The SP/NK1R System-Mediated ROS Generation in GBM Cells through Inhibiting Glutaredoxin Protein

Altered redox balance is among the main contributing factors developing glioblastoma multiforme (GBM), a highly aggressive grade IV brain tumor. Neuropeptide substance P (SP) plays a key role in modifying the cellular redox environment by activating the neurokinin-1 receptor (NK1R). In this study, we aimed to investigate the redox-modulating properties of both SP and a commercially available NK1R antagonist, aprepitant in GBM cells. To detect the effect of aprepitant on the viability of U87 glioblastoma cells, resazurin assay was applied. The level of intracellular ROS was assessed using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. The expression of glutaredoxin, a well-known redox-active protein, was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Concurrently, the activity of glutaredoxin was also analyzed by a commercial kit (ZellBio GmbH). We found that SP increased the intracellular levels of reactive oxygen species (ROS) in U87 GBM cells, and aprepitant remarkably decreased this effect. We also explored the effects of SP/NK1R signaling on the glutaredoxin system as a major cellular redox buffer in GBM cells. SP reduced both expression and enzymatic activity of glutaredoxin, and these effects were significantly decreased by aprepitant. In conclusion, our results suggest a possible involvement of SP/NK1R signaling in GBM pathogenesis through oxidative stress and offering new insight for the application of aprepitant as a redox-modulating strategy in GBM patients.

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A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells

Scientific Reports ◽

10.1038/s41598-021-97632-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Khoi Thieu Ho ◽

Kohei Homma ◽

Jun Takanari ◽

Hanako Bai ◽

Manabu Kawahara ◽

...

Keyword(s):

Cell Function ◽

Redox Balance ◽

Asparagus Officinalis ◽

Hsp70 Expression ◽

Ros Generation ◽

Cellular Redox ◽

Pheochromocytoma Cells ◽

Cell Functions ◽

Progesterone Synthesis ◽

Hsp70 Induction

AbstractHeat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.

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Potential Role of GST-π in Lung Cancer Stem Cell Cisplatin Resistance

BioMed Research International ◽

10.1155/2021/9142364 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Wenjun Wang ◽

Jianping Wei ◽

Xiaoyun Tu ◽

Xiaoqun Ye

Keyword(s):

Lung Cancer ◽

Stem Cells ◽

Protein Expression ◽

A549 Cell ◽

Potential Role ◽

Redox Balance ◽

Adherent Cells ◽

Cellular Redox ◽

Intracellular Ros

Background. Cancer stem cells (CSCs) are responsible for tumorigenesis, chemoresistance, and metastasis. Chemoresistance is a major challenge in the management of lung cancer. Glutathione-sulphur-transferase-π (GST-π) plays an important role in the origin and development of various types of cancer by regulating the cellular redox balance. Recent investigations have demonstrated that GST-π is associated with the chemoresistance of lung CSCs (LCSCs). However, the mechanism of GST-π in lung cancer, particularly in LCSCs, remains unclear. The present study is aimed at exploring the potential role of GST-π in stemness and cisplatin (DDP) resistance of LCSCs. Materials and methods. In the present study, lung cancer cell spheres were established using the A549 cell line, which according to our previous research, was confirmed to exhibit characteristics of stem cells. Next, GST-π protein expression, apoptosis percentage, and intracellular reactive oxygen species (ROS) concentration in A549 adherent cells and A549 cell spheres were analyzed by western blotting and flow cytometry, respectively. Finally, DDP resistance, ROS concentration, and GST-π expression in LCSCs were analyzed following the interference with GST-π using DL-buthionine-(S,R)-sulphoximine and N-acetylcysteine. Results. The results revealed that GST-π was highly expressed in A549 cell spheres compared with A549 adherent cells and was associated with a decreased intracellular ROS concentration (both P < 0.05 ). Regulating GST-π protein expression could alter DDP resistance of LCSCs by influencing ROS. Conclusion. These results suggested that GST-π may be important for LCSC drug resistance by downregulating ROS levels. These findings may contribute to the development of new adjuvant therapeutic strategies for lung cancer.

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Involvement of vertebrate hemoglobin in antioxidant protection: chicken blood as a model

Canadian Journal of Zoology ◽

10.1139/z07-020 ◽

2007 ◽

Vol 85 (3) ◽

pp. 404-412 ◽

Cited By ~ 4

Author(s):

Alcir Luiz Dafre ◽

Tiago A.S. Brandão ◽

Evaldo Reischl

Keyword(s):

Redox Balance ◽

Evolutionary Trend ◽

Cellular Redox ◽

Reactive Protein ◽

Cellular Events ◽

Protein Thiols ◽

Antioxidant Function ◽

Chicken Erythrocytes ◽

Redox Buffer ◽

Chicken Blood

Redox balance can be described as the equilibrium between oxidative and reductive forces within the cell. These forces control several cellular events, including the modulation of redox-sensitive receptors and signaling pathways. In cells, glutathione is the major non-protein thiol and is considered the main redox buffer. The ratio between the oxidized (GSSG) and reduced (GSH) forms reflects the cellular redox balance. Reactive protein thiols, including vertebrate hemoglobin (Hb), have been proposed as effective antioxidants that can contribute to the redox balance. To further explore this possibility, chicken ( Gallus gallus (L., 1758)) blood was used as a model system. The use of known oxidants (hydroperoxides, diamide, and a system generating reactive oxygen species) originated a pattern of glutathiolation in chicken erythrocytes that was fully reversed after removal of the oxidant, this being consistent with a physiological response. The glutathiolation sequence correlates to kinetic data on chicken Hb cysteine reactivity. The major chicken hemoglobin (Hb A) is responsible for most of the glutathiolated protein where the presence of externally positioned and fast-reacting cysteines is a contributing factor. The antioxidant potential of fast-reacting Hb cysteines is in line with the conservation of cysteine residues in Hb stereochemical positions in more than 95% of the available avian Hb sequences. This may represent an evolutionary trend for the antioxidant function of externally positioned and reactive cysteines in abundant proteins.

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The Combination of PEITC (Phenehyl Isothiocyanate) with a Histone Deacetylase Inhibitor (HDACi) Has Synergistic Antileukemia Activity by Overcoming a Redox-Mediated Resistance Pathway.

Blood ◽

10.1182/blood.v114.22.1739.1739 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1739-1739

Author(s):

Yumin Hu ◽

Gang Chen ◽

Yu Jia ◽

Hui Zhang ◽

Peng Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Transcription Factor ◽

Histone Deacetylase ◽

Cell Lines ◽

Redox Balance ◽

Ros Generation ◽

Combination Effect ◽

Antioxidant Genes ◽

Cellular Redox ◽

Cellular Defense

Abstract Abstract 1739 Poster Board I-765 Introduction Histone deacetylase inhibitors (HDACI) have limited but well established clinical activity in human leukemia. Results of a phase 1 trial of vorinostat in AML indicate that a gene signature composed mainly of antioxidants was associated with clinical resistance to vorinostat (Blood 2008;111:1060-60). This study suggested that generation of reactive oxygen species (ROS) appears to be a mechanism of action of vorinostat whereas increase of antioxidants may correlate with vorinostat resistance. The aims of this study were to further investigate the underlying molecular mechanisms and test the combination effect of vorinostat and redox modulation agents. Methods and results The parental HL60 and the pan-HDACI resistant HL60/LR were used to compare the redox parameters in this pair of cell lines. Real time PCR and western blot analysis demonstrated that a variety of glutathione related antioxidant defense enzymes were substantially increased in HL60/LR compared to its parental HL60, which is consistent with the clinical findings cited above. Most importantly, Nrf2, a master transcription factor that activates the transcription of cellular defense and antioxidant genes was also upregulated in HL60/LR. Confocal microscopy study showed that vorinostat treatment of HL60 cells caused translocation of Nrf2 from cytosol to nucleus. Furthermore, its downstream antioxidant genes including GST (glutathione S- Transferase), GSR (glutathione reductase), GCLC (glutathione synthase) and SOD (superoxide dismutase) were upregulated, demonstrating that the cellular defense against oxidative stress was induced by vorinostat. Overexpression of Nrf2 in HEK293 cells prevented ROS generation induced by vorinostat. Knock-down of Nrf2 by siRNA in colon cancer cell HCT116 caused increase of ROS production and cytotoxicity induced by vorinostat. These findings further demonstrated the role of Nrf2 in protecting cells from oxidative stress caused by vorinostat. We also observed that vorinostat substantially activated a ROS generating enzyme NADPH Oxidase (NOX) in various AML cell lines including HL60, U937 and ML1. Vorinostat induced ROS in both HL60 and the mitochondrial deficient cell line HL60-C6F. This indicates that NOX is a major source of ROS generation induced by vorinostat. As a result, modulation of antioxidant response may potentiate the cytotoxic activity of vorinostat. In order to modulate cellular redox balance and overcome the resistance to vorinostat, PEITC, a compound known to deplete cellular glutathione was used to test its combination effect with vorinostat. We found that a subtoxic concentration of PEITC (1-2.5 uM) substantially potentiated cytotoxicity of vorinostat in a dose-dependent manner in various AML cell lines, as demonstrated by Annexin-PI assay after 48 hrs and MTT assay after 72 hrs. Treatment with subtoxic concentrations of vorinostat (1.5 uM) and PEITC (1-2.5 uM) for 48 hrs also resulted in synergistic cytotoxicity in primary leukemia cells obtained from AML patient samples as demonstrated by Annexin-PI assay. Parallel results were also obtained with other HDACI such as MGCD0103. Conclusions Our study indicates that NADPH Oxidase is likely a major source of ROS generation induced by vorinostat. Nrf2, a master transcription factor and its downstream antioxidant genes, which protect cells from oxidative stress, contributes to leukemia cellular resistance to vorinostat. Modulation of cellular redox balance such as depletion of glutathione by PEITC significantly potentiates the anti-leukemia activity of vorinostat. Our study provides important information for further development of a mechanism-based combination strategy to maximize the potential of vorinostat and other HDACI and provides an alternative mechanism of the anti-leukemia activity of HDACI. Disclosures No relevant conflicts of interest to declare.

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Effect of A Standardized Extract of Asparagus Officinalis Stem on HSP 70 Induction and Redox Balance in Bovine Cumulus-Granulosa Cells

10.21203/rs.3.rs-640401/v1 ◽

2021 ◽

Author(s):

Ho Khoi ◽

Kohei Homma ◽

Jun Takanari ◽

Hanako Bai ◽

Manabu Kawahara ◽

...

Keyword(s):

Heat Shock ◽

Stress Condition ◽

Redox Balance ◽

Asparagus Officinalis ◽

Hsp70 Expression ◽

Ros Generation ◽

Hsp 70 ◽

Cellular Redox ◽

Heat Stress Condition ◽

Hsp70 Induction

Abstract Heat shock protein 70 (HSP70) is a well-known heat shock (HS)-induced protein that acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to acquire stress-resistant ability to cells, HS causes toxicity to cells by increasing reactive oxygen species (ROS). Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the by-product of asparagus, was found to induce HSP70 expression without HS and regulate cellular redox balance in the cells. However, the effect of EAS on the function of reproductive cells remains unknown. In the present study, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa(CG) cells. EAS significantly increased HSP70 expression, whereas no effect was observed in HSP27 and − 90 under non-heat stress condition. EAS decreased ROS generation and DNA damage, and increased glutathione (GSH) synthesis both under non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression. EAS also increased progesterone (P4) levels in CG cells. HSP70 inhibitor significantly decreased GSH and increased ROS, as well as decreased HSF1, Nrf2, and Keap1 in the presence of EAS. These results suggest that EAS regulates redox balance through HSP70 in bovine CG cells.

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Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors

International Journal of Molecular Sciences ◽

10.3390/ijms22157872 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7872

Author(s):

Malin Tordis Meyer ◽

Christoph Watermann ◽

Thomas Dreyer ◽

Steffen Wagner ◽

Claus Wittekindt ◽

...

Keyword(s):

Redox Balance ◽

Matrix Proteins ◽

Parotid Tumor ◽

Gene Expressions ◽

Tissue Samples ◽

Cellular Redox ◽

Tumor Subtypes ◽

Parotid Tumors ◽

Aggressive Tumors

Salivary gland cancers are rare but aggressive tumors that have poor prognosis and lack effective cure. Of those, parotid tumors constitute the majority. Functioning as metabolic machinery contributing to cellular redox balance, peroxisomes have emerged as crucial players in tumorigenesis. Studies on murine and human cells have examined the role of peroxisomes in carcinogenesis with conflicting results. These studies either examined the consequences of altered peroxisomal proliferators or compared their expression in healthy and neoplastic tissues. None, however, examined such differences exclusively in human parotid tissue or extended comparison to peroxisomal proteins and their associated gene expressions. Therefore, we examined differences in peroxisomal dynamics in parotid tumors of different morphologies. Using immunofluorescence and quantitative PCR, we compared the expression levels of key peroxisomal enzymes and proliferators in healthy and neoplastic parotid tissue samples. Three parotid tumor subtypes were examined: pleomorphic adenoma, mucoepidermoid carcinoma and acinic cell carcinoma. We observed higher expression of peroxisomal matrix proteins in neoplastic samples with exceptional down regulation of certain enzymes; however, the degree of expression varied between tumor subtypes. Our findings confirm previous experimental results on other organ tissues and suggest peroxisomes as possible therapeutic targets or markers in all or certain subtypes of parotid neoplasms.

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Antioxidant or Apoptosis Inhibitor Supplementation in Culture Media Improves Post-Thaw Recovery of Murine Spermatogonial Stem Cells

Antioxidants ◽

10.3390/antiox10050754 ◽

2021 ◽

Vol 10 (5) ◽

pp. 754

Author(s):

Sang-Eun Jung ◽

Hui-Jo Oh ◽

Jin-Seop Ahn ◽

Yong-Hee Kim ◽

Bang-Jin Kim ◽

...

Keyword(s):

Stem Cells ◽

Functional Activity ◽

Culture Media ◽

Quantitative Polymerase Chain Reaction ◽

Spermatogonial Stem Cells ◽

Ros Generation ◽

Apoptosis Assay ◽

Polymerase Chain ◽

Apoptosis Inhibitor

We postulated that supplementation of antioxidant or apoptosis inhibitor in post-thaw culture media of spermatogonial stem cells (SSCs) alleviates reactive oxygen species (ROS) generation and apoptosis. Our aim was to develop an effective culture media for improving post-thaw recovery of SSCs. To determine the efficacy of supplementation with hypotaurine (HTU), α-tocopherol (α-TCP), and Z-DEVD-FMK (ZDF), we assessed the relative proliferation rate and SSC functional activity and performed a ROS generation assay, apoptosis assay, and western blotting for determination of the Bax/Bcl-xL ratio, as well as immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization. The relative proliferation rates with HTU 400 μM (133.7 ± 3.2%), α-TCP 400 μM (158.9 ± 3.6%), and ZDF 200 μM (133.1 ± 7.6%) supplementation were higher than that in the DMSO control (100 ± 3.6%). ROS generation was reduced with α-TCP 400 μM (0.8-fold) supplementation in comparison with the control (1.0-fold). Early apoptosis and Bax/Bcl-xL were lower with α-TCP 400 μM (2.4 ± 0.4% and 0.5-fold) and ZDF 200 μM (1.8 ± 0.4% and 0.3-fold) supplementation in comparison with the control (5.3 ± 1.4% and 1.0-fold) with normal characterization and functional activity. Supplementation of post-thaw culture media with α-TCP 400 μM and ZDF 200 μM improved post-thaw recovery of frozen SSCs via protection from ROS generation and apoptosis after cryo-thawing.

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Cisplatin inhibits SIRT3-deacetylation MTHFD2 to disturb cellular redox balance in colorectal cancer cell

Cell Death and Disease ◽

10.1038/s41419-020-02825-y ◽

2020 ◽

Vol 11 (8) ◽

Author(s):

Xingyou Wan ◽

Chao Wang ◽

Zhenyu Huang ◽

Dejian Zhou ◽

Sheng Xiang ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Cell ◽

Redox Balance ◽

Colorectal Cancer Cell ◽

Cellular Redox

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Endogenous formaldehyde scavenges cellular glutathione resulting in cytotoxic redox disruption

10.1101/2020.05.14.090738 ◽

2020 ◽

Author(s):

Carla Umansky ◽

Agustín Morellato ◽

Marco Scheidegger ◽

Matthias Rieckher ◽

Manuela R. Martinefski ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Repair ◽

Fanconi Anemia ◽

Redox Homeostasis ◽

Thiol Group ◽

Cellular Damage ◽

Repair Pathway ◽

Cellular Redox ◽

Redox Active ◽

Development And Aging

AbstractFormaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking. We show here that FA can cause cellular damage beyond genotoxicity by triggering oxidative stress, which is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR). Mechanistically, we determine that endogenous FA reacts with the redox-active thiol group of glutathione (GSH) forming S-hydroxymethyl-GSH, which is metabolized by ADH5 yielding reduced GSH thus preventing redox disruption. We identify the ADH5-ortholog gene in Caenorhabditis elegans and show that oxidative stress also underlies FA toxicity in nematodes. Moreover, we show that endogenous GSH can protect cells lacking the Fanconi Anemia DNA repair pathway from FA, which might have broad implications for Fanconi Anemia patients and for healthy BRCA2-mutation carriers. We thus establish a highly conserved mechanism through which endogenous FA disrupts the GSH-regulated cellular redox homeostasis that is critical during development and aging.

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Augmented peroxisomal ROS buffering capacity renders oxidative and thermal stress cross-tolerance in yeast

Microbial Cell Factories ◽

10.1186/s12934-021-01623-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Nai-Xin Lin ◽

Rui-Zhen He ◽

Yan Xu ◽

Xiao-Wei Yu

Keyword(s):

Thermal Stress ◽

Production Efficiency ◽

Heterologous Protein ◽

Redox Balance ◽

Industrial Applications ◽

Ros Generation ◽

Cell Factory ◽

Heterologous Proteins ◽

Cross Tolerance ◽

Autophagy Pathway

Abstract Background Thermotolerant yeast has outstanding potential in industrial applications. Komagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins. Results Herein, we obtained a thermotolerant K. phaffii mutant G14 by mutagenesis and adaptive evolution. G14 exhibited oxidative and thermal stress cross-tolerance and high heterologous protein production efficiency. The reactive oxygen species (ROS) level and lipid peroxidation in G14 were reduced compared to the parent. Oxidative stress response (OSR) and heat shock response (HSR) are two major responses to thermal stress, but the activation of them was different in G14 and its parent. Compared with the parent, G14 acquired the better performance owing to its stronger OSR. Peroxisomes, as the main cellular site for cellular ROS generation and detoxification, had larger volume in G14 than the parent. And, the peroxisomal catalase activity and expression level in G14 was also higher than that of the parent. Excitingly, the gene knockdown of CAT encoding peroxisomal catalase by dCas9 severely reduced the oxidative and thermal stress cross-tolerance of G14. These results suggested that the augmented OSR was responsible for the oxidative and thermal stress cross-tolerance of G14. Nevertheless, OSR was not strong enough to protect the parent from thermal stress, even when HSR was initiated. Therefore, the parent cannot recover, thereby inducing the autophagy pathway and resulting in severe cell death. Conclusions Our findings indicate the importance of peroxisome and the significance of redox balance in thermotolerance of yeasts.

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