Question

in cell culture experiments...Does exogenous oxidized glutathione (GSSG) protect cancer cells? Is it converted to the reduced form GSH once it's taking up by the cell to protect the cell? i know that both of them is forming the other and the amount of formation of certain form depends on the status of the cell. i'm working on cancer cell lines and when i used both forms to see their effect on the cell survival, both of them shows cell protection with no cytotoxicity. I'm also using a cytotoxic drug ( with electrophilic structure) that possibly reacting with the thiol group of the reduced form GSH and becomes inactivated when i combine it too with the oxidized form GSSG it also shows no cytotoxicity.. Can you please help me find an explanation to this result?

Answer 1

Glutathione (GSH) is a tripeptide (L--glutamyl-L-cysteinyl-glycine) with multiple functions in living organisms. As a carrier of an active thiol group in the form of a cysteine residue, it acts as an antioxidant either directly by interacting with reactive oxygen/nitrogen species (ROS and RNS, resp.) and electrophiles or by operating as a cofactor for various enzymes [5–8]. Glutathione is moderately stable in the intracellular milieus because intracellular peptidases can cleave peptide bonds formed by the carboxyl groups of amino acids, but typically not the -carboxyl groups. The reduced and oxidized forms of glutathione (GSH and GSSG) act in concert with other redox-active compounds (e.g., NAD(P)H) to regulate and maintain cellular redox status. The former is quantitatively described by the redox potential, calculated according to the Nernst equation. In most cells and tissues, the estimated redox potential for the GSH/GSSG couple ranges from ?260?mV to ?150?mV (cited after GSH is synthesized in a two-step process catalyzed by L-glutamate cysteine L-ligase, (also called -glutamyl-L-cysteine ligase or -glutamylcysteine synthase), and glutathione synthase (GLS, EC 6.3.2.3). GSH is consumed in many ways, such as by oxidation, conjugation, and hydrolysis. GSH can be directly oxidized by ROS and RNS or indirectly during GSH-dependent peroxidase-catalyzed reactions. Conjugation with endogenous and exogenous electrophiles consumes a substantial portion of cellular GSH. In addition, cells may lose GSH due to export of its reduced, oxidized or conjugated forms. Extracellularly, GSH can be hydrolyzed by -L-glutamyl transpeptidase (GGT, EC 2.3.2.2) transferring the -glutamyl functional group to water during hydrolysis to form free glutamate. The enzyme may also transfer the -glutamyl moiety of GSH to amino acids and peptides. Frequently, products of GSH hydrolysis are taken up by cells either as individual amino acids, or as dipeptides. The intra- and extracellular GSH levels are determined by the balance between its production, consumption, and transportation. Due to important physiological functions of GSH, these processes are tightly regulated. The activities of the enzymes involved in GSH metabolism are controlled at transcriptional, translational, and posttranslational levels.

Since GSH participates not only in antioxidant defense systems, but also in many metabolic processes, its role cannot be overestimated. Therefore, it is not surprising that the GSH system has attracted the attention of pharmacologists as a possible target for medical interventions. The main efforts in this field have been applied to decreasing or increasing GSH levels in organisms. General strategies involve specific inhibition of GLCL, a key enzyme of GSH biosynthesis, and depletion of cellular reserves by externally added electrophiles (usually for research purposes). The use of buthionine sulfoximine (BSO) is probably the most popular approach to depleting GSH. BSO was first synthesised as the D,L-form and later as the L-BSO enantiomer. Usually a mixture of D- and L-BSO is used in experiments. GSH levels may be enhanced by supplementation with precursors, mainly cysteine in the form of different esters. However, during the the last decade a new approach for the regulation of GSH-utilizing enzymes has emerged. It is evident that many of these are induced at the transcriptional level by mild oxidative stress, which involves binding of the Nrf2 transcription factor to the antioxidant response element (ARE) (also called the electrophile response element; EpRE) in the promoter region of genes encoding certain enzymes, particularly GLCL and glutathione S-transferases. Glutathione has several additional functions in cells. For example, it is (i) a reserve form of cysteine, (ii) stores and transports nitric oxide, (iii) participates in the metabolism of estrogens, leukotrienes, and prostaglandins, the reduction of ribonucleotides to deoxyribonucleotides, the maturation of iron-sulfur clusters of diverse proteins, (iv) involved in the operation of certain transcription factors (particularly those involved in redox signalling), and (v) the detoxification of many endogenous compounds and xenobiotics The present review will focus on the molecular mechanisms of operation of the GSH system, with special attention to regulatory pathways controlling the expression of the enzymes involved. Information on GSH biosynthesis, hydrolysis and utilization, intracellular compartmentalization, and interorgan transfer will be highlighted. Special sections will deal with GSH functions, such as antioxidant properties and relationship to specific enzymes. On the basis of these mechanisms, some potential approaches for medical interventions will also be evaluated.