Cellular Antioxidant Response Research Articles

Selenocysteine tRNA methylation promotes oxidative stress resistance in melanoma metastasis.

Selenocysteine-containing proteins play a central role in redox homeostasis. Their translation is a highly regulated process and is dependent on two tRNASec isodecoders differing by a single 2'-O-ribose methylation called Um34. Here we characterized FTSJ1 as the Um34 methyltransferase and show that its activity is required for efficient selenocysteine insertion at the UGA stop codon during translation. Specifically, loss of Um34 leads to ribosomal stalling and decreased UGA recoding. FTSJ1-deficient cells are more sensitive to oxidative stress and show decreased metastatic colonization in xenograft models of melanoma metastasis. We found that FTSJ1 mediates efficient translation of selenoproteins essential for the cellular antioxidant response. Our findings uncover a role for tRNASec Um34 modification in oxidative stress resistance and highlight FTSJ1 as a potential therapeutic target specific for metastatic disease.

Arsenic and Chromium Induced Toxicity on Zebrafish Kidney: Mixture Effects on Oxidative Stress and Involvement of Nrf2-Keap1-ARE, DNA Repair, and Intrinsic Apoptotic Pathways.

In polluted water, cooccurrences of two carcinogens, arsenic (As) and chromium (Cr), are extensively reported. Individual effects of these heavy metals have been reported in kidney of fishes, but underlying molecular mechanisms are not well established. There is no report on combined exposure of As and Cr in kidney. Thus, the present study investigated and compared individual and combined effects of As and Cr on zebrafish (Danio rerio) kidney treating at their environmentally relevant concentrations for 15, 30, and 60 days. Increased ROS levels, lipid peroxidation, GSH level, and decreased catalase activity implied oxidative stress in treated zebrafish kidney. Damage in histoarchitecture in treated groups was also noticed. The current study involved gene expression study of Nrf2, an important transcription factor of cellular stress responses along with its negative regulator Keap1 and downstream antioxidant genes nqo1 and ho1. Results indicated activation of Nrf2-Keap1 pathway after combined exposure. Expression pattern of ogg1, apex1, polb, and creb1 revealed the inhibition of base excision repair pathway in treatments. mRNA expression of tumor suppressor genes p53 and brca2 was also altered. Expressional alteration in bax, bcl2, caspase9, and caspase 3 indicated apoptosis (intrinsic pathway) induction, which was maximum in combined group. Inhibition of DNA repair and induction of apoptosis indicated that the activated antioxidant system was not enough to overcome the damage caused by As and Cr. Overall, this study revealed additive effects of As and Cr in zebrafish kidney after chronic exposure focusing cellular antioxidant and DNA damage responses.

Melatonin alleviates LPS-induced depression-like behavior in mice by inhibiting ferroptosis by regulating RNA methylation-mediated SIRT6/Nrf2/HO-1 pathway.

The objective of this study is to investigate the impact of ferroptosis on depression and elucidate the molecular mechanism underlying melatonin's inhibitory effect on ferroptosis in the treatment of depression. In this study, a depression-like behavior model was induced in mice using LPS, and the effect of melatonin on depression-like behavior was evaluated through behavioral experiments (such as forced swimming test (FST) and sucrose preference test (SPT)). Additionally, molecular biological techniques (including real-time fluorescence quantitative PCR, Western blotting, immunoprecipitation) were employed to detect the expression levels and interactions of METTL3, SIRT6 and ferroptosis-related genes in mouse brain tissue. Furthermore, both in vitro and in vivo experiments were conducted to verify the regulatory effect of melatonin on Nrf2/HO-1 pathway and explore its potential molecular mechanism for regulating ferroptosis. Melatonin was found to significantly ameliorate depression-like behavior in mice, as evidenced by reduced immobility time in the forced swimming test and increased sucrose intake in the sucrose preference test. Subsequent investigations revealed that melatonin modulated SIRT6 stability through METTL3-mediated ubiquitination of SIRT6, leading to its degradation. As a deacetylase, SIRT6 plays a pivotal role in cellular metabolism regulation and antioxidative stress response. This study elucidated potential signaling pathways involving Nrf2/HO-1 through which SIRT6 may exert its effects. The findings suggest that melatonin can improve depressive behavior by suppressing ferroptosis and protecting neurons through its antioxidant properties. Additionally, targeting the Nrf2/HO-1 pathway via METTL3 and NEDD4 regulation may be a potential therapeutic approach for depression.

Exploring the cytoprotective role of mesenchymal stem Cell-Derived exosomes in chronic liver Fibrosis: Insights into the Nrf2/Keap1/p62 signaling pathway

Hepatic fibrosis is a common pathology present in most chronic liver diseases. Autophagy is a lysosome-mediated intracellular catabolic and recycling process that plays an essential role in maintaining normal hepatic functions. Nuclear factor erythroid 2-like 2 (Nrf2) is a transcription factor responsible for the regulation of cellular anti-oxidative stress response. This study was designed to assess the cytoprotective effect of mesenchymal stem cell-derived exosomes (MSC-exos) on endothelial-mesenchymal transition (EMT) in Carbon Tetrachloride (CCL4) induced liver fibrosis. Rats were treated with 0.1 ml of CCL4 twice weekly for 8 weeks, followed by administration of a single dose of MSC-exos. Rats were then sacrificed after 4 weeks, and liver samples were collected for gene expression analyses, Western blot, histological studies, immunohistochemistry, and transmission electron microscopy. Our results showed that MSC-exos administration decreased collagen deposition, apoptosis, and inflammation. Exosomes modulate the Nrf2/Keap1/p62 pathway, restoring autophagy and Nrf2 levels through modulation of the non-canonical pathway of Nrf2/Keap1/p62. Additionally, MSC-exos regulated miR-153-3p, miR-27a, miR-144 and miRNA-34a expression. In conclusion, the present study shed light on MSC-exos as a cytoprotective agent against EMT and tumorigenesis in chronic liver inflammation.

Dissecting the mechanism of synergistic interactions between Aspergillus fumigatus and the microalgae Synechocystis sp. PCC6803 under Cd(II) exposure: insights from untargeted metabolomics

Co-culturing fungi and microalgae may effectively remediate wastewater containing Cd and harvest microalgae. Nevertheless, a detailed study of the mechanisms underlying the synergistic interactions between fungi and microalgae under Cd(II) exposure is lacking. In this study, Cd(II) exposure resulted in a significant enhancement of antioxidants, such as glutathione (GSH), malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide dismutase (SOD) compared to the control group, suggesting that the cellular antioxidant defense response was activated. Extracellular proteins and extracellular polysaccharides of the symbiotic system were increased by 60.61 % and ,24.29 %, respectively, after Cd(II) exposure for 72 h. The adsorption behavior of Cd(II) was investigated using three-dimensional fluorescence excitation-emission matrix (3D-EEM), fourier transform infrared spectroscopy (FTIR), and scanning electron microscope (SEM). Metabolomics results showed that the TCA cycle provided effective material and energy supply for the symbiotic system to resist the toxicity of Cd(II); Proline, histidine, and glutamine strengthened the synergistic adsorption capacity of the fungus and microalgae. Overall, the theoretical foundation for a deep comprehension of the beneficial interactions between fungi and microalgae under Cd(II) exposure and the role of the fungal-algal symbiotic system in the management of heavy metal pollution is provided by this combined physiological and metabolomic investigation.

Assessing the phytotoxic effects of class A foam application on northern Europe tree species: a short-term study on implications for wildfire management and ecosystem health

Wildfires are an ever-increasing issue due to the driving forces of climate change. Weather events that lead to higher wildfire potential are likely to increase and thus new fire management methods via more sustainable fire suppressant class A foams rather than retardants are being developed. However, despite their adherence to regulations, foam impact on targeted ecosystems, namely forests and forest trees is poorly studied. We aimed to investigate how three tree species (Pinus sylvestris, Alnus glutinosa and Picea abies) will react to a one-time class A foam application. Two separate trials were conducted. During the first the foam was applied to seeds and during the other – to 1-year-old seedlings. Tree growth and physiological status were evaluated. Stress criteria for cellular damage, non-antioxidant and antioxidant stress response and photosynthesis efficacy were measured. Results showed an obvious species effect, as all three reacted differently. The dose effect was also notable, with the higher application rate leading to a proportionally bigger response. Overall, pines were negatively impacted, spruce were positively affected, and alders didn’t experience a notable change. This leads us to conclude that pending the limitation of this experiment the tested foam while phytotoxic in some cases, is unlikely to affect tree survival rates under field conditions and any physiological responses are likely transient in nature.

Evidence for TGF-β1/Nrf2 Signaling Crosstalk in a Cuprizone Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic and degenerative disease that impacts central nervous system (CNS) function. One of the major characteristics of the disease is the presence of regions lacking myelin and an oxidative and inflammatory environment. TGF-β1 and Nrf2 proteins play a fundamental role in different oxidative/inflammatory processes linked to neurodegenerative diseases such as MS. The evidence from different experimental settings has demonstrated a TGF-β1-Nrf2 signaling crosstalk under pathological conditions. However, this possibility has not been explored in experimental models of MS. Here, by using the cuprizone-induced demyelination model of MS, we report that the in vivo pharmacological blockage of the TGF-β1 receptor reduced Nrf2, catalase, and TGFβ-1 protein levels in the demyelination phase of cuprizone administration. In addition, ATP production, locomotor function and cognitive performance were diminished by the treatment. Altogether, our results provide evidence for a crosstalk between TGF-β1 and Nrf2 signaling pathways under CNS demyelination, highlighting the importance of the antioxidant cellular response of neurodegenerative diseases such as MS.

The Role of Nrf2 in the Regulation of Mitochondrial Function and Ferroptosis in Pancreatic Cancer.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) represents the master regulator of the cellular antioxidant response and plays a critical role in tumorigenesis. This includes a preventive effect of Nrf2 on cell death through ferroptosis, which represents an essential mechanism of therapy resistance in malignant tumors, such as pancreatic ductal adenocarcinoma (PDAC) as one of the most aggressive and still incurable tumors. Addressing this issue, we provide an overview on Nrf2 mediated antioxidant response with particular emphasis on its effect on mitochondria as the organelle responsible for the execution of ferroptosis. We further outline how deregulated Nrf2 adds to the progression and therapy resistance of PDAC, especially with respect to the role of ferroptosis in anti-cancer drug mediated cell killing and how this is impaired by Nrf2 as an essential mechanism of drug resistance. Our review further discusses recent approaches for Nrf2 inhibition by natural and synthetic compounds to overcome drug resistance based on enhanced ferroptosis. Finally, we provide an outlook on therapeutic strategies based on Nrf2 inhibition combined with ferroptosis inducing drugs.

The Role of NRF2 in Trinucleotide Repeat Expansion Disorders.

Trinucleotide repeat expansion disorders, a diverse group of neurodegenerative diseases, are caused by abnormal expansions within specific genes. These expansions trigger a cascade of cellular damage, including protein aggregation and abnormal RNA binding. A key contributor to this damage is oxidative stress, an imbalance of reactive oxygen species that harms cellular components. This review explores the interplay between oxidative stress and the NRF2 pathway in these disorders. NRF2 acts as the master regulator of the cellular antioxidant response, orchestrating the expression of enzymes that combat oxidative stress. Trinucleotide repeat expansion disorders often exhibit impaired NRF2 signaling, resulting in inadequate responses to excessive ROS production. NRF2 activation has been shown to upregulate antioxidative gene expression, effectively alleviating oxidative stress damage. NRF2 activators, such as omaveloxolone, vatiquinone, curcumin, sulforaphane, dimethyl fumarate, and resveratrol, demonstrate neuroprotective effects by reducing oxidative stress in experimental cell and animal models of these diseases. However, translating these findings into successful clinical applications requires further research. In this article, we review the literature supporting the role of NRF2 in the pathogenesis of these diseases and the potential therapeutics of NRF2 activators.

Sulforaphane upregulates the mRNA expression of NRF2 and NQO1 in non-dialysis patients with chronic kidney disease

Sulforaphane (SFN), found in cruciferous vegetables, is a known activator of NRF2 (master regulator of cellular antioxidant responses). Patients with chronic kidney disease (CKD) present an imbalance in the redox state, presenting reduced expression of NRF2 and increased expression of NF-κB. Therefore, this study aimed to evaluate the effects of SFN on the mRNA expression of NRF2, NF-κB and markers of oxidative stress in patients with CKD. Here, we observed a significant increase in the mRNA expression of NRF2 (p = 0.02) and NQO1 (p = 0.04) in the group that received 400 μg/day of SFN for 1 month. Furthermore, we observed an improvement in the levels of phosphate (p = 0.02), glucose (p = 0.05) and triglycerides (p = 0.02) also in this group. On the other hand, plasma levels of LDL-c (p = 0.04) and total cholesterol (p = 0.03) increased in the placebo group during the study period. In conclusion, 400 μg/day of SFN for one month improves the antioxidant system and serum glucose and phosphate levels in non-dialysis CKD patients.

Abstract P48: Advancing Personalized Anti-Cancer Pharmacotherapy: Magnetic Bioprinting of a Ductal Drug Screening Platform for Salivary Gland Mucoepidermoid Carcinoma

Abstract Mucoepidermoid carcinoma (MEC) is the most common malignant neoplasm of the salivary glands (SG), and it arises from “reserve” stem cells within the ductal network. Recently, our research team has consistently generated acinar-like epithelial SG organoids using a magnetic three-dimensional bioprinting (M3DB) platform. However, 3D ductal constructs with lumenized and functional features have not been established by biofabrication strategies due to challenges related to the lack of epithelial polarity and reproducibility. Consequently, our research unit is developing an in vitro high throughput drug screening platform with bio-printed ductal constructs as avatars of MEC ductal units towards a more effective and personalized anti-cancer pharmacotherapy. M3DB, multiplex immunofluorescence, western blot, and functional assays were performed. M3DB induced the formation of ring-like organoid 3D constructs after the magnetic bioassembly of MEC HTB41 cells overnight. These bio-printed constructs had a lumen formation efficiency of ~70% and contained functional E-cadherin+ cell clusters expressing ZO-1 with mainly K14+ and K5+ ductal progenitor populations with high pro-mitotic activity (Ki-67). These bio-printed ductal-like constructs were then proteomic and functionally evaluated in the presence or absence of 5-fluorouracil (5-FU), a chemotherapy drug. Proteomic assessments revealed changes in cellular antioxidant response (Nrf2) as well as DNA damage repair (γ-H2AX) after 5-FU exposure, potentially safeguarding cells from damage and halting cancer cell cycle progression. Tumor cell invasion capabilities within co-culture in mili-fluidic platforms, and functional evaluation with cholinergic and adrenergic agonists and swelling assays confirmed the 5-FU-induced cytotoxicity, decreased invasion potential, and ductal hypofunction. In summary, our team successfully established novel bio-printed MEC ductal-like avatar platforms that will pave the way for personalized pharmacotherapy and precision oncology in SG malignant tumors. Citation Format: Yamin Oo, Tien T.T. Truong, Toan V. Phan, Khurshid Ahmed, Teerapat Rodboon, Risa Chaisuparat, Glauco R. Souza, Joao N. Ferreira. Advancing Personalized Anti-Cancer Pharmacotherapy: Magnetic Bioprinting of a Ductal Drug Screening Platform for Salivary Gland Mucoepidermoid Carcinoma [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P48.

Quantifying redox transcription factor dynamics as a tool to investigate redox signalling

A critical feature of the cellular antioxidant response is the induction of gene expression by redox-sensitive transcription factors. In many cells, activating these transcription factors is a dynamic process involving multiple redox steps, but it is unclear how these dynamics should be measured. Here, we show how the dynamic profile of the Schizosaccharomyces pombe Pap1 transcription factor is quantifiable by three parameters: signal amplitude, signal time and signal duration. In response to increasing hydrogen peroxide concentrations, the Pap1 amplitude decreased while the signal time and duration showed saturable increases. In co-response plots, these parameters showed a complex, non-linear relationship to the mRNA levels of four Pap1-regulated genes. We also demonstrate that hydrogen peroxide and tert-butyl hydroperoxide trigger quantifiably distinct Pap1 activation profiles and transcriptional responses. Based on these findings, we propose that different oxidants and oxidant concentrations modulate the Pap1 dynamic profile, leading to specific transcriptional responses. We further show how the effect of combination and pre-exposure stresses on Pap1 activation dynamics can be quantified using this approach. This method is therefore a valuable addition to the redox signalling toolbox that may illuminate the role of dynamics in determining appropriate responses to oxidative stress.

Abstract 748: Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial

Abstract Introduction: Higher consumption of dietary sulforaphane (SFN), a phytochemical found in many cruciferous vegetables, has been shown to be associated with lower risk of lung cancer (LC). SF activates the NRF2-pathway, which initiates cellular detoxification, antioxidant, and anti-inflammatory responses. To date, no chemoprevention trial for LC has examined the impact of SFN on LC-related gene expression (GE) as an endpoint in nasal and bronchial tissue. The objective of our study was to examine whether oral intake of SF for 12 months impacts the GE of previously identified LC- and premalignant lesions (PML)-related genes in bronchial and nasal brushing samples. Methods: A randomized, placebo-controlled clinical trial (NCT03232138) was conducted in former smokers with ≥ 30 pack-years and quitting smoking for 1-10 years in Western Pennsylvania, USA. Forty-three subjects were randomly assigned to the SFN treatment (n = 21) or placebo group (n = 22) in 2018-2022. Each participant was instructed to take 4 Avmacol® or identical placebo capsules BID for 12 months. Each Avmacol® capsule contained 15 mg of glucoraphanin, which yielded a daily internal dose of 120 μmol SFN. Nasal swabs and bronchial brushings were obtained at baseline and after 12 months. Among the 37 participants who completed the study, RNA was extracted from their nasal swabs and bronchial brushings. We constructed single sample gene signature scores (GSS) using the GSVA algorithm for the following: 1) the NRF2-pathway, 2) genes associated with LC risk in bronchial biopsies, and 3) genes associated with PML. The genes in these sets were stratified by their up- or down-regulation in LC or PML. We evaluated the association between the interaction of SFN supplementation over time (pre-/post-treatment) on GSS using linear mixed models, adjusted for the fixed effects of age at baseline, RNA integrity number (RIN), packyears of smoking, and random effect of participant. Results: GE data was available for 31 participant pairs with bronchial and/or nasal samples. Within nasal tissue, SFN treatment increased both the GSSs of the NRF2-pathway (p=0.0089) and the downregulated LC-risk genes (p=0.0047), and decreased the upregulated LC-risk genes (p=0.012) over time, whereas SFN had no significant impact on GSSs of the NRF2-pathway and LC-risk genes in the bronchial tissue. No association between SFN treatment over time and the PML-related GSS was observed in nasal or bronchial tissue. Conclusion: Our initial analysis indicates that SFN treatment over 12 months impacted LC-associated GE in nasal tissue but not in bronchial tissue from former smokers. The discrepancy results may be due to small sample size and additional studies are warranted to confirm if SFN has direct impact on LC-risk related genes in bronchial tissue (NIH grant No. R01CA213123). Citation Format: Kathryn Demanelis, Avrum Spira, Yohana Kefella, Renwei Wang, Jennifer Adams-Haduch, Thomas W. Kensler, Jennifer E. Beane, David O. Wilson, Jian-Min Yuan. Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 748.

NRF2 regulates EGF stability through OTUD4 in lung adenocarcinoma

NRF2 (NFE2L2) is a transcription factor mainly for regulating cellular antioxidant response and therefore promotes tumor progression. The target genes of NRF2 also play important roles in cellular processes including glucose metabolism, de novo serine synthesis, iron metabolism, etc. Here, by modulating NRF2 expression in lung adenocarcinoma (LUAD) cells, we showed that NRF2 regulated EGF expression at protein level. Furthermore, EGF was identified as a ubiquitinated protein. We predicted three deubiquitinases of EGF, and OTUD4 had the highest correlation with NRF2 in LUAD among the three. OTUD4 expression was reduced upon NRF2 knocking-down and recovered upon NRF2 rescuing in A549 cells. Then a potential binding site for NRF2 in OTUD4 promoter was searched out. By binding with OTUD4 promoter, NRF2 transcriptionally activated OTUD4, thus promoted EGF deubiquitination and enhanced its stability. More importantly, OTUD4 and NRF2 expression was found being correlated in LUAD patients. The data collectively revealed a novel mechanism of NRF2 regulating on EGF stability through OTUD4 in LUAD.

CYLD induces high oxidative stress and DNA damage through class I HDACs to promote radiosensitivity in nasopharyngeal carcinoma

Abnormal expression of Cylindromatosis (CYLD), a tumor suppressor molecule, plays an important role in tumor development and treatment. In this work, we found that CYLD binds to class I histone deacetylases (HDAC1 and HDAC2) through its N-terminal domain and inhibits HDAC1 activity. RNA sequencing showed that CYLD-HDAC axis regulates cellular antioxidant response via Nrf2 and its target genes. Then we revealed a mechanism that class I HDACs mediate redox abnormalities in CYLD low-expressing tumors. HDACs are central players in the DNA damage signaling. We further confirmed that CYLD regulates radiation-induced DNA damage and repair response through inhibiting class I HDACs. Furthermore, CYLD mediates nasopharyngeal carcinoma cell radiosensitivity through class I HDACs. Thus, we identified the function of the CYLD-HDAC axis in radiotherapy and blocking HDACs by Chidamide can increase the sensitivity of cancer cells and tumors to radiation therapy both in vitro and in vivo. In addition, ChIP and luciferase reporter assays revealed that CYLD could be transcriptionally regulated by zinc finger protein 202 (ZNF202). Our findings offer novel insight into the function of CYLD in tumor and uncover important roles for CYLD-HDAC axis in radiosensitivity, which provide new molecular target and therapeutic strategy for tumor radiotherapy.

Exploring the cellular antioxidant mechanism against cytotoxic silver nanoparticles: a Raman spectroscopic analysis.

Silver nanoparticles (AgNPs) hold great promise for several different applications, from colorimetric sensors to antimicrobial agents. Despite their widespread incorporation in consumer products, limited understanding of the detrimental effects and cellular antioxidant responses associated with AgNPs at sublethal concentrations persists, raising concerns for human and ecological well-being. To address this gap, we synthesized AgNPs of varying sizes and evaluated their cytotoxicity against human dermal fibroblasts (HDF). Our study revealed that toxicity of AgNPs is a time- and size-dependent process, even at low exposure levels. AgNPs exhibited low short-term cytotoxicity but high long-term impact, particularly for the smallest NPs tested. Raman microspectroscopy was employed for in-time investigations of intracellular molecular variations during the first 24 h of exposure to AgNPs of 35 nm. Subtle protein and lipid degradations were detected, but no discernible damage to the DNA was observed. Signals associated with antioxidant proteins, such as superoxide dismutase (SOD), catalase (CAT) and metallothioneins (MTs), increased over time, reflecting the heightened production of these defense agents. Fluorescence microscopy further confirmed the efficacy of overexpressed antioxidant proteins in mitigating ROS formation during short-term exposure to AgNPs. This work provides valuable insights into the molecular changes and remedial strategies within the cellular environment, utilizing Raman microspectroscopy as an advanced analytical technique. These findings offer a novel perspective on the cytotoxicity mechanism of AgNPs, contributing to the development of safer materials and advice on regulatory guidelines for their biomedical applications.

Alterations in sperm RNAs persist after alcohol cessation and correlate with epididymal mitochondrial dysfunction.

Chronic preconception paternal alcohol use adversely modifies the sperm epigenome, inducing fetoplacental and craniofacial growth defects in the offspring of exposed males. A crucial outstanding question in the field of paternal epigenetic inheritance concerns the resilience of the male germline and its capacity to recover and correct sperm-inherited epigenetic errors after stressor withdrawal. We set out to determine if measures of the sperm-inherited epigenetic program revert to match the control treatment 1 month after withdrawing the daily alcohol treatments. Using a voluntary access model, we exposed C57BL/6J males to 6% or 10% alcohol for 10 weeks, withdrew the alcohol treatments for 4 weeks, and used RNA sequencing to examine gene expression patterns in the caput section of the epididymis. We then compared the abundance of sperm small RNA species between treatments. In the caput section of the epididymis, chronic alcohol exposure induced changes in the transcriptional control of genetic pathways related to the mitochondrial function, oxidative phosphorylation, and the generalized stress response (EIF2 signaling). Subsequent analysis identified region-specific, alcohol-induced changes in mitochondrial DNA copy number across the epididymis, which correlated with increases in the mitochondrial DNA content of alcohol-exposed sperm. Notably, in the corpus section of the epididymis, increases in mitochondrial DNA copy number persisted 1 month after alcohol cessation. Analysis of sperm noncoding RNAs between control and alcohol-exposed males 1 month after alcohol withdrawal revealed a ∼100-fold increase in mir-196a, a microRNA induced as part of the nuclear factor erythroid 2-related factor 2 (Nrf2)-driven cellular antioxidant response. Our data reveal that alcohol-induced epididymal mitochondrial dysfunction and differences in sperm noncoding RNA content persist after alcohol withdrawal. Further, differences in mir-196a and sperm mitochondrial DNA copy number may serve as viable biomarkers of adverse alterations in the sperm-inherited epigenetic program.

Overexpression of Nrf2 in Mesenchymal Stem Cells Promotes Migration and Invasion of B- Acute Lymphoblastic Leukemia Cells

Background and objective:B -Acute lymphoblastic leukemia (B-ALL) is one of the most difficult cancers to treat in adults, especially recurrence and drug resistance are still the biggest challenges for B-ALL. In recent years, a new concept has been proposed, that is, the interaction between bone marrow microenvironment and leukemic cells can reduce the sensitivity of leukemic cells to chemotherapy. The components of bone marrow microenvironment (BMM) have been shown to mediate the occurrence and development of leukemia. There is growing evidence that leukemic cells teach the bone marrow microenvironment, change its function and establish leukemia protective interactions with stromal cells and immune cells. Bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BM-MSCs) are stromal cells in leukemia microenvironment, which can promote the progression of leukemia under certain conditions. Nuclear factor erythroid related factor 2 (Nrf2) is an important transcription factor and a regulator of cellular antioxidant response, which regulates the expression of genes encoding antioxidant enzymes to prevent various oxidative changes. It has been confirmed that the high expression of Nrf2 in leukemic cells is related to drug resistance and poor prognosis of leukemia. However, the interaction mechanism of Nrf2 overexpression in MSCs on B-cell acute lymphoblastic leukemia (B-ALL) cells is not clear. We will for the first time propose the mechanism of Nrf2 overexpression in MSCs on the migration and invasion of B-ALL leukemic cells. Methods :Bone marrow nucleated cells were detected in 16 patients with complete remission (B-ALL/CR), 12 patients with relapsed/refractory (B-ALL/R/R) ALL and 10 normal controls. The expression of Nrf2 was detected byreal-time PCR and Western Blot. In vitro cell experiments, real-time PCR and Western Blot were used to detect the expression of Nrf2 in mesenchymal stem cells (MSCs) alone and in the co-culture of MSCs and cell lines (Nalm-6/RS4:11) express the situation Mouse model experiment in vivo, transwell chamber experiment in vitro, three-dimensional (3D) cell culture model, lentivirus transfection, WB and PCR were used to explore the possible mechanism of Nrf2 overexpression in mesenchymal stem cells on the migration and invasion of B-ALL cells. Results: In clinical samples, the expression of Nrf2 in B-ALL-R/R group was significantly higher than that in ALL-CR group and normal control group, suggesting that the expression of Nrf2 was closely related to the recurrence and drug resistance of ALL. In vitro cell experiment, the expression level of Nrf2 in MSCs co-cultured with cell line (NALM-6/RS4:11) was higher than that in MSC cultured alone(P < 0.05). By regulating the overexpression of Nrf2 in MSCs, it can significantly promote the growth, proliferation, migration and invasion of leukemia cells, and promote the expression of CXCR4 in tumor tissues. We further regulated the expression of Nrf2 in MSCs. The results showed that it significantly promoted the migration and invasion of NALM-6/RS4:11 cells. We concluded that the overexpression of Nrf2 in MSCs promotes the migration and invasion of leukemic cells by activating the activity of SDF-1-CXCR4 axis. In vivo experiments, we used tail vein injection of MSCs and B-ALL leukemia cells to explore the protective effect of Nrf2 overexpression in mesenchymal stem cells on leukemia cells and promote extramedullary infiltration of leukemia. The results showed that the infiltration degree of extramedullary organs of mice after combined infusion of two kinds of cells was significantly higher than that of leukemia cells alone, and the survival time of mice was significantly shortened after combined infusion. After statistical treatment, the difference was statistically significant (P < 0.05). Conclusion:The expression of Nrf2 in MSCs may be a key component in mediating the interaction between MSCs and ALL cells. This result may provide a new entry point for deepening all disease progression mechanisms and finding more promising targeted therapies.On the other hand, activation of PI3K/AKTERK1/2 signaling pathway in B-ALL cells further promoted the evolution of B-ALL. This may provide new ideas for findingpotential therapeutic targets for B-ALL extramedullary infi ltration, and help to further solve the current problem of B-ALL relapse and refractory.

Peroxiredoxin-1 is an H2O2 safe-guard antioxidant and signalling enzyme in M1 macrophages

Macrophages are characterised by their high plasticity and ability to adapt their phenotype and functionality in response to environmental cues, resulting in a spectrum of activation states the two extremes of which are M1 and M2. Reactive oxygen species, such as hydrogen peroxide, are among the cues that impact macrophage polarisation. Moreover, high levels of hydrogen peroxide play a role in the phagocytic response executed by M1 macrophages. Therefore, macrophages must balance the need to shield themselves from the harmful effects of hydrogen peroxide bursts with the ability to interpret hydrogen peroxide signals from the surroundings and initiate a cellular response. Peroxiredoxins (PRDX) are proteins capable of performing both roles. Specifically, PRDX1 and PRDX5 have been demonstrated to safeguard macrophages against reactive oxygen species while also impacting their polarisation status. Previously conducted studies did not differentiate between the polarisation state of macrophages or investigate the signalling events triggered by PRDXs. In this study, we utilised bone marrow-derived murine macrophages polarised to the M1 and M2 states. Our findings revealed that the expression of PRDX1 was significantly higher in M1 macrophages than in M2 and unpolarised macrophages. Moreover, we present evidence that in M1 macrophages, PRDX1 interacts with ASK1, its established interaction partner, and also binds to other proteins that regulate the cellular antioxidant response. Interestingly, we found that pharmacological elevation of hydrogen peroxide levels leads to an increase in PRDX1 expression on the mRNA level, but not in the highly related PRDX2 expression. Taken together, our findings suggest that PRDX1 plays a critical role in macrophage antioxidant defence and redox signalling, and provide scope for exploring redox-signalling proteins as highly sought-after candidates for macrophage repolarisation.

An antioxidant feedforward cycle coordinated by linker histone variant H1.2 and NRF2 that drives nonsmall cell lung cancer progression

Nonsmall cell lung cancer (NSCLC) is highly malignant with limited treatment options, platinum-based chemotherapy is a standard treatment for NSCLC with resistance commonly seen. NSCLC cells exploit enhanced antioxidant defense system to counteract excessive reactive oxygen species (ROS), which contributes largely to tumor progression and resistance to chemotherapy, yet the mechanisms are not fully understood. Recent studies have suggested the involvement of histones in tumor progression and cellular antioxidant response; however, whether a major histone variant H1.2 (H1C) plays roles in the development of NSCLC remains unclear. Herein, we demonstrated that H1.2 was increasingly expressed in NSCLC tumors, and its expression was correlated with worse survival. When crossing the H1c knockout allele with a mouse NSCLC model (KrasLSL-G12D/+), H1.2 deletion suppressed NSCLC progression and enhanced oxidative stress and significantly decreased the levels of key antioxidant glutathione (GSH) and GCLC, the catalytic subunit of rate-limiting enzyme for GSH synthesis. Moreover, high H1.2 was correlated with the IC50 of multiple chemotherapeutic drugs and with worse prognosis in NSCLC patients receiving chemotherapy; H1.2-deficient NSCLC cells presented reduced survival and increased ROS levels upon cisplatin treatment, while ROS scavenger eliminated the survival inhibition. Mechanistically, H1.2 interacted with NRF2, a master regulator of antioxidative response; H1.2 enhanced the nuclear level and stability of NRF2 and, thus, promoted NRF2 binding to GCLC promoter and the consequent transcription; while NRF2 also transcriptionally up-regulated H1.2. Collectively, these results uncovered a tumor-driving role of H1.2 in NSCLC and indicate an "H1.2-NRF2" antioxidant feedforward cycle that promotes tumor progression and chemoresistance.