTo study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed.ResultsA total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A.ConclusionsThe methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.

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Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they monitor and maintain low levels of glutamate. Synaptic homeostasis of glutamate involves its removal from the synaptic cleft via high-affinity glutamate transporters, glutamate transporter-1 (GLT-1)/excitatory amino acid transporters (EAAT)2 and glutamate and aspartate transporter (GLAST)/EAAT1, and glutamate-catabolizing enzyme, glutamine synthase. Glutamate transporters have been mostly characterized in rodent astrocytes, due to the lack of a convenient human cell system. We report here that NTera-2 (NT2/D1, a cell line derived from a human teratocarcinoma and known to differentiate into neurons) can also be differentiated by a 4-week treatment with retinoic acid into functional astrocytes (NT2/A). Differentiation was accompanied by decreased cell proliferation and cell-cycle arrest, as measured by flow cytometry, immunostaining for Ki67 and incorporation of 5-bromo-2'deoxyuridine (BrdU). Immunocytochemistry and Western blot analysis showed that NT2/A expressed glial fibrillary acidic protein, vimentin and S100beta. Reverse transcription polymerase chain reaction (PCR) detected mRNA encoding glutamate transporters GLT-1/EAAT2 and GLAST/EAAT1. The expression level of GLAST/EAAT1 was higher than that of GLT-1/EAAT2, which is a typical expression pattern for primary astrocytes. Functionality of the transporters was demonstrated by the uptake of (3)H-glutamate. NT2/A also expressed active glutamine synthase, and treatment with glutamate (up to 1 mM for 24 hr) was non-toxic, suggesting that these cells were capable of converting it to non-toxic metabolites. NT2/A and NT2-derived neurons could be grown as mixed cultures and this may prove to be a useful experimental model to study molecular mechanisms underlying glutamate excitotoxicity.

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Neutrophils represent a potential source of genotoxic reactive oxygen and nitrogen species in the tumor microenvironment. Using Mutatect cell lines, which can form subcutaneous tumors in syngeneic C57BL/6 mice, we have previously established that the number of spontaneously infiltrating neutrophils correlates with the number of mutations at the hypoxanthine phosphoribosyltransferase (Hprt) locus. We now describe the properties of four lines that express different levels of the neutrophil chemokine, interleukin-8 (IL-8), from a tetracycline (TET)-responsive promoter. In a series involving 45 animals, IL-8-expressing lines produced tumors with a higher neutrophil content than the control line. Analysis of the 45 tumors revealed that the neutrophil level again strongly correlated with hprt mutant frequency (MF) (P<.0001, r=0.88). Administration of TET was effective in lowering the neutrophil content of low IL-8-expressing tumors, but not high IL-8-expressing tumors. Although the IL-8 transgene was stable in all lines in vitro, high IL-8-expressing lines completely lost the transgene in vivo whereas low IL-8-expressing lines showed no evidence of transgene instability. These results provide further evidence, based on the study of an endogenous gene (hprt) and an IL-8 transgene, that neutrophils may contribute to genetic instability in tumors.