Effects of glutathione depletion by buthionine sulfoximine on radiosensitization by oxygen and misonidazole in vitro

Buthionine sulfoximine (BSO) has been used to deplete glutathione (GSH) in V79-379A cells in vitro, and the effect on the efficiency of oxygen and misonidazole (MISO) as radiosensitizers has been determined. Treatment with 50 or 500 microM BSO caused a rapid decline in GSH content to less than 5% of control values after 10 hr of exposure (t1/2 = 1.6 hr). Removal of BSO resulted in a rapid regeneration of GSH after 50 microM BSO, but little regeneration was observed over the subsequent 10-hr period after 500 microM. Treatment with either of these two concentrations of BSO for up to 14 hr did not affect cell growth or viability. Cells irradiated in monolayer on glass had an oxygen enhancement ratio (OER) of 3.1. After 10-14 hr pretreatment with 50 microM BSO, washed cells were radiosensitized by GSH depletion at all oxygen tensions tested. The OER was reduced to 2.6, due to greater radiosensitization of hypoxic cells than aerated ones by GSH depletion. GSH depletion had the effect of shifting the enhancement ratio vs pO2 curve to lower oxygen tensions, making oxygen appear more efficient by a factor of approximately 2, based on the pO2 required to give an OER of 2.0. In similar experiments performed with MISO, an enhancement ratio of 2.0 could be achieved with 0.2 mM MISO in anoxic BSO-pretreated cells, compared to 2.7 mM MISO in non-BSO-treated cells. Thus MISO appeared to be more efficient in GSH-depleted cells by a factor of 13.5. These apparent increases in radiosensitizer efficiency in GSH-depleted cells could be explained on the basis of radiosensitization of hypoxic cells by GSH depletion alone (ER = 1.29-1.41). The effect of GSH depletion was approximately equal at all sensitizer concentrations tested, except at high oxygen tensions, where the effect was insignificantly small. These results are consistent with hypoxic cell radiosensitization by GSH depletion and by MISO or oxygen acting by separate mechanisms.     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.     

Enhanced potentiation of cisplatin cytotoxicity in human ovarian carcinoma cells by prolonged glutathione depletion

We have determined the effect of extended glutathione (GSH) depletion on cis-diamminedichloroplatinum(II) (DDP) cytotoxicity in parent and DDP-resistant human ovarian carcinoma cells. Cells were exposed to 50 microM buthionine sulfoximine (BSO) for 48 h and exposed to DDP for the last 24 h of this time. This treatment protocol sensitized 2008 cells to DDP. The dose modification factor (DMF) defined as IC50 control cells/IC50 GSH depleted cells was 1.6 +/- 0.5 (N = 9). DDP-resistant cells selected by acute, high dose DDP exposure were also sensitized by this treatment; the DMF in the 3-6-fold resistant 2008/DDP cells was 2.4 +/- 1.2 (N = 9). The sensitization was not significantly greater in the resistant cells than in the parent cells (P greater than 0.05). When the rebound of GSH following BSO exposure was reexamined, the GSH levels were found to rise rapidly following trypsinizing and plating. BSO treatment following DDP exposure had no effect on DDP cytotoxicity in 2008 and 2008/DDP cells. These results indicate that simply depleting GSH prior to DDP exposure is not sufficient for sensitizing these cells to DDP. In contrast to the potentiation of nitrogen mustard cytotoxicity, exposure to GSH depletion must be maintained during DDP treatment for enhancement of DDP cytotoxicity to occur.     

Nonxenobiotic manipulation and sulfur precursor specificity of human endothelial cell glutathione

Confluent human umbilical vein endothelial (HUVE) cells were readily (within 1 h) depleted of their glutathione (GSH) by diethylmaleate (0.1-1.0 mM), but dose-dependent cell detachment was noted. Buthionine sulfoximine (BSO, 25 microM) depleted cell GSH with sigmoidal kinetics, showing an initial half-life of depletion of 4-6 h and greater than 95% depletion by 48 h without morphological changes to the cells. However, BSO-dependent depletion of cell GSH was only partially reversible by cell washing and reincubation with complete medium. Likewise, incubation of the cells in sulfur-free medium depleted cell GSH again without morphological changes to the cells. However, unlike with BSO, these cells readily resynthesized GSH when resupplied with complete medium, fresh plasma, or whole blood, with a characteristic overloading of cell GSH (up to 200%) by 12 h. By use of the sulfur-free medium, it was shown that both cystine and cysteine are effective precursors to GSH synthesis in HUVE cells in culture and that cystine is the most likely precursor in vivo. During cystine-supported resynthesis of GSH, high levels of cysteine accumulated in the cells (up to 10% of total soluble free thiol). Physiologically relevant concentrations of extracellular GSH were not as effective as cystine or cysteine in stimulating GSH biosynthesis, whereas nonphysiologically high (mM) concentrations resulted in substantial elevation of GSH levels above those of control cells in a BSO-insensitive manner. These findings provide a simple methodology for the manipulation of HUVE cell GSH in studies of endothelial-specific oxidant toxicity and the sulfur dependence of the biochemistry and turnover of GSH in these human cells.     

Buthionine sulfoximine induced growth inhibition in human lung carcinoma cells does not correlate with glutathione depletion

Treatment of A549 human lung carcinoma cells with L-buthionine-[S,R]-sulfoximine (BSO) results concomitantly in cellular glutathione (GSH) depletion and growth inhibition. The nature of BSO effects on cell growth and the relationships between BSO inhibition of cell growth and BSO effects on cellular GSH levels were determined in this study. A dose dependent effect of BSO on cell growth was observed, but this effect was found not to correlate with BSO effects on cellular GSH levels. Treatment with BSO for 60 h at concentrations of 5 and 10 mM was found to deplete cellular GSH at similar rates and to an undetectable level (below 0.5 nmol/mg protein). However, cessation of growth occured in 10 mM BSO whereas growth continued at better than one half the control rate in 5 mM BSO. The results suggest there may be a distinct threshold level of intracellular G GSH (on the order of or less than 0.5 nmol/mg protein) required for cell growth and for cells to protect themselves from the antiproliferative effects of BSO. At a concentration of 10 mM, BSO inhibited both DNA and protein synthesis and arrested growth of A549 cells throughout rather than at a specific phase of the cell cycle. BSO inhibition of growth was not, as indicated by colony-forming efficiency (CFE) and electron microscopy studies, accompanied by indications of cytotoxic effects. A stimulatory effect of 0.1 mM BSO on the growth of A549 cells was found also.Abbreviations BSO L-buthionine-[S,R]-sulfoximine - GSH Glutathione (reduced form) - GSSG Glutathione disulfide - DTNB 5,5-dithiobis (2-nitrobenzoate) - PBS Phosphate buffered saline - BSA Bovine serum albumin - PI Propidium iodide - CFE Colony-forming efficiency - EM Electron microscopy     

Depletion of glutathione after gamma irradiation modifies survival

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D0. To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM. Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM. Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM. In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.     

The role of glutathione in the aerobic radioresponse. I. Sensitization and recovery in the absence of intracellular glutathione

The effect of changes in both the intracellular glutathione (GSH) concentration and the concentration of extracellular reducing equivalents on the aerobic radiosensitization was studied in three cell lines: CHO-10B4, V79, and A549. Intracellular GSH was metabolically depleted after the inhibition of GSH synthesis by buthionine sulfoximine (BSO), while the extracellular environment was controlled through the replacement of growth medium with a thiol-free salt solution and in some experiments by the exogenous addition of either GSH or GSSG. Each of the cell lines examined exhibited an enhanced aerobic radioresponse when the intracellular GSH was extensively depleted (GSH less than 1 nmol GSH/10(6) cells after 1.0 mM BSO/24 h treatment) and the complexity of the extracellular milieu decreased. Although the addition of oxidized glutathione (5 mM GSSG/30 min) to cells prior to irradiation was without effect, much or all of the induced radiosensitivity was overcome by the addition of reduced glutathione (5 mM GSH/15 min). However, the observation that the exogenous GSH addition restores the control radioresponse without increasing the intracellular GSH concentration was entirely unexpected. These results suggest that a number of factors exert an influence on the extent of GSH depletion and determine the extent of aerobic radiosensitization. Furthermore, the interaction of exogenous GSH with--but without penetrating--the cell membrane is sufficient to result in radiorecovery.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.     

Free radical scavenger depletion in post-ischemic reperfusion brain damage

In the present study the influence of pretreatment with various GSH depletors such as buthionine sulfoximine (BSO) and diethylmaleate (DEM) was investigated in rats following cerebral postischemic reperfusion. Moreover, the effect of diethyldithiocarbamic acid (DDC), inhibitor of endogenous Cu,Zn-SOD, was evaluated. A significant depletion (40% of control value) of GSH levels was observed 24 h after DEM administration; after 48 h the value reached control levels. BSO showed maximal GSH depletion (59%) 24 h after administration and it was constant for almost 48 h. DDC administration caused a marked decrease (60%) of Cu,Zn-SOD activity 4 h after the injection and induced a marked decrease in percentage of survival with respect to control (untreated, ischemic) rats, when administered 4 h before ischemia. BSO and DEM prolonged the survival time of animals when administered 24 h before ischemia. This last paradoxical effect is unclear at present, but it might be due to an influence on glutamate cascade.     

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.     

Glutathione depletion by DL-buthionine-SR-sulfoximine (BSO) potentiates X-ray-induced chromosome lesions after liquid holding recovery

The impact of intracellular glutathione depletion on chromosome damage induced by X irradiation under aerobic conditions was investigated in two different cell lines, Ehrlich ascites tumor cells (EATC) and Chinese hamster ovary cells (CHO-K1). Thiol-depleted cell cultures in plateau phase were obtained by prolonged incubation in growth medium containing DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase. Cells were then assayed using the procedures of G. L. Ellmann (Arch. Biochem. Biophys. 82, 70-77 (1959)), F. Tietze (Anal. Biochem. 27, 502-522 (1969)), and J. Sedlack and R.H. Lindsay (Anal. Biochem. 25, 192-205 (1968)) for non-protein bound SH (NPSH), glutathione (GSH), and total SH (TSH). In both cell lines GSH was reduced to less than 10% of controls at higher BSO concentrations around 1 mM, whereas TSH and NPSH were affected to only 40-60%. In EATC pretreated with up to 1 mM BSO for 72 h, increased levels of spontaneously occurring micronuclei were found. At BSO concentrations above 200 microM, both cell lines showed a potentiation of chromosome lesions scored as micronuclei and induced under aerobic X irradiation when liquid holding recovery in the original nutrient-depleted medium was performed; the extent of chromosome damage eventually reached that which could be obtained by application of beta-arabinofuranosyladenine (beta-araA), known to inhibit DNA repair processes by blocking DNA polymerases. It is therefore suggested that GSH depletion causes impairment of repair of lesions leading to chromosome deletions and subsequently to micronuclei. In contrast to CHO cell cultures, EATC showed a reversion of the potentiation effect as indicated by a decrease in the micronucleus content during prolonged incubation in the presence of BSO in the millimolar range. This effect could not be correlated to the remaining GSH content of less than 10% but may be due to some accumulation of unknown NPSH components. Since addition of L-cysteine to EATC cultures pretreated with BSO decreased the micronucleus content, cysteine/cystine or a related thiol within the NPSH fraction may be involved in the reestablishment of repair. Thus at least in one cell line, a rather complex response to BSO administration indicated that not only GSH but also other thiols may determine the level of chromosome damage after liquid holding recovery.     

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.     

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.     

Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures

A system based on Arabidopsis thaliana suspension cultures was established for the analysis of glutathione (GSH) synthesis in the presence of hydrogen peroxide. Mild oxidative stress was induced by use of the catalase inhibitor, aminotriazole, and its development was monitored by measurement of the oxidative inactivation of aconitase. Addition of 2 mM aminotriazole resulted in a 25% decrease in activity of aconitase over 4 h. During the subsequent 10 h, no further decrease in aconitase activity was measured despite a sustained inhibition of catalase. In combination with our failure to detect significant increases in the level of lipid peroxidation, another marker indicative of oxidative injury, these data suggest that although hydrogen peroxide initially leaked into the cytosol, its accumulation was limited by a cytosolic catalase-independent mechanism. A 4-fold increase in the level of GSH, which was almost exclusively in the reduced form, was observed under the same treatment. To determine to what extent this increase in reduced GSH played a role in limiting the accumulation of hydrogen peroxide in the cytosol, we inhibited GSH synthesis with buthionine sulfoximine (BSO), a specific inhibitor of [gamma]-glutamylcysteine synthetase. No significant oxidative injury was detected as a result of treatment with 50 [mu]M BSO alone, and furthermore, this treatment had no effect on cell viability, However, addition of 2 mM aminotriazole to cells preincubated with 50 [mu]M BSO for 15 h led to a rapid loss of aconitase activity (75% in 4 h), and significant accumulation of products of lipid peroxidation. Within 72 h, cell viability was lost completely. After removal of BSO from the growth medium, GSH levels recovered to normal over a period of 20 h. Addition of 2 mM aminotriazole to cells at different time points during this recovery period demonstrated a strong correlation between the level of reduced GSH and the degree of protection against oxidative injury. These data strongly suggest that the induction of GSH synthesis by an oxidative stimulus plays a crucial role in determining the susceptibility of cells to oxidative stress.     

Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose- and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) does not have protective effects. Co-localization studies show that p-ERK-1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS-dependent activation of the ERK-1/2 signalling pathway in glial cells. These data may be of relevance in Parkinson's disease, where GSH depletion and glial dysfunction have been documented.     

Simultaneous inhibition of glutathione- and thioredoxin-dependent metabolism is necessary to potentiate 17AAG-induced cancer cell killing via oxidative stress

17-Allylamino-17-demethoxygeldanamycin (17AAG) is an experimental chemotherapeutic agent believed to form free radicals in vivo, and cancer cell resistance to 17AAG is believed to be a thiol-dependent process. Inhibitors of thiol-dependent hydroperoxide metabolism [L-buthionine-S,R-sulfoximine (BSO) and auranofin] were combined with the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) to determine if 17AAG-mediated cancer cell killing could be enhanced. When 2DG (20mM, 24h), BSO (1mM, 24h), and auranofin (500nM, 3h) were combined with 17AAG, cell killing was significantly enhanced in three human cancer cell lines (PC-3, SUM159, MDA-MB-231). Furthermore, the toxicity of this drug combination was significantly greater in SUM159 human breast cancer cells, relative to HMEC normal human breast epithelial cells. Increases in toxicity seen with this drug combination also correlated with increased glutathione (GSH) and thioredoxin (Trx) oxidation and depletion. Furthermore, treatment with the thiol antioxidant NAC (15mM, 24h) was able to significantly protect from drug-induced toxicity and ameliorate GSH oxidation, Trx oxidation, and Trx depletion. These data strongly support the hypothesis that simultaneous inhibition of GSH- and Trx-dependent metabolism is necessary to sensitize human breast and prostate cancer cells to 2DG+17AAG-mediated killing via enhancement of thiol-dependent oxidative stress. These results suggest that simultaneous targeting of both GSH and Trx metabolism could represent an effective strategy for chemosensitization in human cancer cells.     

Transient alteration of cellular redox buffering before irradiation triggers apoptosis in head and neck carcinoma stem and non-stem cells

Background

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation.

Methodology/Principal Findings

Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10% of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria.

Conclusions

This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials of endogenous redox modulation to enhance the cytotoxic effects of radiotherapy.     

The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation

The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.