Glutathione Monoethyl Ester Research Articles

Stealth and Biocompatible Gold Nanoparticles through Surface Coating with a Zwitterionic Derivative of Glutathione.

Gold nanoparticles (AuNPs) hold promise in biomedicine, but challenges like aggregation, protein corona formation, and insufficient biocompatibility must be thoroughly addressed before advancing their clinical applications. Designing AuNPs with specific protein corona compositions is challenging, and strategies for corona elimination, such as coating with polyethylene glycol (PEG), have limitations. In this study, we introduce a commercially available zwitterionic derivative of glutathione, glutathione monoethyl ester (GSHzwt), for the surface coating of colloidal AuNPs. Particles coated with GSHzwt were investigated alongside four other AuNPs coated with various ligands, including citrate ions, tiopronin, glutathione, cysteine, and PEG. We then undertook a head-to-head comparison of these AuNPs to assess their behavior in biological fluid. GSHzwt-coated AuNPs exhibited exceptional resistance to aggregation and protein adsorption. The particles could also be readily functionalized with biotin and interact with streptavidin receptors in human plasma. Additionally, they exhibited significant blood compatibility and noncytotoxicity. In conclusion, GSHzwt provides a practical and easy method for the surface passivation of AuNPs, creating "stealth" particles for potential clinical applications.

Negative regulation of chitosan-induced stomatal closure by glutathione in Arabidopsis thaliana.

Chitosan (CHT) is a deacylated derivative of chitin and improves growth and yield performance, activates defensive genes, and also induces stomatal closure in plants. Glutathione (GSH) has significant functions in the growth, development, defense systems, signaling, and gene expression. GSH negatively regulates abscisic acid-, methyl jasmonate-, and salicylic acid-induced stomatal closure. However, the negative regulation by GSH of CHT-induced stomatal closure is still unknown. Regulation of CHT-induced stomatal closure by GSH in guard cells was investigated using two GSH-deficient mutants, cad2-1 and chlorina 1-1 (ch1-1), and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). The cad2-1 and ch1-1 mutations and CDNB treatment enhanced CHT-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the guard cells of cad2-1 and ch1-1 and complemented the stomatal phenotype of the mutants. These results indicate that GSH negatively regulates CHT-induced stomatal closure in Arabidopsis thaliana.

Transient and Sustained Ganglion Cell Light Responses Are Differentially Modulated by Intrinsically Produced Reactive Oxygen Species Acting upon Specific Voltage-Gated Na+ Channel Isoforms.

Increasing spike rates drive greater neuronal energy demand. In turn, mitochondrial ATP production leads to the generation of reactive oxygen species (ROS) that can modulate ion channel gating. Does ROS production autoregulate the excitability of a neuron? We investigated the links between retinal ganglion cell (RGC) excitability and spike activity-driven ROS production in male and female mice. Changes to the light-evoked and current-evoked spike patterns of functionally identified αRGC subtypes, along with their NaV channel-gating properties, were recorded during experimentally induced decreases and increases of intracellular ROS. During periods of highest spike rates (e.g., following light onset in ON sustained RGCs and light offset in OFF sustained RGCs), these αRGC subtypes responded to reductions of ROS (induced by catalase or glutathione monoethyl ester) with higher spike rates. Increases in ROS (induced by mercaptosuccinate, antimycin-A, or H2O2) lowered spike rates. In ON and OFF transient RGCs, there were no changes in spike rate during ROS decreases but increased ROS increased spiking. This suggests that endogenous ROS are intrinsic neuromodulators in RGCs having high metabolic demands but not in RGCs with lower energy needs. We identified ROS-induced shifts in the voltage-dependent gating of specific isoforms of NaV channels that account for the modulation of ON and OFF sustained RGC spike frequency by ROS-mediated feedback. ROS-induced changes to NaV channel gating, affecting activation and inactivation kinetics, are consistent with the differing spike pattern alterations observed in RGC subtypes. Cell-autonomous generation of ROS during spiking contributes to tuning the spike patterns of RGCs.SIGNIFICANCE STATEMENT Energy production within retinal ganglion cells (RGCs) is accompanied by metabolic by-products harmful to cellular function. How these by-products modulate the excitability of RGCs bears heavily on visual function and the etiology of optic neuropathies. A novel hypothesis of how RGC metabolism can produce automodulation of electrical signaling was tested by identifying the characteristics and biophysical origins of changes to the excitability of RGCs caused by oxidizing by-products in the retina. This impacts our understanding of the pathophysiology of RGC dysfunction, supporting an emerging model in which increases in oxidizing chemical species during energy production, but not necessarily bioenergetic failure, lead to preferential degeneration of specific subtypes of RGCs, yielding loss of different aspects of visual capacity.

Oxidative stress in the mitochondrial matrix underlies ischemia/reperfusion-induced mitochondrial instability

Ischemia and reperfusion affect multiple elements of cardiomyocyte electrophysiology, especially within the mitochondria. We previously showed that in cardiac monolayers, upon reperfusion after coverslip-induced ischemia, mitochondrial inner membrane potential (ΔΨ) unstably oscillates between polarized and depolarized states, and ΔΨ instability corresponds with arrhythmias. Here, through confocal microscopy of compartment-specific molecular probes, we investigate the mechanisms underlying the post-ischemic ΔΨ oscillations, focusing on the role of Ca2+ and oxidative stress. During reperfusion, transient ΔΨ depolarizations occurred concurrently with periods of increased mitochondrial oxidative stress (5.07 ± 1.71 oscillations/15 min, N = 100). Supplementing the antioxidant system with glutathione monoethyl ester suppressed ΔΨ oscillations (1.84 ± 1.07 oscillations/15 min, N = 119, t-test P = 0.027) with 37% of mitochondrial clusters showing no ΔΨ oscillations (vs. 4% in control, odds ratio = 14.08, Fisher's exact test P < 0.001). We found that limiting the production of reactive oxygen species using cyanide inhibited post-ischemic ΔΨ oscillations (N = 15, t-test P < 10-5). Furthermore, ΔΨ oscillations were not associated with any discernable pattern in cell-wide oxidative stress, nor with the changes in cytosolic or mitochondrial Ca2+. Sustained ΔΨ depolarization followed cytosolic and mitochondrial Ca2+ increase and was associated with increased cell-wide oxidative stress. Collectively, these findings suggest that transient bouts of increased mitochondrial oxidative stress underlie post-ischemic ΔΨ oscillations, regardless of Ca2+ dynamics.

Negative regulation of salicylic acid-induced stomatal closure by glutathione in Arabidopsis thaliana.

Salicylic acid (SA) is a ubiquitous phenolic phytohormone that induces stomatal closure. Glutathione (GSH) negatively regulates stomatal closure induced by other plant hormones such as abscisic acid (ABA) and methyl jasmonate (MeJA). However, the involvement of GSH in SA-induced stomatal closure is still unknown. We investigated the regulation of SA signaling by GSH in guard cells using an Arabidopsis thaliana mutant, cad2-1, which is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase. Application of SA decreased stomatal apertures with decreasing intracellular GSH level in guard cells. Decreasing GSH by the cad2-1 mutation and by a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene, enhanced the SA-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the cad2-1 guard cells and complemented the stomatal phenotype of the mutant. These results indicate that GSH negatively modulates SA-induced stomatal closure in A. thaliana.

A single-atom Cu-N2 catalyst eliminates oxygen interference for electrochemical sensing of hydrogen peroxide in a living animal brain.

Hydrogen peroxide (H2O2) plays essential roles in various physiological and pathological processes. The electrochemical hydrogen peroxide reduction reaction (HPRR) has been recognized as an efficient approach to H2O2 sensing; however, the HPRR has always suffered from low tolerance against the oxygen reduction reaction (ORR), resulting in poor selectivity of the HPRR-based sensing platform. In this study, we find that the electrochemical HPRR occurs preferentially compared to the ORR when isolated Cu atoms anchored on carbon nitride (Cu1/C3N4) are used as a single-atom electrocatalyst, which is theoretically attributed to the lower energy barrier of the HPRR than that of the ORR on a Cu1/C3N4 single-atom catalyst (SAC). With the Cu1/C3N4 SAC as the electrocatalyst, we fabricated microsensors that have a good response to H2O2, but not to O2 or other electroactive neurochemicals. When implanted into a living rat brain, the microsensor shows excellent in vivo sensing performance, enabling its application in real-time quantitative investigation of the dynamics of H2O2 production induced by mercaptosuccinate and glutathione monoethyl ester in a living animal brain.

Age-Dependent Vulnerability to Oxidative Stress of Postnatal Rat Pyramidal Motor Cortex Neurons.

Oxidative stress is one of the main proposed mechanisms involved in neuronal degeneration. To evaluate the consequences of oxidative stress on motor cortex pyramidal neurons during postnatal development, rats were classified into three groups: Newborn (P2–P7); infantile (P11–P15); and young adult (P20–P40). Oxidative stress was induced by 10 µM of cumene hydroperoxide (CH) application. In newborn rats, using the whole cell patch-clamp technique in brain slices, no significant modifications in membrane excitability were found. In infantile rats, the input resistance increased and rheobase decreased due to the blockage of GABAergic tonic conductance. Lipid peroxidation induced by CH resulted in a noticeable increase in protein-bound 4-hidroxynonenal in homogenates in only infantile and young adult rat slices. Interestingly, homogenates of newborn rat brain slices showed the highest capacity to respond to oxidative stress by dramatically increasing their glutathione and free thiol content. This increase correlated with a time-dependent increase in the glutathione reductase activity, suggesting a greater buffering capacity of newborn rats to resist oxidative stress. Furthermore, pre-treatment of the slices with glutathione monoethyl ester acted as a neuroprotector in pyramidal neurons of infantile rats. We conclude that during maturation, the vulnerability to oxidative stress in rat motor neurons increases with age.

Neuroprotective effect of immunomodulatory peptides in rats with traumatic spinal cord injury.

Several therapies have shown obvious effects on structural conservation contributing to motor functional recovery after spinal cord injury (SCI). Nevertheless, neither strategy has achieved a convincing effect. We purposed a combined therapy of immunomodulatory peptides that individually have shown significant effects on motor functional recovery in rats with SCI. The objective of this study was to investigate the effects of the combined therapy of monocyte locomotion inhibitor factor (MLIF), A91 peptide, and glutathione monoethyl ester (GSH-MEE) on chronic-stage spinal cord injury. Female Sprague-Dawley rats underwent a laminectomy of the T9 vertebra and a moderate contusion. Six groups were included: sham, PBS, MLIF + A91, MLIF + GSH-MEE, A91 + GSH-MEE, and MLIF + A91 + GSH-MEE. Two months after injury, motor functional recovery was evaluated using the open field test. Parenchyma and white matter preservation was evaluated using hematoxylin & eosin staining and Luxol Fast Blue staining, respectively. The number of motoneurons in the ventral horn and the number of axonal fibers were determined using hematoxylin & eosin staining and immunohistochemistry, respectively. Collagen deposition was evaluated using Masson's trichrome staining. The combined therapy of MLIF, A91, and GSH-MEE greatly contributed to motor functional recovery and preservation of the medullary parenchyma, white matter, motoneurons, and axonal fibres, and reduced the deposition of collagen in the lesioned area. The combined therapy of MLIF, A91, and GSH-MEE preserved spinal cord tissue integrity and promoted motor functional recovery of rats after SCI. This study was approved by the National Commission for Scientific Research on Bioethics and Biosafety of the Instituto Mexicano del Seguro Social under registration number R-2015-785-116 (approval date November 30, 2015) and R-2017-3603-33 (approval date June 5, 2017).

In vitro effects of glutathione on Transforming Growth Factor beta and Epidermal Growth Factor Receptor genes expression in the protoscoleces and strobilated worms of Echinococcus granulosus

Cystic Echinococcosis (CE) caused by the small taeniid cestode Echinococcus granulosus, is a globally distributed zoonosis. Administration of some chemicals or natural compounds could lead to significant effects on the expression of some developmentally important genes including Transforming Growth Factor beta (TGF-β) and Epidermal Growth Factor Receptor (EGFR) in other parasitic organisms. The main purpose of this study was to describe the effect of glutathione (GSH) on the expression of TGF-β and EGFR genes in different developmental stages of E. granulosus.Protoscoleces of hydatid cysts collected from naturally infected sheep liver were cultured in diphasic CMRL1066 medium. Glutathione Mono-ethyl Ester (GME) at 250 μg/ml concentration was applied on the invaginated protoscoleces (PSCi), evaginated protoscoleces (PSCe) and strobilated worms (SW3) in vitro. TGF-β and EGFR genes expression were evaluated by using Real Time qPCR analysis compared to the controls.In response to GME treatment TGF-β expression was affected, however no significant effect was observed in EGFR expression. The results indicate a significant difference of TGF-β expression in the intact protoscoleces and the strobilated worms comparing to the controls. In intact invaginated protoscoleces TGF-β expression was significantly increased (p < 0.01) while in the strobilated worms a significant decrease was observed comparing to no-treatment controls (p < 0.001). None of the three developmental stages of E. granulosus demonstrated significant changes in EGFR expression.The results indicated that administration of GSH modified TGF-β expression in the protoscoleces and strobilar stages of E. granulosus. To improve our understanding of the physiology and biochemistry of the parasite more in depth in vitro and in vivo studies on the morphological and molecular effects of glutathione on the parasite is recommended. Further investigation on the gene profiles in other stages including microcysts and germinal layer cells is also suggested. This paves the way for the effective treatment and control of cystic echinococcosis.

Ultrasmall Gold Nanoparticles Coated with Zwitterionic Glutathione Monoethyl Ester: A Model Platform for the Incorporation of Functional Peptides.

Ultrasmall gold nanoparticles (AuNPs) are an emerging class of nanomaterials exhibiting distinctive physicochemical, molecular, and in vivo properties. Recently, we showed that ultrasmall AuNPs encompassing a zwitterionic glutathione monoethyl ester surface coating (AuGSHzwt) were highly resistant to aggregation and serum protein interactions. Herein, we performed a new set of biointeraction studies to gain a more fundamental understanding into the behavior of both pristine and peptide-functionalized AuGSHzwt in complex media. Using the model Strep-tag peptide (WSHPQFEK) as an integrated functional group, we established that AuGSHzwt could be conjugated with increasing numbers of Strep-tags by simple ligand exchange, which provides a generic approach for AuGSHzwt functionalization. It was found that the strep-tagged AuGSHzwt particles were highly resistant to nonspecific protein interactions and retained their targeting capability in biological fluid, displaying efficient binding to Streptactin receptors in nearly undiluted serum. However, AuGSHzwt functionalized with multiple Strep-tags displayed somewhat lower resistance to protein interactions and lower levels of binding to Streptactin than monofunctionalized AuGSHzwt under given conditions. These results underscore the need for optimizing ligand density onto the surface of ultrasmall AuNPs for improved performance. Collectively, our findings support ultrasmall AuGSHzwt as an attractive platform for engineering functional, protein-mimetic nanostructures capable of specific protein recognition within the complex biological milieu.

EF24 Suppresses Cholangiocellular Carcinoma Progression, Inhibits STAT3 Phosphorylation, and Induces Apoptosis via ROS-Mediated Oxidative Stress.

Therapeutic options for advanced stage cholangiocellular carcinoma (CCC) are very limited as of today and patients carry an exceptionally poor overall prognosis. In recent years, increasing evidence has been accumulated to suggest that malignant cells widely show increased intrinsic ROS levels and exhibit altered redox profiles as compared to normal counterparts, opening up potential avenues for therapeutic intervention. This study provides preclinical experimental evidence of therapeutic activity of the curcumin analog EF24 in cholangiocarcinoma models. In CCC cell lines, EF24 inhibited cell viability and induced apoptosis through excessive ROS generation. Moreover, administration of EF24 led to depletion of total intracellular GSH levels, induced mitochondrial depolarization, and abrogated STAT3 phosphorylation. Of interest, these effects were readily averted by treating the cells with exogenous antioxidants such as N-acetyl cysteine (NAC) or glutathione monoethyl ester (GEE). In vivo, EF24, solubilized using a cyclodextrin formulation, significantly suppressed the growth of tumor xenografts without exhibiting any toxic adverse effects. Immunohistochemical analysis of extracted tumor tissues demonstrated reduced nuclear staining for Ki-67 and downregulation of phospho-STAT3 as well as strong staining for oxidative stress biomarker 8-OHdG. Therefore, the data presented here suggest EF24 as potential therapeutic compound against CCC which might act at least to some extent through ROS-induced oxidative damage, subsequently inducing apoptosis. Further evaluation of this approach should be carried out in future follow-up studies.

Characterization and Regulation of Carrier Proteins of Mitochondrial Glutathione Uptake in Human Retinal Pigment Epithelium Cells.

PurposeTo characterize two mitochondrial membrane transporters 2-oxoglutarate (OGC) and dicarboxylate (DIC) in human RPE (hRPE) and to elucidate their role in the regulation of mitochondrial glutathione (mGSH) uptake and cell death in oxidative stress.MethodsThe localization of OGC and DIC proteins in confluent hRPE, polarized hRPE monolayers and mouse retina was assessed by immunoblotting and confocal microscopy. Time- and dose-dependent expression of the two carriers were determined after treatment of hRPE with H2O2, phenyl succinate (PS), and butyl malonate (BM), respectively, for 24 hours. The effect of inhibition of OGC and DIC on apoptosis (TUNEL), mGSH, and mtDNA was determined. Silencing of OGC by siRNA knockdown on RPE cell death was studied. Kinetics of caspase 3/7 activation with OGC and DIC inhibitors and effect of cotreatment with glutathione monoethyl ester (GSH-MEE) was determined using the IncuCyte live cell imaging.ResultsOGC and DIC are expressed in hRPE mitochondria and exhibited a time- and dose-dependent decrease with stress. Pharmacologic inhibition caused a decrease in OGC and DIC in mitochondria without changes in mtDNA and resulted in increased apoptosis and mGSH depletion. GSH-MEE prevented apoptosis through restoration of mGSH. OGC siRNA exacerbated apoptotic cell death in stressed RPE which was inhibited by increased mGSH from GSH-MEE cotreatment.ConclusionsCharacterization and mechanism of action of two carrier proteins of mGSH uptake in RPE are reported. Regulation of OGC and DIC will be of value in devising therapeutic strategies for retinal disorders such as AMD.

GSH depletion and consequent AKT inhibition contribute to the Nrf2 knockdown-induced decrease in proliferation in glioblastoma U251 cells.

Nuclear factor erythroid2-related factor2 (Nrf2), a pivotal transcription regulator that controls the expression of numerous antioxidant and cytoprotective genes, was recently defined as a proto-oncogene. However, the role and mechanism of Nrf2 in glioma pathoetiology remain unclear. In the present study, we first evaluated the expression patterns of Nrf2 in normal human astrocytes and 3glioblastoma (GBM) cell lines (U251, U87 and A172) and found that all 3GBM cell lines overexpressed Nrf2, with the highest level observed in the U251 cells. We further assessed the biological effects of Nrf2 in U251 cells by specific knockdown of Nrf2 using lentivirus‑mediated RNA interference. We discovered that Nrf2 deficiency led to a decrease in U251 cell proliferation and caused intracellular redox imbalance [diminished glutathione (GSH) levels and increased reactive oxygen species (ROS) levels]. Both N-acetylcysteine and glutathione monoethyl ester (GMEE) supplementation completely eliminated the increased levels of ROS that were present in the Nrf2‑deficient U251 cells. However, only GMEE supplementation both reversed Nrf2 deficiency-induced cell growth arrest and restored intracellular GSH levels. Moreover, AKT and ERK1/2 signaling were both impaired in the Nrf2-knockdown U251 cells, but GMEE supplementation restored AKT signaling but not ERK1/2 signaling, and blocking AKT signaling with an AKT-specific inhibitor greatly diminished the GMEE-induced Nrf2-deficient cell proliferation. In conclusion, our findings revealed novel functions for Nrf2 in the regulation of redox status and cell proliferation, and that intracellular GSH levels and AKT signaling are required for this process, a new viewpoint by which to comprehend the role and underlying mechanism of Nrf2 in tumorigenesis.

Stimulation of cell proliferation by glutathione monoethyl ester in aged bone marrow stromal cells is associated with the assistance of TERT gene expression and telomerase activity.

The proliferation and differentiation potential of aged bone marrow stromal cells (BMSCs) are significantly reduced. In order to improve the performance of the aged BMSCs, these cells were treated with 2mM glutathione monoethyl ester (GSH-MEE) for 24h. Proliferation rate, telomerase activity, telomere length, and differentiation to cholinergic neuron-like cells (CNLCs) were observed to increase. Though, the expression level of telomerase reverse transcriptase gene increased, but CTC1 and TEN1 genes from Ctc1-Stn1-Ten1 complex encoding proteins with regulatory function significantly decreased. Trypan blue exclusion assay was used to analyze the proliferation and, while telomere length, its several related gene expressions, and telomerase activity were measured using the real time reverse transcription-polymerase chain reaction and polymerase chain reaction enzyme-linked immunosorbent assay techniques, respectively. CNLCs differentiation potential was evaluated by estimating the percentage of choline acetyltransferase immunereactive cells.The results suggested that GSH-MEE could improve aged rat BMSC properties and would be of potential benefit for enhancing the performance of aged people's BMSCs.

Light-harvesting complexes in photosystem II regulate glutathione-induced sensitivity of Arabidopsis guard cells to abscisic acid

Light-harvesting complexes (LHCs) in photosystem II (PSII) regulate glutathione (GSH) functions in plants. To investigate whether LHCs control GSH biosynthesis that modifies guard cell abscisic acid (ABA) sensitivity, we evaluated GSH content, stomatal aperture, reactive oxygen species (ROS), weight loss and plant growth using a ch1-1 mutant that was defective of LHCs and compared this with wild-type (WT) Arabidopsis thaliana plants. Glutathione monoethyl ester (GSHmee) increased but 1-chloro-2,4 dinitrobenzene (CDNB) decreased the GSH content in the guard cells. The guard cells of the ch1-1 mutants accumulated significantly less GSH than the WT plants. The guard cells of the ch1-1 mutants also showed higher sensitivity to ABA than the WT plants. The CDNB treatment increased but the GSHmee treatment decreased the ABA sensitivity of the guard cells without affecting ABA-induced ROS production. Dark and light treatments altered the GSH content and stomatal aperture of the guard cells of ch1-1 and WT plants, irrespective of CDNB and GSHmee. The ch1-1 mutant contained fewer guard cells and displayed poor growth, late flowering and stumpy weight loss compared with the WT plants. This study suggests that defective LHCs reduced the GSH content in the guard cells and increased sensitivity to ABA, resulting in stomatal closure.

Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.

The xCT Antiporter Is a Therapeutic Target in the ABC Subtype of DLBCL

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the adult population and can be subdivided into two main subtypes, i.e. GCB-DLBCL and ABC-DLBCL. While both subtypes are derived from normal germinal center (GC) B cells, they differ in B cell maturation stage, transformation pathway, and clinical behavior. When treated with either the combination chemotherapy CHOP or the immuno-chemotherapy R-CHOP, the survival outcome of ABC-DLBCL patients is typically much worse than that of GCB-DLBCL patients. Although the molecular mechanisms underlying this survival disparity remain poorly understood, an attractive hypothesis is that there exist subtype-specific resistance mechanisms directed against the chemo-therapy drugs in the original CHOP formulation. In support of this notion, our previous study has revealed that Doxorubicin (Dox), the main cytotoxic ingredient in CHOP, has subtype-specific mechanisms of cytotoxicity in DLBCLs due to differences in its subcellular distribution pattern. In particular, Dox-induced cytotoxicity in ABC-DLBCLs is largely dependent on oxidative stress rather than DNA damage response. Based on these findings, we hypothesize that agents capable of disturbing the redox balance in ABC-DLBCL cells could potentiate the therapeutic activity of first line lymphoma therapy.As the major route of cystine uptake from extracellular space, the xCT cystine/glutamate antiporter controls the rate-limiting step for glutathione (GSH) synthesis in several types of cancer cells, including CLL. We focused the current study on xCT because its protein stability is known to be positively regulated by a splicing variant of CD44 and we have recently published that expression of CD44 and CD44V6 are poor prognosticators for DLBCL. Indeed, we found that surface CD44 is exclusively expressed in ABC-DLBCL (6/6) but not GCB-DLBCL (0/5) cell lines. In addition, the xCT proteins in two ABC-DLBCL cell lines, Riva and SuDHL2, are extraordinarily stable, with half-lives exceeding 24 hours. As such, transient transfection using siRNA oligos was ineffective in reducing the endogenous xCT protein in ABC-DLBCL cell lines. To circumvent this issue, we turned to a clinically approved anti-inflammatory drug, sulfasalazine (SASP), which is a validated xCT inhibitor in its intact form. When Riva and SuDHL2 cells were treated overnight with the IC50 dose of SASP, the endogenous GSH pool was drastically reduced, leading to significant increase in intracellular ROS, p38 and JNK activation, and progressive apoptosis. Unexpectedly, we found that Dox-treated cells had significantly elevated GSH levels, possibly the result of an antioxidant response to Dox-triggered ROS accumulation. This increase in GSH was completely suppressed when the IC25 dosage of SASP was included in the Dox treatment. As expected, SASP/Dox combination significantly enhanced Dox-triggered ROS accumulation and synergistically promoted cell death in Riva and SuDHL2 cells. Mechanistically, p38 activation and cell death induced by SASP/Dox combination could be markedly attenuated by pretreatment with glutathione monoethyl ester, demonstrating the critical role of oxidative stress. Furthermore, cytotoxicity triggered by SASP/Dox could also be suppressed by the p38 inhibitor, SB203580. We have developed stable cell lines expressing xCT shRNA to confirm the results obtained with SASP. In vivo interactions between SASP and Dox are also being evaluated in xenograft-based ABC-DLBCL models.In summary, we report here for the first time a critical role of xCT in sustaining in vivo GSH production in ABC-DLBCL cells. More importantly, pharmacologic inhibition of xCT function in ABC-DLBCL cells not only prevented Dox-induced endogenous GSH increase, but also potentiated Dox-induced ROS accumulation and cytotoxicity in a p38-dependent manner. With additional evidence from ongoing experiments, our study aims to provide a mechanistic basis for development of novel therapies that target either xCT or redox homeostasis to improve treatment outcomes for ABC-DLBCLs. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Evaluation of a Potential GSH Ester Based PET/SPECT Imaging Probe DT(GSHMe)₂ to Detect Gamma Glutamyl Transferase Over Expressing Tumors.

Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor imaging using PET/SPECT.

Trolox and ascorbic acid reduce direct and indirect oxidative stress in the IPEC-J2 cells, an in vitro model for the porcine gastrointestinal tract.

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H2DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pre-treatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets.

Biarmed poly(ethylene glycol)-(pheophorbide a)2 conjugate as a bioactivatable delivery carrier for photodynamic therapy.

In the study presented here, we developed a bioreducible biarmed methoxy poly(ethylene glycol)-(pheophorbide a)2 (mPEG-(ss-PhA)2) conjugate for cancer-cell-specific photodynamic therapy (PDT). PhA molecules were chemically conjugated with biarmed linkages at one end of the mPEG molecule via disulfide bonds. Under aqueous conditions, the amphiphilic mPEG-(ss-PhA)2 conjugate self-assembled to form core-shell-structured nanoparticles (NPs) with good colloidal stability. The mPEG-(ss-PhA)2 NPs exhibited intramolecular and intermolecular self-quenching effects that enabled the NPs to remain photoinactive in a physiological buffer. However, the dissociation of the NP structure was effectively induced by the cleavage of the disulfide bonds in response to intracellular reductive conditions, triggering the rapid release of PhA molecules in a photoactive form. In cell-culture systems, in addition to significant phototoxicity and intracellular uptake, we observed that the dequenching processes of PhA in the mPEG-(ss-PhA)2 NPs highly depended on the expression of intracellular thiols and that supplementation with glutathione monoethylester facilitated more rapid PhA release and enhanced the PhA phototoxicity. These findings suggest that the bioreducible activation mechanism of mPEG-(ss-PhA)2 NPs in cancer cells can maximize the cytosolic dose of active photosensitizers to achieve high cytotoxicity, thereby enhancing the treatment efficacy of photodynamic cancer treatment.