Glutathione Overproduction Research Articles

Glutathione-Sensitive Photosensitizer-Drug Conjugates Target the Mitochondria to Overcome Multi-Drug Resistance in Cancer.

Multi-drug resistance (MDR) is a major cause of cancer therapy failure. Photodynamic therapy (PDT) is a promising modality that can circumvent MDR and synergize with chemotherapies, based on the generation of reactive oxygen species (ROS) by photosensitizers. However, overproduction of glutathione (GSH) by cancer cells scavenges ROS and restricts the efficacy of PDT. Additionally, side effects on normal tissues are unavoidable after PDT treatment. Here, to develop organic systems that deliver effective anticancer PDT and chemotherapy simultaneously with very little side effects, three GSH-sensitive photosensitizer-drug conjugates (CyR-SS-L) are designed and synthesized. CyR-SS-L localized in the mitochondria then is cleaved into CyR-SG and SG-L parts by reacting with and consuming high levels of intracellular GSH. Notably, CyR-SG generates high levels of ROS in tumor cells instead of normal cells and be exploited for PDT and the SG-L part is used for chemotherapy. CyR-SS-L inhibits better MDR cancer tumor inhibitory activity than indocyanine green, a photosensitizer (PS) used for PDT in clinical applications. The results appear to be the first to show that CyR-SS-L may be used as an alternative PDT agent to be more effective against MDR cancers without obvious damaging normal cells by the combination of PDT, GSH depletion, and chemotherapy.

Glutathione overproduction mediates lymphoma initiating cells survival and has a sex-dependent effect on lymphomagenesis

Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.

Regulation of cadmium uptake in the hyperaccumulator Solanum nigrum is mediated by glutathione

Solanum nigrum is a common hyperaccumulator of the heavy metal cadmium (Cd). Our study aims to investigate the impact of glutathione regulation, specifically glutathione overproduction or reduction, on the growth of S. nigrum and its soil remediation potential. To achieve this, plants were subjected to treatments using a glutathione inhibitor (buthionine sulfoximine) and a glutathione promoter (L-2-oxothiazolidine-4-carboxylic acid [OTC]). We assessed plant height, root length and biomass to evaluate the influence of Cd and glutathione regulation on plant growth. Furthermore, we analyzed chlorophyll content. Cd and malondialdehyde (MDA) contents were measured to determine the effect of glutathione regulation on Cd accumulation and lipid peroxidation. Our findings revealed that glutathione overproduction significantly increased the biomass of S. nigrum, resulting in an approximately 12% increase. Additionally, it led to a ca. 10% rise in chlorophylls content. The application of exogenous OTC enhanced reduced glutathione (GSH) content, accompanied by enhanced Cd accumulation. GSH can be converted to oxidized glutathione, which aids in combatting and mitigating Cd toxicity. The MDA content in plants also increased. The overproduction of glutathione holds promise for enhancing heavy metal accumulation and tolerance in hyperaccumulator plants. It provides a theoretical basis for improving hyperaccumulator plants genetically.

Engineering thio-/seleno-ether linkers into zwitterionic small molecule nano-prodrugs for traceable cancer theranostics

Given the over-production of glutathione (GSH) and hydrogen peroxide (H2O2) in tumor cells, redox-responsive linkers such as disulfide bonds have been widely used to develop site-specific prodrug system, but may suffer from their poor sensitivity that results in low efficacy. Herein, we report the development of a class of redox-triggered small molecule prodrugs (RhB-S(Se)-CPT), by conjugating zwitterionic rhodamine B (RhB) and camptothecin (CPT) with thio-/seleno-ether linkers. The amphiphilic prodrug can self-assemble into stable nanoparticles in water, and zwitterionic RhB is located on the surface of RhB-S(Se)-CPT nanoparticles, tracking the distribution and kinetics of drug delivery in real time, and reducing the non-specific binding to in vivo components, thus decreasing the side effects of nanomedicines. The thio-/seleno-ether linkers enable selective and rapid release of CPT in response to a redox heterogeneous intratumoral microenvironment. Interestingly, selenoether bonds exhibit significant advantages over thioether in terms of CPT release rate, intracellular toxicity and therapeutic efficacy against large tumors (∼ 200 mm3) in mice. These results provide insights to the rational design of subsequent redox-responsive nanoprodrug system, which may contribute to the material selection and structural design of next-generation nanomedicines for tumor theranostics.

Stimuli-Responsive Nanomaterials for Smart Tumor-Specific Phototherapeutics.

Phototherapy, a type of photoresponsive regulation of biological activities, together with additional stimuli-responsive features, offers significant potential for enhancing the precision and efficacy of cancer treatments. To achieve tumor-specific therapeutics, numerous studies have focused on the development of smart phototherapeutic nanomaterials (PNMs) that can respond to endogenous pathological characteristics (e. g., mild acidity, the overproduction of glutathione, the overproduction of hydrogen peroxide, the overexpression of specific surface receptors, etc.) present in the tumor and/or exogenous stimuli. Such responsiveness can effectively improve the physicochemical properties, cellular uptake, tumor-targeting performance, and pharmacokinetic profile of PNMs. Herein, we will systematically discuss recent advances in this field. Moreover, potential challenges and future directions in the development of stimuli-responsive PNMs are also presented to support the development of this emerging cutting-edge research area.

Anhydrobiosis in yeasts: Glutathione synthesis by yeast Ogataea (Hansenula) polymorpha cells after their dehydration-rehydration

The possibility of using active dry microbial preparations in biotechnological processes is essential for the development of new modern industrial technologies. In this study, we show the possibility of obtaining such preparations of the genetically engineered yeast strain Ogataea (Hansenula) polymorpha with glutathione overproduction. Special pre-treatment involving the gradual rehydration of dry cells in water vapour led to the restoration/reactivation of almost 100% of dehydrated cells. Furthermore, dry cells do not lose their viability during storage at room temperatures. Application of dry cells as the inoculum provides the same levels of glutathione synthesis as that of a native yeast culture.

Anhydrobiosis in yeast: Glutathione overproduction improves resistance to dehydration of a recombinant Ogataea (Hansenula) polymorpha strain

Abstract We show for the first time that a recombinant strain of yeast Ogataea (Hansenula) polymorpha is at least as tolerant to dehydration-rehydration treatment as the wild type strain. It is believed that this unusual characteristic of this recombinant yeast strain is linked with its ability to overproduce glutathione. Based on plasma membrane permeability analysis, we hypothesise that glutathione, in addition to its powerful antioxidative protective effects on membrane lipids, may also protect membrane proteins and/or nucleic acids. The combination of yeast cell dehydration with immobilisation and subsequent preliminary slow rehydration in water vapour gave good results in terms of recombinant strain cell viability in the dry state. Correspondingly, this recombinant strain can be used efficiently in the industrial production of glutathione.

Rotenone induces nephrotoxicity in rats: oxidative damage and apoptosis

Previous studies have examined rotenone toxicity on the human central nervous system, especially in the pathogenesis of Parkinson’s disease, but few have investigated the effects of rotenone on the kidney. Here, rotenone-induced nephrotoxicity was evaluated by determining morphological, biochemical, oxidative stress-related, and apoptotic factor alterations in rat renal tissue. Morphological and biochemical analyzes showed that rotenone administration to rats damaged renal tissue. Western blot results revealed that rotenone-induced oxidative damage, causing overproduction of glutathione, malonaldehyde, and reactive oxygen species (ROS), and inhibiting superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. Rotenone also decreased the mitochondrial membrane potential and increased voltage-dependent anion channel (VDAC), caspase-3, and caspase-9 protein levels, indicating an association of apoptosis with renal damage. Our results suggest that glutathione, malonaldehyde, and ROS may be signals of rotenone-induced oxidative damage, and that the mitochondrial pathway plays a key role in apoptosis of renal cells following rotenone administration.

Enhanced incorporation yield of cysteine for glutathione overproduction by fed-batch fermentation of Saccharomyces cerevisiae

In the following work a high cell density fed-batch process with Saccharomyces cerevisiae coupled with a high efficient incorporation of cysteine for glutathione (GSH) overproduction was developed. Therefore, a feeding strategy based on the respiratory quotient (RQ) was applied to ensure high biomass (96.1g/l). Furthermore, the optimal cysteine concentration and time of cysteine addition were investigated. Low concentrations of cysteine at late fermentation phases resulted in relatively high incorporation yields of about 0.40mol/mol and maintained the physiology of cultivated yeast. By changing the cysteine feeding from standard single shot to continuous addition, an often observed cell specific toxicity, triggered by high cysteine concentrations, could be prevented and the cysteine incorporation yield (0.54±0.01mol/mol) and GSH content (1650.7±42.8mg/l; 1.76±0.08%) were maximized, respectively. The developed process was transferred from laboratory into pilot plant scale. Further, the reduced cell specific toxicity enabled the development of a repeated fed-batch procedure with a suitable performance concerning cysteine incorporation yield (0.40±0.1mol/mol), biomass (84.2±1.2g/l) and GSH content (1304.7±61.4mg/l).

Study on Screening of Glutathione over-Production Strain and its Fermentation Conditions

This study was to screen strains for glutathione over-production. A high GSH-producing strain HSJB1 was isolated from soil.According to the morphological, physiological and biochemical characteristics of cells, this strain was primarily identified as Saccharomyces cerevisiae. The glutathion fermentation conditions of this strain by shake flask culture were studied.The optimal fermentation medium was as follows: glucose 20g/L, (NH4)2SO4 5g/L, peptone 12.5 g/L, KH2PO4 9.0 g/L, MgSO4•7H2O 1.0 g/L, NaCl 0.2 g/L, ZnSO4•H2O 0.01 g/L. The optimal culture conditions were as follows :pH 5，broth quantity 30ml/250ml, temperature 28°C，inoculum 10%. Under the optimal conditions, the glutathione productivity and biomass were 53.60 mg/L and 3.8 g/L, respectively, which were 11.2% and 11.8% higher than those in the original conditions, respectively.

Glutathione depletion and overproduction both initiate degeneration of nigral dopaminergic neurons

Parkinson’s disease is a neurodegenerative disorder characterized by severe motor deficits mainly due to degeneration of dopaminergic neurons in the substantia nigra. Decreased levels of the cell’s most important anti-oxidant, glutathione, have been detected in nigral neurons of Parkinson patients, but it is unknown if they are the cause or merely the consequence of the disease. To elucidate if glutathione depletion causes selective degeneration of nigral dopaminergic neurons, we down-regulated glutathione synthesis in different brain areas of adult rats by a viral vector-based RNAi approach. Decreased glutathione synthesis resulted in progressive degeneration of nigral dopaminergic neurons, while extra-nigral and striatal neurons were significantly less vulnerable. Degeneration of dopaminergic neurons was accompanied by progressive protein aggregate formation and functional motor deficits and was partially rescued by α-synuclein. That the survival of nigral dopaminergic neurons depends on the precise control of glutathione levels was further demonstrated by significant degeneration induced through moderate overproduction of glutathione. Over-expression of either of the two subunits of glutamate–cysteine ligase induced aberrant glutathiolation of cellular proteins and significant degeneration of dopaminergic neurons. Thus, while glutathione depletion was demonstrated to be a selective trigger for dopaminergic neuron degeneration, a glutathione replacement approach as a potential treatment option for Parkinson’s patients must be considered with great care. In conclusion, our data demonstrate that survival of nigral dopaminergic neurons crucially depends on a tight regulation of their glutathione levels and that the depleted glutathione content detected in the brains of Parkinson’s disease patients can be a causative insult for neuronal degeneration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-010-0791-x) contains supplementary material, which is available to authorized users.

Combined amino acids modulation with H 2 O 2 stress for glutathione overproduction in Candida utilis

Strategies of amino acids addition coupled with H 2 O 2 stresses were developed for glutathione (GSH) overproduction in high cell density (HCD) cultivation of Candida utilis . Based on the fact that glycine shows two functions of promoting cells growth as well as GSH production, precursor amino acids modulations of feeding glycine at 4 mmol/l/h at exponential phase and adding precursor amino acids (glutamic acid 42 mmol/l, glycine 40 mmol/l, and cysteine 36 mmol/) at stationary phase were conducted. As a result, cell density reached 114.8 g/l at 45 h and glutathione yield of 2136 mg/l was achieved at 60 h, which was 12.5 and 90.2% higher than the control, respectively. Furthermore, the novel strategies of amino acids modulation combined with H2O2 additions (24 mmol/l at 21 h, 26 mmol/l at 29 h, 28 mmol/l at 37 h and 30 mmol/l at 45 h) were adopted to maximize glutathione production. Final glutathione yield reached 2448 mg/l after 60 h cultivation, suggesting the strategies developed as being feasible for GSH overproduction. Keywords: Amino acids, glutathione (GSH), high cell density (HCD) cultivation, Candida utilis , H 2 O 2 stresses African Journal of Biotechnology Vol. 9(33), pp. 5399-5406, 16 August, 2010

Optimization of sodium dedecyl sulfate (SDS) addition coupled with adenosine triphosphate (ATP) regeneration for glutathione overproduction in high density cultivation of Candida utilis

In the previous study, glutathione production was elevated by adding precursor amino acids and adenosine triphosphate (ATP) in high cell density cultivation of Candida utilis. Furthermore, in the present research, glutathione production was further improved by optimizing sodium dedecyl sulfate (SDS) addition coupled with ATP regeneration. Results indicated that with 2g/l ATP added at 60h and 0.8g/l SDS added repeatedly at 60, 63, 66 and 69h, final glutathione yield reached 2286mg/l after 72h cultivation. Moreover, by applying the novel strategies of regenerating ATP by feeding glucose at 6g/(lh) from 60 to 72h coupled with impulse SDS treatments, a total glutathione yield of 2485mg/l was achieved at 72h, which was 8.7% higher than with addition of ATP and SDS, suggesting application of the proposed strategies as being feasible for glutathione overproduction on industrial scales.

Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells

After protracted low level arsenic exposure, the normal human prostate epithelial cell line RWPE-1 acquires a malignant phenotype with DNA hypomethylation, indicative of disrupted methyl metabolism, and shows arsenic adaptation involving glutathione overproduction and enhanced arsenic efflux. Thus, the interplay between methyl and glutathione metabolism during this progressive arsenic adaptation was studied. Arsenic-treated cells showed a time-dependent increase in LC50 and a marked increase in homocysteine (Hcy) levels. A marked suppression of S-adenosylmethionine (SAM) levels occurred with decreased methionine adenosyltransferase 2A (converts methionine to SAM) expression and increased negative regulator methionine adenosyltransferase B, suggesting reduced conversion of Hcy to SAM. Consistent with Hcy overproduction, activity and expression of S-adenosylhomocysteine hydrolase (converts S-adenosylhomocysteine to Hcy) were both increased. Expression of cystathionine beta-synthase, a key gene in the transsulfuration pathway, and various glutathione production genes were increased, resulting in a 5-fold increase in glutathione. Arsenic efflux increased along with expression of ATP-binding cassette protein C1, which effluxes arsenic as a glutathione conjugate. Evidence of genomic DNA hypomethylation was observed during early arsenic exposure, indicating that the disruption in methyl metabolism had a potential impact related to oncogenesis. Thus, cellular arsenic adaptation is a dynamic, progressive process that involves decreased SAM recycling and concurrent accumulation of Hcy, which is channeled via transsulfuration to increase glutathione and enhance arsenic efflux but may also impact the carcinogenic process.

Overproduction of glutathione by l-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.

Combined Proteome and Metabolite-profiling Analyses Reveal Surprising Insights into Yeast Sulfur Metabolism

Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism. When combined with proteome studies, metabolite-profiling analyses revealed unanticipated insights into the yeast sulfur pathway. In response to cadmium, the observed overproduction of glutathione, essential for the detoxification of the metal, can be entirely accounted for by a marked drop in sulfur-containing protein synthesis and a redirection of sulfur metabolite fluxes to the glutathione pathway. A kinetic analysis showed sequential and dramatic changes in intermediate sulfur metabolite pools within the first hours of the treatment. Strikingly, whereas proteome and metabolic data were positively correlated under cadmium conditions, proteome and metabolic data were negatively correlated during other growth conditions, i.e. methionine supplementation or sulfate starvation. These differences can be explained by alternative mechanisms in the regulation of Met4, the activator of the sulfur pathway. Whereas Met4 activity is controlled by the cellular cysteine content in response to sulfur source and availability, the present study suggests that Met4 activation under cadmium conditions is cysteine-independent. The results clearly indicate that the metabolic state of a cell cannot be safely predicted based solely on proteomic and/or gene expression data. Combined metabolome and proteome studies are necessary to draw a comprehensive and integrated view of cell metabolism.

Overproduction of glutathione and its derivatives by genetically engineered microbial cells

In order to improve the biotechnological potentials of Escherichia coli cells to produce glutathione, S- d-lactoylglutathione and other γ-glutamyl compounds, the genes for enzymes [γ-L-glutamyl-L-cysteine synthetase (GSH A) in E. coli B, glutathione synthetase (GSH B) in E. coli B, glyoxalase I (GLO I) in Pseudomonas putida] were cloned and amplified in E. coli. E. coli B cells transformed with both GSH A and GSH B genes exhibited a high activity in the synthesis of glutathione and other γ-glutamyl compounds in bioreactor systems containing immobilized cells. E. coli C600 cells transformed with GLO I gene of P. putida showed a high GLO I activity and were used for the preparation of S- d-lactoylglutathione and other glutathione thiol esters.