Buthionine Sulfoximine Treatment Research Articles

Glutathione is required for nitric oxide-induced chilling tolerance by synergistically regulating antioxidant system, polyamine synthesis, and mitochondrial function in cucumber (Cucumis sativus L.)

In this paper, we discussed the physiological mechanism of enhanced chilling tolerance with combined treatment of nitric oxide (NO) and reduced glutathione (GSH) in cucumber seedlings. With prolonged low temperature (10 °C/6 °C), oxidative stress improved, which was manifested as an increase the hydrogen peroxide (H2O2) and malondialdehyde (MDA), causing cell membrane damage, particularly after 48 h of chilling stress. Exogenous sodium nitroprusside (SNP, NO donor) enhanced the activity of nitric oxide synthase NOS-like, the contents of GSH and polyamines (PAs), and the cellular redox state, thus regulating the activities of mitochondrial oxidative phosphorylation components (CI, CII, CIV, CV). However, buthionine sulfoximine (BSO, a GSH synthase inhibitor) treatment drastically reversed or attenuated the effects of NO. Importantly, the combination of SNP and GSH treatment had the best effect in alleviating chilling-induced oxidative stress by upregulating the activities of antioxidant enzyme, including superoxidase dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) and improved the PAs content, thereby increased activities of CI, CII, CIII, CIV, and CV. This potentially contributes to the maintenance of oxidative phosphorylation originating from mitochondria. In addition, the high activity of S-nitrosoglutathione reductase (GSNOR) in the combined treatment of SNP and GSH possibly mediates the conversion of NO and GSH to S-nitrosoglutathione. Our study revealed that the combined treatment with NO and GSH to synergistically improve the cold tolerance of cucumber seedlings under prolonged low-temperature stress.

Feasibility study of multimodal imaging for redox status and glucose metabolism in tumor

Understanding the tumor redox status is important for efficient cancer treatment. Here, we noninvasively detected changes in the redox environment of tumors before and after cancer treatment in the same individuals using a novel compact and portable electron paramagnetic resonance imaging (EPRI) device and compared the results with glycolytic information obtained through autoradiography using 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). Human colon cancer HCT116 xenografts were used in the mice. We used 3-carbamoyl-PROXYL (3CP) as a paramagnetic and redox status probe for the EPRI of tumors. The first EPRI was followed by the intraperitoneal administration of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, or X-ray irradiation of the tumor. A second EPRI was performed on the following day. Autoradiography was performed after the second EPRI. After imaging, the tumor sections were evaluated by histological analysis and the amount of reducing substances in the tumor was measured. BSO treatment and X-ray irradiation significantly decreased the rate of 3CP reduction in tumors. Redox maps of tumors obtained from EPRI can be compared with tissue sections of approximately the same cross section. BSO treatment reduced glutathione levels in tumors, whereas X-ray irradiation did not alter the levels of any of the reducing substances. Comparison of the redox map with the autoradiography of [18F]FDG revealed that regions with high reducing power in the tumor were active in glucose metabolism; however, this correlation disappeared after X-ray irradiation. These results suggest that the novel compact and portable EPRI device is suitable for multimodal imaging, which can be used to study tumor redox status and therapeutic efficacy in cancer, and for combined analysis with other imaging modalities.

Nanoparticle-Mediated Delivery of Satraplatin to Overcome Cisplatin Drug Resistance.

Drug resistance and cancer metastasis are the major obstacles for widely used platinum-based chemotherapy. It is acknowledgement that the decreasing intracellular accumulation of anticancer drugs and increasing sulfur-binding detoxification are two major mechanisms related to drug resistance. Herein, we developed a practical and straightforward method for formulating the clinically used anticancer drug satraplatin (JM-216) with D-α-tocopheryl polyethylene glycol succinate (TPGS)-based polymers to create satraplatin-loaded nanoparticles (SatPt-NPs). The experimental results demonstrate that SatPt-NPs exhibited comparable efficacy to A2780 in treating the A2780 cisplatin-resistant ovarian cancer cell line (A2780DDP), indicating their significant potential in overcoming drug resistance. Additionally, buthionine sulfoximine (BSO) is capable of depleting intracellular glutathione (GSH), resulting in reduced detoxification. After BSO treatment, the IC50 value of SatPt-NPs changed from 0.178 to 0.133 μM, which remained relatively unchanged compared to cisplatin. This suggests that SatPt-NPs can overcome drug resistance by evading GSH detoxification. Therefore, SatPt-NPs have the ability to inhibit drug resistance in tumor cells and hold tremendous potential in cancer treatment.

Glutathione Depression Alters Cellular Mechanisms of Skeletal Muscle Fatigue in Early Stage of Recovery and Prolongs Force Depression in Late Stage of Recovery.

The effects of reduced glutathione (GSH) on skeletal muscle fatigue were investigated. GSH was depressed by buthionine sulfoximine (BSO) (100 mg/kg body weight/day) treatment for 5 d, which decreased GSH content to ~10%. Male Wistar rats were assigned to the control (N = 18) and BSO groups (N =17). Twelve hours after BSO treatment, the plantar flexor muscles were subjected to fatiguing stimulation (FS). Eight control and seven BSO rats were rested for 0.5 h (early stage of recovery), and the remaining were rested for 6 h (late stage of recovery). Forces were measured before FS and after rest, and physiological functions were estimated using mechanically skinned fibers. The force at 40 Hz decreased to a similar extent in both groups in the early stage of recovery and was restored in the control but not in the BSO group in the late stage of recovery. In the early stage of recovery, sarcoplasmic reticulum (SR) Ca2+ release was decreased in the control greater than in the BSO group, whereas myofibrillar Ca2+ sensitivity was increased in the control but not in the BSO group. In the late stage of recovery, SR Ca2+ release decreased and SR Ca2+ leakage increased in the BSO group but not in the control group. These results indicate that GSH depression alters the cellular mechanism of muscle fatigue in the early stage and delays force recovery in the late stage of recovery, due at least in part, to the prolonged Ca2+ leakage from the SR.

RNAi-mediated gene knockdown of progesterone 5\u03b2-reductases in Digitalis lanata reduces 5\u03b2-cardenolide content

Key messageStudying RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) in 5β-cardenolide formation.Progesterone 5β-reductases (P5βR) are assumed to catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-cardenolides. P5βRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5βRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5βR1 (AY585867.1) and DlP5βR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5β-cardenolide formation, we here established several RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5βR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5βR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5βR2-RNAi lines but not in Dl P5βR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5βR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).

Glutathione Enhances Auxin Sensitivity in Arabidopsis Roots.

Root development is regulated by the tripeptide glutathione (GSH), a strong non-enzymatic antioxidant found in plants but with a poorly understood function in roots. Here, Arabidopsis mutants deficient in GSH biosynthesis (cad2, rax1, and rml1) and plants treated with the GSH biosynthesis inhibitor buthionine sulfoximine (BSO) showed root growth inhibition, significant alterations in the root apical meristem (RAM) structure (length and cell division), and defects in lateral root formation. Investigation of the molecular mechanisms of GSH action showed that GSH deficiency modulated total ubiquitination of proteins and inhibited the auxin-related, ubiquitination-dependent degradation of Aux/IAA proteins and the transcriptional activation of early auxin-responsive genes. However, the DR5 auxin transcriptional response differed in root apical meristem (RAM) and pericycle cells. The RAM DR5 signal was increased due to the up-regulation of the auxin biosynthesis TAA1 protein and down-regulation of PIN4 and PIN2, which can act as auxin sinks in the root tip. The transcription auxin response (the DR5 signal and expression of auxin responsive genes) in isolated roots, induced by a low (0.1 µM) auxin concentration, was blocked following GSH depletion of the roots by BSO treatment. A higher auxin concentration (0.5 µM) offset this GSH deficiency effect on DR5 expression, indicating that GSH deficiency does not completely block the transcriptional auxin response, but decreases its sensitivity. The ROS regulation of GSH, the active GSH role in cell proliferation, and GSH cross-talk with auxin assume a potential role for GSH in the modulation of root architecture under stress conditions.

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Two strategies were used: the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO‐treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH‐related proteome has been observed with an upregulation of the regulatory subunit of glutamate–cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.

Cysteine preservation confers resistance to glutathione-depleted cells against ferroptosis via CDGSH iron sulphur domain-containing proteins (CISDs)

Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by the suppression of phospholipid hydroperoxide-glutathione peroxidase (GPX4) activity and a corresponding elevation in lipid peroxidation products. While the inhibition of the xCT-mediated uptake of cystine commonly causes ferroptosis, the sensitivity of cells to the inhibition of the glutathione (GSH) synthesis by buthionine sulfoximine (BSO) varies considerably. The objective of this study was to clarify the underlying mechanism responsible for these differential responses to the targeted inhibition of either the cysteine (Cys) supply to or GSH synthesis in the cells. While intracellular GSH levels were depleted when mouse hepatoma Hepa 1-6 cells were treated with BSO, intracellular Cys levels rather increased and viability of the cells remained unchanged, suggesting that Cys has a role in this resistance to ferroptosis under conditions of GSH depletion. When the cells were treated with pioglitazone (PGZ), a potent inhibitor of CDGSH iron sulphur domain-containing proteins (CISDs), PGZ alone had no effect on either cell viability or GSH levels but induced ferroptosis under conditions of GSH depletion by the BSO treatment. In the case of the co-treatment with PGZ and BSO, ferrous iron and the levels of lipid peroxides were robustly increased in the cells, but neither endoplasmic reticulum stress nor apoptosis was evident. Collectively, CISDs appeared to exert an anti-ferroptotic function by suppressing free iron toxicity and the subsequent lipid peroxidation with assistance provided by Cys.

SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism.

SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism to sustain cell proliferation, the role of SLC25A32 in cancer cell growth remains unexplored.Our results indicate that the SLC25A32 gene is highly amplified in different tumors and that amplification correlates with increased mRNA expression and reduced patients´ survival. siRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of cell lines sensitive and resistant to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine revealed that SLC25A32 knock-down does not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition results in a respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen Species (ROS) and depletion of reduced glutathione (GSH), which impairs cancer cell proliferation. Moreover, buthionine sulfoximine (BSO) treatment further sensitizes cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescues cancer cell proliferation upon SLC25A32 down-regulation.Our results indicate that the reduction of mitochondrial FAD concentrations by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth.

Vulnerability of glutathione-depleted Crassostrea gigas oysters to Vibrio species

Glutathione (GSH) is a major cellular antioxidant molecule participating in several biological processes, including immune function. In this study, we investigated the importance of GSH to oysters Crassostrea gigas immune response. Oysters were treated with the GSH-synthesis inhibitor buthionine sulfoximine (BSO), and the function of immune cells and mortality were evaluated after a bacterial challenge with different Vibrio species. BSO caused a moderate decrease (20–40%) in GSH levels in the gills, digestive gland, and hemocytes. As expected, lower GSH decreased survival to peroxide exposure. Hemocyte function was preserved after BSO treatment, however, oysters became more susceptible to challenges with Vibrio anguillarum, V. alginolyticus, or V. harveyi, but not with V. parahaemolyticus and V. vulnificus, indicating a species-specific vulnerability. Our study indicates that in natural habitats or in mariculture farms, disturbances in GSH metabolism may pre-dispose oysters to bacterial infection, decreasing survival.

The Arabidopsis APR2 positively regulates cadmium tolerance through glutathione-dependent pathway

Cadmium (Cd) is a dangerous environmental pollutant with high toxicity to plants. The adenosine 5′-phosphosulfate reductase 2 (APR2) is the dominant APRs in Arabidopsis and plays an important role in reductive sulfate assimilation pathway. However, whether the involvement of plant APRs in Cd stress response is largely unclear. Herein, we report that APR2 functions in Cd accumulation and tolerance in Arabidopsis. The transcript levels of APR2 were markedly induced by Cd exposure. Transgenic plants overexpressing APR2 improved Cd tolerance, whereas knockout of APR2 reduced Cd tolerance. APR2-overexpressing plants with increased Cd accumulation and tolerance showed higher glutathione (GSH) and phytochelatin (PC) levels than the wild type and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure. Moreover, exogenous GSH application effectively rescued Cd hypersensitivity in APR2-knockout plants. Further analysis showed that buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) treatment completely eliminated the enhanced Cd tolerance phenotypes of APR2-overexpressing plants, implying that APR2-mediated enhanced Cd tolerance is GSH dependent. In addition, over-expression of the APR2 led to elevated expressions of the GSH/PC synthesis-related genes under Cd stress. Taken together, our results indicated that APR2 regulated Cd accumulation and tolerance possibly through modulating GSH-dependent antioxidant capability and Cd-chelation machinery in Arabidopsis. APR2 could be exploited for engineering heavy metal-tolerant plants in phytoremediation.

Exposure to a competitive social environment activates an epigenetic mechanism that limits pheomelanin synthesis in zebra finches.

Competitive environments promote high testosterone levels, produce oxidative stress and, consequently, impair cellular homeostasis. The regulation of genes involved in the synthesis of the pigment pheomelanin in melanocytes seems to help to maintain homeostasis against environmental oxidative stress. Here, we experimentally increased social interactions in some zebra finch (Taeniopygia guttata) males by keeping them in groups of six birds during feather growth, while others were kept alone, to test if melanocytes show epigenetic lability under a competitive social environment. As these changes may depend on the oxidative status, we administrated buthionine sulfoximine (BSO) to decrease the antioxidant capacity of some birds. The competitive environment downregulated a gene involved in pheomelanin synthesis (Slc7a11) by changing the level of DNA methylation in feather melanocytes. In other genes involved in pheomelanin synthesis (Slc45a2, MC1R and AGRP), DNA methylation was also affected, but no changes in expression were detected. Exposure to the competitive environment did not affect systemic oxidative stress and damage, indicating that a protective epigenetic mechanism that changes the expression of Slc7a11 may have been activated. However, no changes to the pigmentation phenotype of birds were found, probably due to the short duration or low intensity of the competitive environment. BSO treatment did not affect the epigenetic mechanism, suggesting that the antioxidant capacity of birds was high enough to deal with the competitive environment. An epigenetic mechanism limiting pheomelanin synthesis therefore becomes activated under exposure to a competitive environment in male zebra finches, which may help to avoid damage caused by competitive interactions.

Increased oxidative stress in the cerebellum and peripheral immune cells leads to exaggerated autism-like repetitive behavior due to deficiency of antioxidant response in BTBR T + tf/J mice

Autism is a neurodevelopmental disorder that affects social cognitive abilities resulting in communication or sensory deficits, and stereotyped behaviors in millions of people worldwide. Oxidant-antioxidant imbalance contributes significantly to the neurobehavioral dysregulations and severity of symptoms in patients with autism, however it has not been explored earlier whether it affects autism-like behavior directly. Therefore, we investigated oxidant-antioxidant balance in peripheral immune cells (neutrophils and CD3+ T cells) and cerebellum of BTBR T + tf/J (BTBR) mice which show autism-like behavior and the social C57BL/6 J (C57) mice. Further, we utilized buthionine sulfoximine (BSO), a glutathione depleting agent to assess the impact of oxidant-antioxidant dysregulation on autism-like behavior. Our study shows that BTBR mice have increased lipid/protein oxidation products in cerebellum and neutrophils/CD3+ T cells along with increased NADPH oxidase (NOX2) and inducible nitric oxide synthase (iNOS) expression. This was concurrent with lower levels of glutathione and enzymatic antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the cerebellum and peripheral immune cells. BSO administration led to further lowering of glutathione with a concurrent upregulation of iNOS, and NOX2 in cerebellum and peripheral immune cells. However, there was deficiency of an adaptive antioxidant response which was associated with exaggerated repetitive behaviors in BTBR mice. On the other hand, C57 mice also had increased oxidative stress after BSO treatment, however there was an enzymatic antioxidant response both in cerebellum and periphery. Overall, this study suggests that BTBR mice have increased oxidative stress with a deficient enzymatic antioxidant response that is associated with autism-like repetitive behaviors.

Abstract 1453: SLC25A32 sustains cancer cell proliferation by regulating flavin adenine dinucleotide (FAD) metabolism

Abstract SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism for generation of nucleotides, lipids and for maintenance of redox homeostasis to sustain growth, the role of SLC25A32 in cancer cell survival remains unexplored. Our results indicate that the SLC25A32 gene is highly amplified in different tumors and that its amplification correlates with increased mRNA expression and with reduced patient survival. SiRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of sensitive and resistant cells to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine upon SLC25A32 knock-down did not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition resulted in respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen Species (ROS) and depletion of reduced glutathione (GSH), impairing cancer cell proliferation. Moreover, Buthionine sulfoximine (BSO) treatment further sensitized cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescued cancer cell proliferation upon SLC25A32 down-regulation. Our results indicate that inhibition of mitochondrial FAD transport by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth. Citation Format: Valeria Santoro, Ilya Kovalenko, Kim Vriens, Stefan Christen, Andreas Bernthaler, Andrea Haegebarth, Sarah-Maria Fendt, Sven Christian. SLC25A32 sustains cancer cell proliferation by regulating flavin adenine dinucleotide (FAD) metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1453.

Buthionine sulfoximine is a multitarget inhibitor of trypanothione synthesis in Trypanosomacruzi.

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosomacruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

The replication of spring viraemia of carp virus can be regulated by reactive oxygen species and NF-κB pathway

Different viruses could induced ROS generation to alter intracellular redox state in the host cells, and unbalanced redox state was suggested to have various effects on viral replication. In this study, we investigated the influence of reactive oxygen species (ROS) on replication of spring viraemia of carp virus (SVCV) in fish cells. After SVCV infection, there existed a time-dependent increase in ROS generation. The present results revealed that antioxidant N-acetyl-l-cysteine (NAC) resulted in a lower ROS levels and increased SVCV replication in EPC cell. In contrast, a GSH synthesis inhibitor buthionine sulfoximine (BSO) induced ROS generation and decreased SVCV replication. In addition, activation of NF-κB suppressed SVCV replication by using two inhibitors of cytokine-induced IκBα phosphorylation. More importantly, enhancement of the activity of NF-κB was found in BSO treatment, which indicated that dropped SVCV replication likely occurred via ROS activation of NF-κB. Overall, our results revealed that the SVCV infection and replication could generate ROS and be affected by the redox state, where this progression was associated with the alteration in NF-κB pathway induced by oxidative stress.

Gallic acid inhibits the growth of calf pulmonary arterial endothelial cells through cell death and glutathione depletion.

Gallic acid (GA) exhibits a number of cellular effects, including apoptosis, which is associated with oxidative stress. The present study investigated the effects of GA on calf pulmonary arterial endothelial cell (CPAEC) growth and death, along with the levels of reactive oxygen species (ROS) and glutathione (GSH). GA treatment inhibited the growth of CPAECs at 24h, and the half‑maximal inhibitory concentration (IC50) value of GA was ~30µM. GA treatment also induced cell death, which was accompanied by a loss of mitochondrial membrane potential (ΔѰm). GA treatment in CPAECs resulted in decreased ROS levels, including O2•‑, whereas the number of GSH‑depleted cells increased. Neither a pan‑caspase inhibitor (benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone) nor buthionine sulfoximine treatment affected GA‑induced cell growth inhibition, cell death, ROS and GSH levels in CPAECs, whereas co‑treatment with N‑acetyl‑cysteine (NAC) resulted in enhanced cell growth inhibition, cell death and ΔѰm loss in these cells. Although NAC treatment did not significantly influence ROS levels in GA‑treated CPAECs, it significantly enhanced GSH depletion in these cells. In conclusion, GA inhibited the growth of CPAECs via cell death, which was associated with GSH depletion rather than alterations to ROS levels.

Opposite Effects of Mechanical Action of Fluid Flow on Proangiogenic Factor Secretion From Human Adipose-Derived Stem Cells With and Without Oxidative Stress.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+ /CD29+ /CD44+ /CD34- /CD106- /CD45- ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration-stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G-CSF, HGF, Leptin, IL-8, PDGF-BB, Angiopoietin-2, and Follistatin from mechanically-stimulated ASCs via cyclooxygenase-2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL-8, leptin, Angiopoitein-2, and PDGF-BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration-stimulating activity from GSH-depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration-stimulating activity from mechano-stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH-depletion had independent proangiogenic-stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal-derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158-2167, 2017. © 2016 Wiley Periodicals, Inc.

Betaine homocysteine S-methyltransferase emerges as a new player of the nuclear methionine cycle

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with d-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded.

Assessing Oxidative Stress in Tumors by Measuring the Rate of Hyperpolarized [1-13C]Dehydroascorbic Acid Reduction Using 13C Magnetic Resonance Spectroscopy

Rapid cancer cell proliferation promotes the production of reducing equivalents, which counteract the effects of relatively high levels of reactive oxygen species. Reactive oxygen species levels increase in response to chemotherapy and cell death, whereas an increase in antioxidant capacity can confer resistance to chemotherapy and is associated with an aggressive tumor phenotype. The pentose phosphate pathway is a major site of NADPH production in the cell, which is used to maintain the main intracellular antioxidant, glutathione, in its reduced state. Previous studies have shown that the rate of hyperpolarized [1-13C]dehydroascorbic acid (DHA) reduction, which can be measured in vivo using non-invasive 13C magnetic resonance spectroscopic imaging, is increased in tumors and that this is correlated with the levels of reduced glutathione. We show here that the rate of hyperpolarized [1-13C]DHA reduction is increased in tumors that have been oxidatively prestressed by depleting the glutathione pool by buthionine sulfoximine treatment. This increase was associated with a corresponding increase in pentose phosphate pathway flux, assessed using 13C-labeled glucose, and an increase in glutaredoxin activity, which catalyzes the glutathione-dependent reduction of DHA. These results show that the rate of DHA reduction depends not only on the level of reduced glutathione, but also on the rate of NADPH production, contradicting the conclusions of some previous studies. Hyperpolarized [1-13C]DHA can be used, therefore, to assess the capacity of tumor cells to resist oxidative stress in vivo. However, DHA administration resulted in transient respiratory arrest and cardiac depression, which may prevent translation to the clinic.