Glutathione depletion prevents diet-induced obesity and enhances insulin sensitivity

Excessive accumulation of reactive oxygen species (ROS) in adipose tissue has been implicated in the development of insulin resistance and type 2 diabetes. However, emerging evidence suggests a physiologic role of ROS in cellular signaling and insulin sensitivity. In this study, we demonstrate that pharmacologic depletion of the antioxidant glutathione in mice prevents diet-induced obesity, increases energy expenditure and locomotor activity, and enhances insulin sensitivity. These observations support a beneficial role of ROS in glucose homeostasis and warrant further research to define the regulation of metabolism and energy balance by ROS.     

Glutathione depletion in lung cells by low-molecular-weight aldehydes

Use of oxygenates in gasoline in the United States may increase atmospheric levels of aldehydes. To assist in health assessments of inhalation exposure to aldehydes, we studied glutathione (GSH) depletion by low-molecular-weight n-alkanals and 2-alkenals, ubiquitous air pollutants, in adult rat lung (ARL) cells by laser cytometry. For each homologous series, the effective aldehyde concentration that depleted GSH by 50% (EC₅₀) in ARL cells correlates with published values for the median lethal dose of the chemicals and with Hammett/Taft electronic parameters, * for n-alkanals and _p* for 2-alkenals. n-Alkanals (EC₅₀, 110–400 mmol/L) were 1000 times less effective in depleting GSH than were 2-alkenals (EC₅₀, 2–180 mol/L), of which acrolein was the most potent. Ability of the 2-alkenals to deplete GSH follows the second-order rate constant for adduct formation. Ability of n-alkanals to deplete GSH follows chain length. Within a homologous series of low-molecular-weight aldehydes, structure–activity relationships are useful for predicting the toxicity of the aldehydes in vitro and in vivo.     

Glutathione depletion in vivo enhances contraction and attenuates endothelium-dependent relaxation of isolated rat aorta

Ten-day administration of the glutamate-cysteine ligase inhibitor L-buthionine-[S,R]-sulfoximine (BSO; 20 or 30 mM in drinking water) to adult male Sprague-Dawley rats induced 50-60% glutathione depletion (p<0.001) and elevated aortic ring reactive oxygen species release and tissue and plasma H2O2 concentrations (p<0.001) compared to control animals (CON) that consumed normal drinking water. In contrast to previous studies using tail cuff plethysmography methods, BSO had no significant effect on systolic blood pressure assessed by indwelling femoral artery catheters in conscious animals (10-day values, 119+/-3 mn Hg vs 122+/-4 mm Hg in CON vs BSO, respectively). Thoracic aorta rings were excised for in vitro assessment of vasomotor function. BSO shifted the phenylephrine (PE) dose-response curve to the left (p=0.003), lowering the EC50 for PE contraction (from -6.752+/-0.056 to -7.056+/-0.055 log units; p=0.001). Endothelium-dependent relaxation to acetylcholine (ACh) was significantly blunted (p=0.019) and the EC50 for ACh relaxation was significantly increased (from -7.428+/-0.117 to -7.129+/-0.048 log units; p=0.02) in BSO vs CON. Endothelium-independent vasorelaxation to sodium nitroprusside was similar in BSO and CON groups. Thoracic aorta immunoblot analyses revealed increases in endothelial nitric oxide synthase, superoxide dismutase 1 and 2, and soluble guanylate cyclase in BSO vs CON (all p<0.01). Thus, enhanced PE contraction, blunted endothelium-dependent relaxation, and adaptations in nitric oxide bioavailability pathways provide the first evidence of chronic, in vivo BSO-induced, oxidative stress-mediated direct effects on the vasomotor function of arteries.     

Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells

The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be capable of performing isomerization or reduction reactions, it must be maintained in a reduced state. Here we show that most of the oxidoreductases are predominantly reduced in vivo. Following oxidative stress the oxidoreductases are quickly reduced, demonstrating that a robust reductive pathway is in place in mammalian cells. Using ERp57 as a model we show that the reductive pathway is cytosol-dependent and that the component responsible for the reduction of the oxidoreductases is the low molecular mass thiol glutathione. In addition, ERp57 is not reduced following oxidative stress when inhibitors of glutathione synthesis or glutathione reduction are added to cells. Glutathione directly reduces ERp57 at physiological concentrations in vitro, and biotinylated glutathione forms a mixed disulfide with ERp57 in microsomes. Our results demonstrate that glutathione plays a direct role in the isomerization of disulfide bonds by maintaining the mammalian oxidoreductases in a reduced state.     

Glutathione depletion greatly reduces neocarzinostatin cytotoxicity in Chinese hamster V79 cells

The role of the intracellular thiol glutathione in the reductive activation of neocarzinostatin was investigated in Chinese hamster V79 cells. The cells were pretreated with agents that either lower (buthionine sulfoximine or diethyl maleate) or elevate (oxothiazolidine carboxylate) intracellular glutathione levels. These cells were then exposed to 1-5 micrograms/ml neocarzinostatin for 1 h and assayed for survival. Depletion of glutathione to levels at or below the limit of detection resulted in a marked reduction in neocarzinostatin cytotoxicity, while increasing glutathione levels to 250% of control values had little or no effect on neocarzinostatin toxicity. High performance liquid chromatography analysis of cysteine in untreated and glutathione-depleted cells showed cysteine levels lower than 0.2 microM, indicating that cysteine does not play a major role in the reductive activation of neocarzinostatin in untreated or glutathione-depleted cells. When intracellular cysteine levels were artificially elevated by oxothiazolidine carboxylate treatment of glutathione-depleted cells, neocarzinostatin toxicity was about two-thirds that seen in cells with normal glutathione levels. In cell-free systems, others have shown that reducing agents such as 2-mercaptoethanol are necessary for the activation of neocarzinostatin to a species that will cleave DNA. In this study, we have identified glutathione as the major cellular reducing agent for the activation of neocarzinostatin in a mammalian cell line.     

Verapamil enhances the efficiency of DNA-mediated gene transfer in mammalian cells

Treatment of Ltk^? cells with the calcium antagonists, verapamil and diltiazem, but not nifedipin, causes a 3-fold enhancement of the frequency of transfer of the cloned gene for herpes simplex virus thymidine kinase (HSV-tk). The frequency of phenotypic expression of the HSV-tk DNA was 20 to 34 times higher than that of genotypic transformation. Phenotypic expression was also 2.3 to 2.6 times increased when 20 μg/ml of verapamil was present during calcium phosphate-mediated DNA transfection.     

Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo

Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3–7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors.Malignant gliomas are the most common and aggressive type of primary brain tumor in adults. Current therapy includes surgery for tumor resection, followed by radiotherapy and/or concomitant adjuvant chemotherapy with temozolomide (TMZ) or chloroethylating nitrosoureas (CNUs). However, these protocols have limited success, and patients diagnosed with glioma have a dismal prognosis, with a median survival of 15 months and a 5-year survival rate of ~2%.¹ Several molecular mechanisms for cell resistance to these agents have been described. Because both are alkylating agents, the repair enzyme O₆-methylguanine-DNA methyltransferase (MGMT) is certainly a first barrier that is associated with increased tumor resistance.^{2, 3} The p53 status has also been proposed to act in an opposite manner in glioma cell resistance to TMZ or CNUs. Although p53 mutation is shown to be more resistant to TMZ treatment, owing to the induction of cell death,⁴ the p53 protein protects glioma cells after CNU treatment, most likely by improving other DNA repair systems.⁵Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors, such as ovarian, lung and testicular cancer,⁶ and it is used for adjuvant therapy in gliomas.⁷ Cisplatin is a molecule formed by one platinum ion that is surrounded by four ligands at the cis position: two chloride atoms and two amine molecules. The mechanism of action of cisplatin is mainly based on DNA damage. Once inside the cell, cisplatin becomes activated by the substitution of one or two chloride atoms by water, a process known as aquation. Owing to this process, the drug becomes positively charged and interacts with the DNA molecule, inducing the formation of DNA adducts. Activated cisplatin preferentially binds to purine bases in the nucleophilic N7 sites, where the majority of adducts occur between two guanines on the same strand, whereas ~3–5% of cisplatin adducts react with purines at the opposite strands, forming interstrand crosslinks (ICLs). The DNA lesions, in turn, trigger a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis.⁸Although relatively efficient, resistance to cisplatin, either intrinsic or acquired, during cycles of therapy is common, and overcoming tumor resistance remains the major challenge for cisplatin anticancer therapy. Cellular cisplatin resistance is a multifactorial phenomenon that may include decreased drug uptake, enhanced DNA repair capacity and higher glutathione (GSH) concentration.⁹GSH is a highly abundant, low-molecular-weight peptide in the cell, and it is well known for its critical importance in maintaining the cellular oxidative balance as a free radical scavenger. Additionally, GSH has a protective role against xenobiotic agents once its highly reactive thiol group binds and inactivates those agents. In fact, the GSH content and glutathione S-transferase (GST) have long been associated with cisplatin resistance in numerous cell lines and tumor tissues.^{9, 10, 11}Considering these possible pathways, it is not clear, however, which one determinates cisplatin resistance in glioma cells. Aiming to better understand the molecular mechanisms of resistance to this drug, four human glioma cell lines with different p53 status were investigated. We showed that cellular resistance was found to be independent of p53 as well as of the DNA repair capacity of the cells. On the other hand, the GSH levels within the cell were shown to act as a decisive resistance barrier to cisplatin, reducing the induction of DNA damage in the treated cells. Also, both in an in vitro and in vivo model depletion of GSH by an inhibitor (buthionine sulfoximine, BSO) sensitized the glioma cell lines to cisplatin. Interestingly, BSO also potentiated TMZ cytotoxicity. Thus, combination with BSO, cisplatin and TMZ turned out to be an extremely powerful approach to improve cytotoxicity in glioma, thus providing an exciting alternative for glioma treatment.     

Glutathione content and adaptation to endogenously induced energy depletion in Mv1Lu cells

Transfection of genes that code for enzymes of energy metabolism provides alternative models to study the adaptive response to energy restriction induced by endogenous changes instead of by unfavorable environmental conditions. Overexpression of the glycolytic enzyme fructose-2,6-bisphosphatase reduced the content of fructose 2,6-bisphosphate, inducing energy limitation in the mink lung epithelial cell line Mv1Lu. This metabolic stress reduced the ATP available in transfected cells by 20%, which downregulated active ion transport and protein turnover. Ion homeostasis and cell function require concomitant reductions in cell membrane ion permeability and protein damage. Our results indicate that glutathione content linked these features of the adaptive response to the endogenously induced metabolic downregulation.     

Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.     

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitively growth-suppressed accumulated the mRNA of IgG₁ which is reported stable, and IgG₁ production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM ₁ chain and cultured at nonpermissive temperature to enhance production of ₁. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.Abbreviations IL6 recombinant human interleukin-6 - TGF- recombinant human TGF-₁ - X63.653-P3X63 Ag8.653 myeloma     

Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells

CHIP is a cochaperone of Hsp70 that inhibits Hsp70-dependent refolding in vitro. However, the effect of altered expression of CHIP on the fate of unfolded proteins in mammalian cells has not been determined. Surprisingly, we found that overexpression of CHIP in fibroblasts increased the refolding of proteins after thermal denaturation. This effect was insensitive to geldanamycin, an Hsp90 inhibitor, and required the tetratricopeptide repeat motifs but not the U-box domain of CHIP. Inhibition of Hsp70 chaperone activity abolished the effects of CHIP on protein folding, indicating that the CHIP-mediated events were Hsp70 dependent. Hsp40 competitively inhibited the CHIP-dependent refolding, which is consistent with in vitro data indicating that these cofactors act on Hsp70 in the ATP-bound state and have opposing effects on Hsp70 ATPase activity. Consistent with these observations, CHIP overexpression did not alter protein folding in the setting of ATP depletion, when Hsp70 is in the ADP-bound state. Concomitant with its effects on refolding heat-denatured substrates, CHIP increased the fraction of nascent chains coimmunoprecipitating with Hsc70, but only when sufficient ATP was present to allow Hsp70 to cycle rapidly. Our data suggest that, consistent with in vitro studies, CHIP attenuates the Hsp70 cycle in living cells. The impact of this effect on the fate of unfolded proteins in cells, however, is different from what might be expected from the in vitro data. Rather than resulting in inhibited refolding, CHIP increases the folding capacity of Hsp70 in eukaryotic cells.     

Glutathione depletion increases chemiluminescence emission and lipid peroxidation in the heart

Diamide, CDNB and phorone were used to deplete glutathione in retrogradely perfused rat hearts. Following glutathione depletion the spontaneous chemiluminescence increased by 70%, irrespective of the agent used. The glutathione depletion and the chemiluminescence emission were associated to an increase of malondialdehyde content in the heart, as determined by HPLC. Under these conditions the heart function was impaired and histological examination showed a coagulative myocytolysis, a pattern already described in human and experimental pathology, where a key role is attributed to a Ca2+ homeostasis impairment.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.Key words: topoisomerase II, mitosis, G₂, conditional knockdown, S phase, mitotic catastrophe     

Camptothecin enhances random integration of transfected DNA into the genome of mammalian cells

In order to study the involvement of DNA topoisomerase I (top1) in recombination, we examined the effect of the anti-neoplastic drug camptothecin, which selectively poisons top1 by trapping top1-cleavable complexes on integration of exogenic vector into the genome of mammalian cells. We transfected mouse F9 teratocarcinoma cells as well as Chinese hamster V79 cells with a plasmid carrying a selectable neo gene treated with camptothecin, and determined the frequency of neo+ (G418(R)) colonies. We found that treatment with camptothecin for as short a time as 4 h after electroporation resulted in a 4- to 33-fold stimulation of plasmid integration into the recipient genome via non-homologous recombination. These results imply that top1-cleavable complexes trapped by camptothecin could be potentially recombinogenic structures and could stimulate non-homologous recombination in vivo, promoting the integration of transfected plasmids into mammalian genome.     

Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. In this study, we explored the potential role of polyamines in roscovitine-induced apoptosis in HCT116 colon cancer cells. Roscovitine induced apoptosis by activating mitochondrial pathway caspases and modulating the expression of Bcl-2 family members. Depletion of polyamines by treatment with difluoromethylornithine (DFMO) increased roscovitine-induced apoptosis. Transient silencing of ornithine decarboxylase, polyamine biosynthesis enzyme and special target of DFMO also increased roscovitine-induced apoptosis in HCT116 cells. Interestingly, additional putrescine treatment was found pro-apoptotic due to the presence of non-functional ornithine decarboxylase (ODC). Finally, roscovitine altered polyamine catabolic pathway and led to decrease in putrescine and spermidine levels. Therefore, the metabolic regulation of polyamines may dictate the power of roscovitine induced apoptotic responses in HCT116 colon cancer cells.     

5-Azacytidine enhances transient expression of a transfected gene in cultured mammalian cells

The level of transient expression of the Escherichia coli β-galactosidase (β-Gal) gene transfected into three mammalian cell lines was enhanced by culturing the cells with 1–5 μM 5-azacytidine (5-azaC) after the transfection. This enhancement was similarly observed in four different transfection procedures tested. The enhancement of β-Gal expression was primarily due to the increase in the level of its mRNA. Since 5-azaC did not exhibit any obvious effect on the levels of endogenous gene products examined, it is suggested that 5-azaC may preferentially affect the transient expression of an exogenous gene.