Peroxiredoxin Research Articles

PRDX1 negatively regulates bleomycin-induced pulmonary fibrosis via inhibiting the epithelial-mesenchymal transition and lung fibroblast proliferation in vitro and in vivo

BackgroundPulmonary fibrosis is a major category of end-stage changes in lung diseases, characterized by lung epithelial cell damage, proliferation of fibroblasts, and accumulation of extracellular matrix. Peroxiredoxin 1 (PRDX1), a member of the peroxiredoxin protein family, participates in the regulation of the levels of reactive oxygen species in cells and various other physiological activities, as well as the occurrence and development of diseases by functioning as a chaperonin.MethodsExperimental methods including MTT assay, morphological observation of fibrosis, wound healing assay, fluorescence microscopy, flow cytometry, ELISA, western blot, transcriptome sequencing, and histopathological analysis were used in this study.ResultsPRDX1 knockdown increased ROS levels in lung epithelial cells and promoted epithelial-mesenchymal transition (EMT) through the PI3K/Akt and JNK/Smad signalling pathways. PRDX1 knockout significantly increased TGF-β secretion, ROS production, and cell migration in primary lung fibroblasts. PRDX1 deficiency also increased cell proliferation, cell cycle circulation, and fibrosis progression through the PI3K/Akt and JNK/Smad signalling pathways. BLM treatment induced more severe pulmonary fibrosis in PRDX1-knockout mice, mainly through the PI3K/Akt and JNK/Smad signalling pathways.ConclusionsOur findings strongly suggest that PRDX1 is a key molecule in BLM-induced lung fibrosis progression and acts through modulating EMT and lung fibroblast proliferation; therefore, it may be a therapeutic target for the treatment of BLM-induced lung fibrosis.

Alterations in the expression pattern of RBC membrane associated proteins (RMAPs) in whole body γ-irradiated Sprague Dawley rats

Purpose Peripheral blood serum/plasma proteins are frequently studied for their potential use as radiation exposure biomarkers. Here we report RBC membrane associated proteins (RMAPs), which show alterations in expression level following whole-body γ-irradiation of rats at sub-lethal/lethal doses. Materials and Methods RBCs from peripheral blood of Sprague Dawley rats were segregated using the Ficoll-Hypaque method, and membrane fractions were hypotonically isolated at various time points (6 h, 24 h, 48 h) after γ-irradiation at 2 Gy, 5 Gy, and 7.5 Gy doses. Following purification of proteins from these fractions, two-dimensional electrophoresis (2-DE) was carried out. Treatment induced differentially expressed protein spots (≥2 fold increase/decrease) were picked up, trypsinized, and identified using LC-MS/MS analysis. Western immunoblots using protein specific antibodies were used to confirm the results. Gene ontology and interactions of these proteins were also studied. Results From a number of differentially expressed radiation-responsive 2-DE protein spots detected, eight were identified unequivocally using LC-MS/MS. Out of these, actin, cytoplasmic 1 (ACTB) showed detectable yet insignificant variation (<50%) in expression. In contrast, peroxiredoxin-2 (PRDX2) and 26S proteasome regulatory subunit RPN11 (PSMD14) were the two most prominently over-expressed proteins. Five more proteins, namely tropomyosin alpha-3 chain (TPM3), exosome component 6 (EXOSC6), isoform 4 of tropomyosin alpha-1 chain (TPM1), serum albumin (ALB), and the 55 kDa erythrocyte membrane protein (P55) showed distinct alteration in their expression at different time-points and doses. ALB, EXOSC6, and PSMD14 were the most responsive at 2 Gy, albeit at different time-points. While EXOSC6 and PSMD14 showed maximum over-expression (5-12 fold) at 6 h post-irradiation, ALB expression increased progressively (4 up to 7 fold) from 6 h to 48 h. TPM1 showed over-expression (2-3 fold) at all doses and time-points tested. TPM3 showed a dose-dependent response at all time-points studied; with no variation at 2 Gy, ∼2 fold increase at 5 Gy, and 3-6 fold at the highest dose used (7.5 Gy). The p55 protein was over-expressed (∼2.5 fold) only transiently at 24 h following the lethal (7.5 Gy) dose. Conclusion This is the first study to report γ-radiation induced alterations in the RBC membrane associated proteins. We are further evaluating the potential of these proteins as radiation biomarkers. Due to the abundance and easy use of RBCs, this approach can prove very useful for detecting ionizing radiation exposure.

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines.

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3β inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the "cadherin switch". These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies.

Ficus deltoidea Leaf Alters Oxidative Stress, Protein Homeostasis and Ubiquitin-Proteasome Pathways in Fatty Acid-Induced Cell Line.

Ficus deltoidea (mistletoe fig) is a shrub well known among locals in Malaysia primarily for its treatment of toothaches, colds and wounds. The aim of this study is to determine the potential of leaves, sourced from three different varieties of F. deltoidea, to exhibit antioxidant activity, a reduction of lipid concentration, and protein expression in steatosis-induced liver cell lines. The leaves of three F. deltoidea varieties, namely Ficus deltoidea var. angustifolia, Ficus deltoidea var. trengganuensis and Ficus deltoidea var. kunstleri, were subjected to water extraction. The resulting crude extracts were fractionated using water and ethyl acetate. Palmitic acid was used to induce lipid accumulation (steatosis) in human liver (WRL68) cells, before all the samples were tested for their lipid-reducing activity. Several proteomic approaches were incorporated. The changes in protein expression were determined using 2-dimensional gel electrophoresis separation, whereas identification of our protein spots of interest was carried out via matrix-assisted laser desorption/ionization time-of-flight. Ficus deltoidea var. kunstleri alone demonstrated the ability to reduce lipids at the highest tested concentration (200 µg/mL) and was, therefore, used for subsequent experiments. Treatment with Ficus deltoidea var. kunstleri was found to restore redox status by increasing superoxide dismutase and glutathione peroxidase amounts and decreasing malondialdehyde formation. Six proteins were successfully identified; these were heat shock protein beta-1 (HSPB1), proteasome subunit alpha type 1 (PSMA1), glutathione S-transferase omega 1 (GSTO1), peroxiredoxin-1 (PRDX1), histone H2B (HIST1H2BD) and ubiquitin c-terminal hydrolase L3 (UCHL3). Through bioinformatics analysis, it was found that these proteins were significantly involved in specific pathways such as oxidative stress (PRDX1 and GSTO1), protein homeostasis (HSPB1) and degradation (UCHL3 and PSMA1). F. deltoidea pretreatment was shown to reduce lipid accumulation, thus improving the redox status and protein homeostasis. This suggests the role of F. deltoidea as a preventive mechanism in non-alcohol fatty liver disease.

The contribution of glutathione peroxidases to chloroplast redox homeostasis in Arabidopsis

Oxidizing signals mediated by the thiol-dependent peroxidase activity of 2-Cys peroxiredoxins (PRXs) plays an essential role in fine-tuning chloroplast redox balance in response to changes in light intensity, a function that depends on NADPH-dependent thioredoxin reductase C (NTRC). In addition, plant chloroplasts are equipped with glutathione peroxidases (GPXs), thiol-dependent peroxidases that rely on thioredoxins (TRXs). Despite having a similar reaction mechanism than 2-Cys PRXs, the contribution of oxidizing signals mediated by GPXs to the chloroplast redox homeostasis remains poorly known. To address this issue, we have generated the Arabidopsis (Arabidopsis thaliana) double mutant gpx1gpx7, which is devoid of the two GPXs, 1 and 7, localized in the chloroplast. Furthermore, to analyze the functional relationship of chloroplast GPXs with the NTRC-2-Cys PRXs redox system, the 2cpab-gpx1gpx7 and ntrc-gpx1gpx7 mutants were generated. The gpx1gpx7 mutant displayed wild type-like phenotype indicating that chloroplast GPXs are dispensable for plant growth at least under standard conditions. However, the 2cpab-gpx1gpx7 showed more retarded growth than the 2cpab mutant. The simultaneous lack of 2-Cys PRXs and GPXs affected PSII performance and caused higher delay of enzyme oxidation in the dark. In contrast, the ntrc-gpx1gpx7 mutant combining the lack of NTRC and chloroplast GPXs behaved like the ntrc mutant indicating that the contribution of GPXs to chloroplast redox homeostasis is independent of NTRC. Further supporting this notion, in vitro assays showed that GPXs are not reduced by NTRC but by TRX y2. Based on these results, we propose a role for GPXs in the chloroplast redox hierarchy.

CircZNF215 promotes tumor growth and metastasis through inactivation of the PTEN/AKT pathway in intrahepatic cholangiocarcinoma

BackgroundIncreasing evidence shows that circular RNAs (circRNAs), a novel class of noncoding RNAs, play a crucial role in the development of cancers, including intrahepatic cholangiocarcinoma (iCCA). Nevertheless, their functions and exact mechanisms in iCCA progression and metastasis are still unclear. Ipatasertib is a highly selective inhibitor of AKT that inhibits tumor growth by blocking the PI3K/AKT pathway. In addition, phosphatase and tensin homolog (PTEN) can also inhibit the activation of the PI3K/AKT pathway, but it is not clear whether the cZNF215-PRDX-PTEN axis plays a role in the antitumor activity of ipatasertib.MethodsWe identified a new circRNA (circZNF215, termed cZNF215) through high-throughput circRNA sequencing (circRNA-seq). In addition, RT‒qPCR, immunoblot assay, RNA pull-down assay, RNA immunoprecipitation (RIP) assay, and fluorescence in situ hybridization assay (FISH) were used to investigate the interaction of cZNF215 with peroxiredoxin 1 (PRDX1). Coimmunoprecipitation (Co-IP) assays and duolink in situ proximity ligation assays (PLAs) were conducted to analyze the effects of cZNF215 on the interaction between PRDX1 and PTEN. Finally, we tested the potential effects of cZNF215 on the antitumor activity of ipatasertib with in vivo experiments.ResultsWe found that cZNF215 expression was obviously upregulated in iCCA tissues with postoperative metastases and was correlated with iCCA metastasis and poor outcome in patients with iCCA. We further revealed that overexpression of cZNF215 promoted iCCA cell growth and metastasis in vitro and in vivo, while cZNF215 knockdown had the opposite effect. Mechanistic studies suggested that cZNF215 competitively interacted with PRDX1, which blocked the association between PRDX1 and PTEN, subsequently leading to oxidation-induced inactivation of the PTEN/AKT pathway and finally contributing to iCCA progression and metastasis. Additionally, we also revealed that silencing cZNF215 in iCCA cells had the potential to enhance the antitumor effect of ipatasertib.ConclusionsOur study demonstrates that cZNF215 facilitates iCCA progression and metastasis by regulating the PTEN/AKT pathway and may serve as a novel prognostic predictor in patients with iCCA.

Redox-stress response resistance (RRR) mediated by hyperoxidation of peroxiredoxin 2 in senescent cells.

Aging is closely related to redox regulation. In our previous work, we proposed a new concept, "redox-stress response capacity (RRC)," and found that the decline in RRC was a dynamic characteristic of aging. However, the mechanism of RRC decline during aging remains unknown. In this study, using the senescent human fibroblast cell model and Caenorhabditis elegans model, we identified that peroxiredoxin 2 (PRDX2), as a hydrogen peroxide (H2O2) sensor, was involved in mediating RRC. PRDX2 knockdown led to a decline of RRC and accelerated senescence in fibroblasts and prdx-2 mutant C. elegans also showed decreased RRC. The mechanism study showed that the decreased sensor activity of PRDX2 was related to the increase in hyperoxidation of PRDX2 in senescent cells. Moreover, the level of PRDX2 hyperoxidation also increased in old C. elegans. Simultaneous overexpression of both PRDX2 and sulfiredoxin (SRX) rescued the reduced RRC and delayed senescence. The increase in PRDX2 hyperoxidation in senescent cells led to a decrease in its sensor activity, resulting in the decreased cellular response to H2O2, which is similar to the mechanism of insulin resistance due to the lower insulin receptor sensitivity. Treatment of young cells with a high level of H2O2 to induce a higher level of PRDX2-SO3 resulted in mimicking the RRC decline in senescent cells, which is also similar to a model of insulin resistance induced by high levels of insulin. All these results thrillingly indicate that there is an insulin-resistance-like phenomenon in senescent cells, we named it redox-stress response resistance, RRR. RRR in senescent cells is an important new discovery that explains RRC decline during aging and reveals the internal relationship between redox regulation and aging from a new perspective.

Disulfide-Bond-Induced Structural Frustration and Dynamic Disorder in a Peroxiredoxin from MAS NMR.

Disulfide bond formation is fundamentally important for protein structure and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive μs time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfill other favorable contacts.

Melatonin alleviates ovarian function damage and oxidative stress induced by dexamethasone in the laying hens through FOXO1 signaling pathway

Oxidative stress can trigger follicular atresia, and decrease follicles quantity in each development stage, thereby alleviating reproductive activity. The induction of oxidative stress in chickens through intraperitoneal injection of dexamethasone is a reliable and stable method. Melatonin has been shown to mitigate oxidative stress in this model, but the underlying mechanism remains unclear. Therefore, this study aimed to investigate whether melatonin can recover aberrant antioxidant status induced by dexamethasone and the specific mechanism behind melatonin-dependent protection. A total of 150 healthy 40-wk-old Dawu Jinfeng laying hens with similar body weights and laying rates were randomly divided into three groups, with five replicates per group and 10 hens per replicate. The hens in the control group (NS) received intraperitoneal injections of normal saline for 30 d, the dexamethasone group (Dex+NS) received 20 mg/kg dose of dexamethasone for the first 15 d, followed by the 15 d of normal saline treatment. While in the melatonin group (Dex+Mel), dexamethasone (20 mg/kg dose) was injected intraperitoneally in the first 15 d, and melatonin (20 mg/kg/d) was injected in the last 15 d. The results showed that dexamethasone treatment significantly enhanced oxidative stress (P < 0.05), while melatonin not only inhibited the oxidative stress but also notably enhanced the antioxidant enzymes superoxide dismutase (SOD), catalase activity (CAT), glutathione peroxidase (GSH-Px), and antioxidant genes CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 3 (GPX3), and recombinant peroxiredoxin 3(PRDX3) expression (P < 0.05). Melatonin treatment also markedly reduced 8-hydroxy deoxyguanosine(8-OHdG), malondialdehyde (MDA), and reactive oxygen species (ROS) levels (P < 0.05) and apoptotic genes Caspase-3, Bim, and Bax in the follicle. In the Dex+Mel group, the Bcl-2 and SOD1 protein levels were also increased (P < 0.05). Melatonin inhibited the forkhead Box Protein O1(FOXO1) gene and its protein expression (P < 0.05). In general, this investigation revealed that melatonin might decrease oxidative stress and ROS by enhancing antioxidant enzymes and genes, activating the antiapoptotic genes, and inhibiting the FOXO1 pathway in laying hens.

Peroxiredoxin-4 and Dopamine D5 Receptor Interact to Reduce Oxidative Stress and Inflammation in the Kidney.

Aims: Reactive oxygen species are highly reactive molecules generated in different subcellular compartments. Both the dopamine D5 receptor (D5R) and endoplasmic reticulum (ER)-resident peroxiredoxin-4 (PRDX4) play protective roles against oxidative stress. This study is aimed at investigating the interaction between PRDX4 and D5R in regulating oxidative stress in the kidney. Results: Fenoldopam (FEN), a D1R and D5R agonist, increased PRDX4 protein expression, mainly in non-lipid rafts, in D5R-HEK 293 cells. FEN increased the co-immunoprecipitation of D5R and PRDX4 and their colocalization, particularly in the ER. The efficiency of Förster resonance energy transfer was increased with FEN treatment measured with fluorescence lifetime imaging microscopy. Silencing of PRDX4 increased hydrogen peroxide production, impaired the inhibitory effect of FEN on hydrogen peroxide production, and increased the production of interleukin-1β, tumor necrosis factor (TNF), and caspase-12 in renal cells. Furthermore, in Drd5-/- mice, which are in a state of oxidative stress, renal cortical PRDX4 was decreased whereas interleukin-1β, TNF, and caspase-12 were increased, relative to their normotensive wild-type Drd5+/+ littermates. Innovation: Our findings demonstrate a novel relationship between D5R and PRDX4 and the consequent effects of this relationship in attenuating hydrogen peroxide production in the ER and the production of proinflammatory cytokines. This study provides the potential for the development of biomarkers and new therapeutics for renal inflammatory disorders, including hypertension. Conclusion: PRDX4 interacts with D5R to decrease oxidative stress and inflammation in renal cells that may have the potential for translational significance. Antioxid. Redox Signal. 38, 1150-1166.

Bacillus subtilis YtpP and Thioredoxin A Are New Players in the Coenzyme-A-Mediated Defense Mechanism against Cellular Stress.

Coenzyme A (CoA) is an important cellular metabolite that is critical for metabolic processes and the regulation of gene expression. Recent discovery of the antioxidant function of CoA has highlighted its protective role that leads to the formation of a mixed disulfide bond with protein cysteines, which is termed protein CoAlation. To date, more than 2000 CoAlated bacterial and mammalian proteins have been identified in cellular responses to oxidative stress, with the majority being involved in metabolic pathways (60%). Studies have shown that protein CoAlation is a widespread post-translational modification which modulates the activity and conformation of the modified proteins. The induction of protein CoAlation by oxidative stress was found to be rapidly reversed after the removal of oxidizing agents from the medium of cultured cells. In this study, we developed an enzyme-linked immunosorbent assay (ELISA)-based deCoAlation assay to detect deCoAlation activity from Bacillus subtilis and Bacillus megaterium lysates. We then used a combination of ELISA-based assay and purification strategies to show that deCoAlation is an enzyme-driven mechanism. Using mass-spectrometry and deCoAlation assays, we identified B. subtilis YtpP (thioredoxin-like protein) and thioredoxin A (TrxA) as enzymes that can remove CoA from different substrates. With mutagenesis studies, we identified YtpP and TrxA catalytic cysteine residues and proposed a possible deCoAlation mechanism for CoAlated methionine sulfoxide reducatse A (MsrA) and peroxiredoxin 5 (PRDX5) proteins, which results in the release of both CoA and the reduced form of MsrA or PRDX5. Overall, this paper reveals the deCoAlation activity of YtpP and TrxA and opens doors to future studies on the CoA-mediated redox regulation of CoAlated proteins under various cellular stress conditions.

Proteome profiling of endogenous and potential S-nitrosylation in colorectal cancer.

As a common cancer with high incidence rate and mortality, colorectal cancer (CRC) is seriously threatening human health. S-nitrosylation (SNO) proteins mediated by nitric oxide (NO) has important implications in the genesis, progression, and apoptosis of CRC. It's worth noting that the SNO proteins also play an important role in the tumor endocrine and metabolic pathways of CRC. In this study, the protein extracts of human tissues and cell lines were treated by biotin switch technology and magnetic beads enrichment. The proteomic results of endogenous and potential SNO proteins were analyzed by mass spectrometry (MS). Through the comparison and analysis of MS results, Gene Ontology (GO) analysis, and literatures, some endogenous and potential SNO proteins were identified in CRC, which were closely related to the tumor endocrine and metabolic pathways, the apoptotic signaling pathways, protein maturation, and other biological processes of the proliferation and apoptosis of CRC cells. A total of 19 proteins containing potential or endogenous SNO sites were detected in both human cancer tissue and SW 480 cells. Through the cross validation of MS results, GO analysis, and literatures, several SNO proteins were identified frequently in CRC, such as the actin, cytoplasmic 1 (ACTB), peroxiredoxin-4 (PRDX4), protein S100A8 (S100A8), pyruvate kinase PKM (PKM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which were closely related to the tumor endocrine and metabolic pathways and the apoptotic signaling pathways of CRC. Different CRC cells and tissues contained potential and endogenous SNO modified proteins. In addition, some SNO proteins could participate in the proliferation, metastasis and apoptosis of CRC by regulating the tumor endocrine and metabolic pathways.

A TNFα/Miz1-positive feedback loop inhibits mitophagy in hepatocytes and propagates non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a chronic inflammatory disease that can further progress to cirrhosis and hepatocellular carcinoma. However, the key molecular mechanisms behind this process have not been clarified. We analyzed human NASH and normal liver tissue samples by RNA-sequencing and liquid chromatography-mass spectrometry, identifying hepatocyte cytosolic protein Myc-interacting zinc-finger protein 1 (Miz1) as a potential target in NASH progression. We established a Western diet+fructose-induced NASH model in hepatocyte-specific Miz1 knockout and adeno-associated virus type 8-overexpressing mice. Human NASH liver organoids were used to confirm the mechanism, and immunoprecipitation and mass spectrometry were used to detect proteins that could interact with Miz1. We demonstrate that Miz1 is reduced in hepatocytes in human NASH. Miz1 is shown to bind to peroxiredoxin 6 (PRDX6), retaining it in the cytosol, blocking its interaction with mitochondrial Parkin at Cys431, and inhibiting Parkin-mediated mitophagy. In NASH livers, loss of hepatocyte Miz1 results in PRDX6-mediated inhibition of mitophagy, increased dysfunctional mitochondria in hepatocytes, and production of proinflammatory cytokines, including TNFα, by hepatic macrophages. Crucially, the increased production of TNFα results in a further reduction in hepatocyte Miz1 by E3-ubiquitination. This produces a positive feedback loop of TNFα-mediated hepatocyte Miz1 degradation, resulting in PRDX6-mediated inhibition of hepatocyte mitophagy, with the accumulation of dysfunctional mitochondria in hepatocytes and increased macrophage TNFα production. Our study identified hepatocyte Miz1 as a suppressor of NASH progression via its role in mitophagy; we also identified a positive feedback loop by which TNFα production induces degradation of cytosolic Miz1, which inhibits mitophagy and thus leads to increased macrophage TNFα production. Interruption of this positive feedback loop could be a strategy to inhibit the progression of NASH. Non-alcoholic steatohepatitis (NASH) is a chronic inflammatory disease that can further develop into cirrhosis and hepatocellular carcinoma. However, the key molecular mechanism of this process has not been fully clarified. Herein, we identified a positive feedback loop of macrophage TNFα-mediated hepatocyte Miz1 degradation, resulting in PRDX6-mediated inhibition of hepatocyte mitophagy, aggravation of mitochondrial damage and increased macrophage TNFα production. Our findings not only provide mechanistic insight into NASH progression but also provide potential therapeutic targets for patients with NASH. Our human NASH liver organoid culture is therefore a useful platform for exploring treatment strategies for NASH development.

Abstract 4144: Characterization of immune cell phenotypes in patient derived malignant pleural effusion treated with thiostrepton

Abstract Tumor cells generate elevated levels of reactive oxygen species (ROS) and therefore exhibit increased expression, activity, and reliance on critical ROS scavenging pathways, including the mitochondrial peroxiredoxin 3 (PRX3) system. PRX3 is a critical reactive oxygen species (ROS) scavenging enzyme localized to the mitochondrial matrix. Our group has characterized the mechanism of action of the PRX3 inhibitor and pro-oxidant therapeutic thiostrepton (TS). TS covalently crosslinks the peroxidatic and resolving cysteines of PRX3, leading to increased levels of mitochondrial ROS, resulting in tumor cell death. We have characterized the cellular and chemical composition of malignant pleural effusion (MPE) from patients with advanced cancer and evaluated cellular responses to TS. TS targets PRX3 in both adherent tumor cells and in non-adherent immune cell populations in a dose-dependent manner. We further characterized MPE-infiltrated immune cells and cytokines released following TS-treatment. Together, these data provide the first evidence for the activity of TS in MPE and resulting changes to immune cell profiles. TS is the active pharmaceutical ingredient of RSO-021, a new covalent inhibitor of PRX3 currently being tested in the MITOPE phase 1/2 clinical trial in patients with malignant pleural effusion (MPE) due to advanced solid tumors or malignant mesothelioma (NCT05278975). Citation Format: Terri Messier, Roxana Del Rio Guerra, George Naumov, Brian Cunniff. Characterization of immune cell phenotypes in patient derived malignant pleural effusion treated with thiostrepton. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4144.

Synthesis of novel oxazol-5-one derivatives containing chiral trifluoromethyl and isoxazole moieties as potent antitumor agents and the mechanism investigation

In this study, a series of novel oxazol-5-one derivatives containing a chiral trifluoromethyl and isoxazole moiety were synthesized and evaluated for cytotoxic activities. Among them, 5t was the most effective compound against HepG2 liver cancer cells with an IC50 of 1.8 μM. 5t inhibited cell proliferation, migration, invasion, and induced cell cycle arrest and apoptosis in vitro. Nevertheless, the potential anti-hepatocellular carcinoma (HCC) target and mechanism of 5t were unclear. This work aimed to seek the molecular target of 5t against HCC and investigate its mechanism. Liquid chromatography tandem-mass spectrometry was used to identify peroxiredoxin 1(PRDX1) as a possible target of 5t. Cellular thermal shift assay, drug affinity responsive target stability, and molecular docking provided conclusive evidence that 5t targeted PRDX1 and inhibited its enzymatic activity. 5t augmented the level of reactive oxygen species (ROS) and led to ROS-dependent DNA damage, endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis in HepG2 cells. Silencing PRDX1 also resulted in ROS-mediated apoptosis in HepG2 cells. In vivo, 5t inhibited mouse tumor growth by increasing oxidative stress. Briefly, our studies revealed that compound 5t targeted PRDX1 through a ROS-dependent mechanism, highlighting the future development of compound 5t as a novel therapeutic drug for HCC.

EEF1A2 accelerates the protein translation of chemokine in rat myocardial cells induced by ischemia-reperfusion

How to reduce the damage caused by myocardial ischemia-reperfusion (IR) in a timely manner to save patients' lives is still a great clinical challenge. Although dexmedetomidine (DEX) has been reported to protect the myocardium, the regulatory mechanism of gene translation responding to IR injury and DEX protection is poorly understood. In this study, IR rat model with DEX and the antagonist yohimbine (YOH) pretreatment were established, and RNA sequencing was carried out to seek the important regulators in differential expressed genes. A series of cytokines and chemokine as well as eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) were induced by IR compared to control and compromised by DEX pretreatment compared to IR, then reversed by YOH. Immunoprecipitation was conducted to identify that peroxiredoxin 1 (PRDX1) interacted with EEF1A2 and contributed to the recruitment of EEF1A2 on mRNA molecules of cytokines and chemokine. Knockdown of PRDX1 could weaken the enhancive effect of EEF1A2 for gene translation of IL6, CXCL2 and CXCL11 under the IR condition, and indeed reduce cell apoptosis of cardiomyocytes. We also determined that the RNA motif “USCAGDCU” at 5′ UTR could be particularly recognized by PRDX1. Destruction of this motif at the 5’ UTR of IL6, CXCL2 and CXCL11 by CRISPR-CAS9 could result in the loss occupancies of EEF1A2 and PRDX1 on the mRNA of these three genes. Our observations showed the importance of PRDX1 in the reasonable control of cytokine and chemokine expression to prevent excessive inflammatory response to cell damage.

Effects of Date Palm Pollen Supplementations on The Expression of PRDX1 and PRDX6 Genes in Infertile Men: A Controlled Clinical Trial.

Accumulating evidences suggest that date palm pollen (DPP) induces antioxidant activity and improves semen parameters in male rats. However, there is a few scientific evidences in support of the DPP effects on human male fertility. Hence, the effect of oral consumption of DPP on sperm parameters and expression pattern of Peroxiredoxin- 1 (PRDX1) and Peroxiredoxin-6 (PRDX6) genes was evaluated in men with infertility. The current controlled clinical trial included 40 men with infertility (DPP group) and 10 normospermic fertile men as controls. The DPP group received gelatinous capsules of DPP (400 mg/kg) for 74 days. Semen sampling was done before and after treatment in the both groups. Semen analysis and 8-isoprostane concentration assessments were performed by computer-assisted sperm analysis and ELISA methods, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays were employed to explore expression of PRDX1 and PRDX6 genes. DPP consumption significantly improved semen volume (P=0.030), count (P<0.001) and morphology of sperm (P=0.023). Concentration of 8-isoprostane was significantly decreased after intervention in the DPP group (P<0.001). DPP consumption led to a significant elevation in the expression of PRDX1 and PRDX6 genes (P<0.001). Elevated gene expression of PRDX6 and PRDX1 was positively correlated with improved parameters of sperm including count, volume, motility and morphology. Taken together, DPP seems to promote sperm quality through a decrease in reactive oxygen species (ROS) by increasing expression of antioxidant genes. Further large-scale studies are required to challenge this hypothesis (registration number: IRCT2015021221014N2).

Functions of Peroxiredoxins and Their Roles in Autoimmune Diseases.

Significance: Oxidative stress is a common feature of autoimmune diseases. Low levels of reactive oxygen species (ROS) generation are important for various biological processes. Redox homeostasis can be disrupted when there is an imbalance between the production of ROS and the detoxification effect of antioxidants. Peroxiredoxins (PRDXs) are essential regulators of cellular redox signaling. Recent Advances: PRDXs are widely expressed antioxidant enzymes, and their physiological role is mainly to remove excess ROS in cells and reduce oxidative stress. Recent studies have shown that almost all PRDX subtypes are involved in the development of autoimmune diseases. Critical Issues: The pathogenesis of autoimmune diseases is complex, and effective treatments are lacking. Therefore, there is an urgent need to find new therapeutic targets. In this review, we discuss the functions of PRDXs and their pathophysiological roles in several autoimmune diseases. PRDXs may serve as potential targets for the treatment of autoimmune diseases. Future Directions: PRDXs are important in oxidative stress-mediated pathological situations. Future in-depth exploration of the mechanisms involved in regulating PRDXs in autoimmune diseases is needed to develop strategies targeting PRDXs for the treatment of autoimmune diseases. Antioxid. Redox Signal. 40, 329-344.

Correlation between PRDX2 and spermatogenesis under oxidative stress

BackgroundObesity is one of the world's diseases that endanger human health, causing systemic inflammation caused by excessive reactive oxygen damage. An increase in the proportion of obese people with reduced sperm motility has been reported. But the mechanism behind it remains unclear. Peroxiredoxin 2 (PRDX2) is a member of the peroxidase family that effectively removes hydrogen peroxide. This study is to clarify the expression of PRDX2 in the testes of obese mice and lay a foundation for further exploration of the regulatory and protective effects of PRDX2 on spermatogenesis. MethodA model of high-fat-induced obesity in animals was constructed, and the expression of PRDX2 in the testes of the two groups was detected by immunohistochemistry, western blotting, immunofluorescence and other techniques. Hydrogen peroxide (H2O2) and cholesterol were co-cultured in testicular support cells for 48 h to observe the expression of PRDX2. ResultPRDX2 expression was reduced in the testes of the obese group, and immunohistochemistry showed that it was mainly localized to supporting cells. H2O2 inhibits the expression of PRDX2 in Sertoli cells, and high cholesterol upregulates the expression of PRDX2 in Sertoli cells. ConclusionPRDX2 has some antioxidant properties against changes in the testicular environment caused by HFD. And under short-term oxidative stress to enhance its antioxidant capacity. PRDX2 may be involved in maintaining the oxidative balance of the spermatogenesis environment.

Expression and prognostic value of PRDX family in colon adenocarcinoma by integrating comprehensive analysis and in vitro and in vivo validation.

The peroxiredoxin family, a crucial regulator of redox reactions, is strongly associated with various tumorigenesis. However, the role of peroxiredoxin4 (PRDX4) in colon adenocarcinoma (COAD) remains poorly understood. Multicenter databases, including GEPIA, HPA, UALCAN, cBioPortal, cancerSEA, STRING, CCLE, and LinkedOmics, comprehensively analyzed transcriptional expression, prognostic value, genetic alterations, signaling pathways, and associated genes of the PRDXs in COAD patients. Colony formation, transwell, flow cytometry, sphere formation, and xenograft assays were performed to validate further in vitro and in vivo. Members of the PRDX family were differentially expressed in COAD, with each member showing varying degrees of genetic alterations. Intriguingly, only PRDX4 significantly correlated with COAD prognosis and stage. The single-cell sequencing suggested that PRDX4 is positively correlated with proliferation, apoptosis, and invasion, whereas negatively correlated with stemness. Moreover, PRDX4 involved in a series of critical biological processes, such as cell growth. Furthermore, in vivo and in vitro analyses indicated that knocking down PRDX4 inhibits the proliferation and invasion of HCT116 cells while promoting apoptosis and stemness. We identified PRDX4 expression as a novel potential prognostic marker in COAD.