Prdx6 Expression Research Articles

In vivo oxidative stress alters thiol redox status of peroxiredoxin 1 and 6 and impairs rat sperm quality.

Oxidative stress, the imbalance between the production of reactive oxygen species (ROS) and antioxidant activity is a major culprit of male infertility. Peroxiredoxins (PRDXs) are major antioxidant enzymes of mammalian spermatozoa and are thiol oxidized and inactivated by ROS in a dose-dependent manner. Their deficiency and/or inactivation have been associated with men infertility. The aim of this study was to elucidate the impact of oxidative stress, generated by the in vivo tert-butyl hydroperoxide (tert-BHP) treatment on rat epididymal spermatozoa during their maturation process. Adult Sprague-Dawley males were treated with μmoles tert-BHP/kg or saline (control) per day intraperitoneal for 15 days. Lipid peroxidation (2-thibarbituric acid reactive substances assay), total amount and thiol oxidation of PRDXs along with the total amount of superoxide dismutase (SOD), motility and DNA oxidation (8-hydroxy-deoxyguanosine) were determined in epididymal spermatozoa. Total amount of PRDXs and catalase and thiol oxidation of PRDXs were determined in caput and cauda epididymis. While animals were not affected by treatment, their epididymal spermatozoa have decreased motility, increased levels of DNA oxidation and lipid peroxidation along with increased PRDXs (and not SOD) amounts. Moreover, sperm PRDXs were highly thiol oxidized. There was a differential regulation in the expression of PRDX1 and PRDX6 in the epididymis that suggests a segment-specific role for PRDXs. In conclusion, PRDXs are increased in epididymal spermatozoa in an attempt to fight against the oxidative stress generated by tert-BHP in the epididymis. These findings highlight the role of PRDXs in the protection of sperm function and DNA integrity during epididymal maturation.

Proteomics Based Identification of Proteins with Deregulated Expression in B Cell Lymphomas.

Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL.

Delivery of a protein transduction domain-mediated Prdx6 protein ameliorates oxidative stress-induced injury in human and mouse neuronal cells.

Oxidative stress or reduced expression of naturally occurring antioxidants during aging has been identified as a major culprit in neuronal cell/tissue degeneration. Peroxiredoxin (Prdx) 6, a protective protein with GSH peroxidase and acidic calcium-independent phospholipase A2 activities, acts as a rheostat in regulating cellular physiology by clearing reactive oxygen species (ROS) and thereby optimizing gene regulation. We found that under stress, the neuronal cells displayed reduced expression of Prdx6 protein and mRNA with increased levels of ROS, and the cells subsequently underwent apoptosis. Using Prdx6 fused to TAT transduction domain, we showed evidence that Prdx6 was internalized in human brain cortical neuronal cells, HCN-2, and mouse hippocampal cells, HT22. The cells transduced with Prdx6 conferred resistance against the oxidative stress inducers paraquat, H2O2, and glutamate. Furthermore, Prdx6 delivery ameliorated damage to neuronal cells by optimizing ROS levels and overstimulation of NF-κB. Intriguingly, transduction of Prdx6 increased the expression of endogenous Prdx6, suggesting that protection against oxidative stress was mediated by both extrinsic and intrinsic Prdx6. The results demonstrate that Prdx6 expression is critical to protecting oxidative stress-evoked neuronal cell death. We propose that local or systemic application of Prdx6 can be an effective means of delaying/postponing neuronal degeneration.

Peroxiredoxin 6 attenuates ischemia‑ and hypoxia‑induced liver damage of brain‑dead donors.

Oxidative stress induced by ischemia and hypoxia in the livers of donors after brain death (DBD) is associated with poor organ function and low patient survival rates in those receiving DBD liver transplants. Peroxiredoxin 6 (Prdx6) can defend cells against liver damage induced by oxidative stress. The present study aimed to investigate the role of Prdx6 in ischemia- and hypoxia-induced liver damage in DBD livers. Liver tissue samples from ten DBD patients were collected. The control group constituted of six liver samples from patients with liver hemangioma that had accepted tumor excision surgery. Protein expression levels were determined by western blotting, cell viability was assessed using a CCK-8 assay, intracellular reactive oxygen species (ROS) levels were measured using a ROS assay kit, and phospholipase A2 (PLA2) activity was measured using a PLA2 assay kit. In DBD liver samples, Prdx6 expression was downregulated and the nuclear factor-κB (NF-κB) signaling pathway was activated. Furthermore, when human liver L02 cells were exposed to ischemia and hypoxia, the expression of Prdx6 was reduced, causing an increase in reactive oxygen species (ROS); this in turn activated NF-κB signaling and lowered cell viability (P<0.01). In agreement, overexpression of Prdx6 reduced ROS levels and improved cell viability. It was also demonstrated that inhibition of NF-κB increased Prdx6 expression, while inhibition of Prdx6 limited PLA2 activity, exacerbating ischemia- and hypoxia-induced cell damage. This data suggests that Prdx6 and its PLA2 activity have a protective role in DBD livers, the expression of which is regulated by NF-κB. Thus, Prdx6 may be a novel target to alleviate liver damage in DBD.

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre-Implantation Stage.

Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat-shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.

Hydrogen peroxide‐induced redox homeostasis regulation in endothelial cells

Human vasculature is shown to regulate hydrogen peroxide (H2O2) at various levels in normal as well as pathophysiological conditions. Recent research studies have put forward the involvement of H2O2 in regulating cytotoxic and vasoactive response to vasculature. However, being part of redox system, the effect of H2O2 on regulation of vascular and in particular endothelial redox system is not clearly understood. Thus, we investigated H2O2 –induced regulation in redox system during transient and consistent exposure to endothelial cells. Using systems biology approach, we studied the superoxide (O₂‾) production, mitochondrial membrane polarization and gene expression of several redox system enzymes in HUVEC following both transient and consistent H2O2 exposure for 72 hours. Our results, showed a significant down‐regulation in ASK1, CAT, TXNRD1 under continuous H2O2 exposure. But in transient H2O2 exposure followed by remodeling period showed significant up‐regulation in GPX1 and TXNRD1 whereas genes expression of ASK1, SOD1 and PRDX1 returned to homeostasis. In both treatment conditions, exposure to H₂O₂ significantly increased O₂‾ production, which remained significantly higher up to 48 hours and decreased back to control level. Similar trend was also observed in mitochondrial membrane polarization which transitioned from hyperpolarized to normal state over the period of 72 hours in both H₂O₂ treatment conditions. Results from our study indicate that HUVEC actively regulated functional and gene expression changes to reach similar extent of redox homeostasis under both H₂O₂ treatments. These results further the understanding on how endothelial cells endure varying levels of H₂O₂ in pathological conditions.

Evaluation of Peroxiredoxins (PRDX1, PRDX2 and PRDX6) Expression in Patients with Chronic Myeloid Leukemia (CML) Treated with Imatinib in First Line

Introduction: Satisfactory response is present for the majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP) treated with tyrosine kinase inhibitors (ITK) . However, some pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The oxidative stress modulation is tightly related with the physiopathology of various hematologic diseases and can cause cell death, apoptosis and necrosis. Peroxiredoxins (Prdx) are a family of multifunctional antioxidant thioredoxin-dependent peroxidases that protect cells against oxidative stress and modulate signaling cell proliferation pathways and may influence the metabolism of ITKs.The aim of this study was to analyze PRDX1, PRDX2 and PRDX6 levels of CML pts and correlate with cytogenetics and molecular responses.Methods: PRDX1, PRDX2 and PRDX6 expression was evaluated in 20 blood donors, 18 newly diagnosed CML pts and 22 previously treated pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis or during treatment and RNA samples were submitted to the synthesis of complementary DNA (cDNA) using the kit RevertAid™ HMinus First Strand cDNA Synthesis Kit (Fermentas, Life Sciences). For cDNA synthesis, 3 ug of RNA was used and peroxiredoxins expression was evaluated by real-time PCR with Syber Green (Applied Biosystems) and endogenous (β-Actina and GAPDH) controls. The results were analyzed using 2-ΔΔCT. Statistical analysis were made by using Mann Withney’s T test. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RQ-PCR. Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1% (IS).Results: 40 CML pts, 55% male, median age of 53 years (23-84) were evaluated, 60% in chronic phase (CP), 30% in accelerated phase (AP) and 10% in blast crisis (BC). The mean of PRDX transcript levels in the total group was (PRDX1: 0.006 and 10.10 / PRDX2: 0.002 and 16.26 / PRDX6: 0.003 and 49.97) respectively (PRDX1: 1.2 / PRDX2: 0.9 / PRDX6: 15.36). The results showed that there are a significantly difference (p<0.05) in the PRDX gene expression between pts and blood donors. All PRDX expression was reduced in responsive patients, and increase expression in pts resistant to TKI. The median duration of imatinib treatment was 29 months (1-104) and 97% achieved complete hematological response, 75% complete cytogenetic response and 65% major or complete molecular response. The analysis showed that higher levels of PRDX were maybe correlated with a no reduction of BCR-ABL transcripts (p<0.05). As well as, there was may influence of the PRDX levels at diagnosis in the response at 24 months of treatment.Conclusion:Is known that that the increase of ROS in CML leads to an increase of DNA damage, triggering genomic instability and resulting in accumulation of mutations and chromosomal aberrations, and contribute to the mechanism of acquisition of resistance to TKI inhibitors. The decrease of Peroxiredoxins expression observed in the responsive group, could contribute to this process, since the detoxification of these species are compromising and the effects caused by oxidative stress are even more drastic, leading to mutations that could be followed by TKi resistance. The relation between Prdx and CML not yet been elucidated. DisclosuresNo relevant conflicts of interest to declare.

Peroxiredoxin 6 (Prdx6) Is Associated with Changes in Autophagy in Myeloid and Non-Myeloid Cells

Introduction: Prdx6, a member of a new family of peroxidases, is found in neutrophil cytosol associated with p67phox. This protein reduces H2O2 with a conserved cysteine (C47) and also expresses PLA2 activity. In human neutrophils, Prdx6 translocates to plasma membrane after activation and enhances Nox2 activity in a cell-free system of oxidase activity. In transgenic K562 cells and PLB-985 cells, suppression of Prdx6 by siRNA or shRNA decreases Nox2 activity. In these studies, we investigated the association of Prdx6with autophagy in PLB-985 and HEK-293 cells.Methods: PLB-985 and HEK-293 cells were cultured under standard conditions and were transfected with a plasmid producing stable expression of an shRNA targeting Prdx6. PLB-985 cells were matured with the addition of 1.3% DM50 for 4 days. DNA for wild type (WT) Prdx6 and mutants for the Prdx6 active site (C47S) and PLA2active sites (H26A, S32A, S140A) were cloned into pcDNA3.1 which also had silent mutations making all corresponding mRNAs resistant to shRNA targeting.Prdx6 knockdown and vector control cells had stable re-expression of the various proteins when cultured with appropriate antibiotics. Superoxide anion (O2-) was determined by SOD inhibitable chemilluminescence with Digenes (National Diagnosistics). Proteins from lysates of various cell lines were separated by 10-12% SDS-PAGE and blotted onto nitrocellulose. Specific proteins were detected standard antibodies and a chemilluminescent technique with quantitation completed using Image J platform. Autophagy in PLB-985 cells with or without suppression of Prdx6 was completed by expression of LC3-II by Western blot after stimulation with serum opsonized zymsoan (5mg/mL). In HEK-293 cells, autophagy was determined by quantification of LC3-II (western blot) under based conditions with the addition of 10µM Chloraquine or starvation conditions with minimal media.Results: Stable suppression of Prdx6 by shRNA was achieved in both PLB-985 cells (down to 30%) and HEK cells (25%). Re-expression of WT and Prdx6 mutant proteins re-established normal levels. O2-production in response to lµM FMLP was decreased by 44% in KD PLB-985 cells compared to control and re-establishment of oxidase activity depended on PLA2, not Prdx activity.In PLB-985 cells, LC3-II levels increased (seven fold) over the first 30 minutes after exposure to opsonized zymosan, decreasing to baseline at 60 minutes. Prdx6 suppressed PLB-985 cells showed no change in LC3-II after opsonized zymosan. In HEK-293 cells lacking an active oxidase, suppression of Prdx6 resulted in increased autophagy under basal and starvation conditions. Furthermore, in HEK-293 cells, reintroduction of empty vector or Prdx6 mutated at the Prdx active site (C47S) did not suppress autophagy under basal conditions. However, re-introduction of WT and mutant Prdx6 at the PLA2 active site (D140A) did suppress autophagy demonstrating that this effect was associated with Prdx activity. Similar results were obtained under starvation conditions.Conclusions: In neutrophil like cells Prdx6 enhanced Nox2 activity through its PLA2 activity and suppression of Prdx6 decreased autophagy as measured by LC3-II accumulation after exposure to a phagocytic stimulus. In non-myeloid HEK-293 cells, suppression of Prdx6 was associated with increased autophagy under basal and starvation conditions, and this effect was dependent on Prdx activity.Prdx6, then, plays a critical role in neutrophil functions such as Nox2 activity and autophagy. Prdx6 may also be involved in autophagy in non-myeloid cells, and the mechanism for its involvement in autophagy may relate to different activities expressed by this protein. DisclosuresNo relevant conflicts of interest to declare.

Peroxiredoxins-1 and 2 Affect Proliferation and Survival of Lymphoma Cells

Peroxiredoxins (PRDXs) are abundant antioxidant enzymes. In mammals there are six PRDXs. Among them, four (PRDXs 1-4) are functional dimers. PRDXs are oxidoreductases that scavenge peroxides with reducing equivalents from thioredoxin-thioredoxin reductase system. PRDXs function as a defense against redox stress but their role far exceeds peroxides removal. They directly support the activation of several protein tyrosine phosphatases thus suppressing kinase-mediated signaling. PRDXs are usually highly expressed in tumor cells, but there are cases of their epigenetic downregulation. Depending on a particular experimental model and the stage of tumor development, PRDXs can either suppress or support tumor cell growth. The role of peroxiredoxins in B-cell derived tumors has not been studied so far.We have used two Burkitt’s lymphoma cell lines: Raji and Ramos. To isolate protein targets for a novel small molecule peptidomimetic inhibitor (Klossowski, 2012, J Med Chem) with antitumor activity we have used biotin affinity probe labelling approach and mass spectrometry. Covalent crosslinking of PRDXs dimers as well as the activation of MAPK signaling were investigated by Western blotting. We employed quantitative real time PCR and Western blotting to analyze the expression of PRDX1 and PRDX2 in a range of human leukemia and lymphoma cell lines at mRNA and protein levels, respectively. To study the role of PRDX1 and PRDX2 in lymphoma cells, we have employed the shRNA-mediated knockdown approach. To evaluate cell viability and proliferation rate, viable cells were counted in a hemocytometer or, alternatively, propidium iodide-negative cells were counted using BD Accuri flow cytometer. To study cell cycle we analyzed DNA content by propidium iodide staining followed by flow cytometry and evaluated levels of cell cycle-associated markers, e.g. p21 and cyclin E2 using Western blotting. To detect apoptosis we evaluated the levels of cleaved caspase-3, caspase-9, and PARP with Western blotting.We have found that in human Burkitt’s lymphoma cell lines, a novel peptidomimetic inhibitor with antitumor activity targets dimeric 2-cystein peroxiredoxins. In Raji and Ramos cells, the inhibitor covalently crosslinks PRDX dimers, triggers MAPK activation, redox stress, cell cycle arrest in G1 phase, and apoptosis. Next, we have analyzed the levels of PRDX1 and PRDX2 in a range of human leukemia and lymphoma cell lines, and found them upregulated, as compared to normal B cells. To further study the role of PRDX1 and PRDX2 in lymphoma cells, we have reduced the level of PRDX1 in Raji cells expressing significant levels of PRDX2 as well as in a subclone of Raji cells, which does not express PRDX2. The effects of PRDX1 downregulation strongly depend on the level of PRDX2 expression. In the cells which express PRDX2 a decrease in PRDX1 expression results in the suppression of cell proliferation and cell cycle arrest in G1 phase. In Raji cells, which do not express PRDX2, the downregulation of PRDX1 entirely inhibits cell growth and induces apoptosis.We show that in lymphoma cell lines dimeric peroxiredoxins are targets of a small molecule peptidomimetic inhibitor. Our findings indicate that PRDX1 and PRDX2 support lymphoma cell proliferation and survival. Moreover, they can substitute each other, at least to some extent, suggesting overlapping functions of these two cytoplasmic PRDXs in lymphoma cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aberrant sumoylation signaling evoked by reactive oxygen species impairs protective function of Prdx6 by destabilization and repression of its transcription.

Loss of the cytoprotective protein peroxiredoxin 6 (Prdx6) in cells that are aging or under oxidative stress is known to be linked to the pathobiology of many age-related diseases. However, the mechanism by which Prdx6 activity goes awry is largely unknown. Using Prdx6-deficient (Prdx6(-/-) ) cells as a model for aging or redox active cells, human/mouse lens epithelial cells (LECs) facing oxidative stress and aging lenses, we found a significant increase in the levels of small ubiquitin-like modifier (Sumo)1 conjugates. These cells displayed increased levels of Sumo1 and reduced the expression of Prdx6. Specifically, we observed that Prdx6 is a target for aberrant sumoylation signaling, and that Sumo1 modification reduces its cellular abundance. LECs overexpressing Sumo1 showed reduced expression and activity of Prdx6 and its transactivator specificity protein1 (Sp1), mRNA and protein with increased levels of reactive oxygen species; those cells were vulnerable to oxidative stress-induced cell death. A significant reduction in Prdx6, Sp1 protein and mRNA expression was observed in redox active Prdx6(-/-) cells and in aging lenses/LECs. The reduction was correlated with increased expression of Sumo1 and enrichment of the inactive form (dimeric) of Sumo-specific protease(Senp)1. Experiments with Sumo1-fused Prdx6 and Prdx6 promoter-linked to chloramphenicol acetyltransferase reporter gene constructs indicated that Sumo1 dysregulated Prdx6 activity by reducing its abundance and attenuating its transcription; in contrast, the delivery of Senp1 or Prdx6 reversed the process. The data show that reactive oxygen species-evoked aberrant sumoylation signaling affects Prdx6 activity by reducing Prdx6 abundance, as well as transcription. The findings of the present study may provide a foundation for a strategy to repair deleterious oxidative signaling generated by a reduced activity of Prdx6.

Peroxiredoxin 6 in skin carcinogenesis.

Non-melanoma skin cancer represents a serious and globally increasing health problem. Therefore, a detailed knowledge on the mechanisms of skin cancer formation is required to develop innovative, efficient and cost-effective strategies for skin cancer prevention and treatment. A major risk factor for cancer formation is oxidative stress, which results from excessive levels of reactive oxygen species (ROS). This can cause oxidative damage of lipids, proteins and DNA, which enhances the risk of mutations [1]. A major enzyme involved in the detoxification of ROS is peroxiredoxin 6 (Prdx6). As a “moonlighting” enzyme it bears two distinct catalytic activities: a cytosolic, selenium-independent peroxidase activity and a lysosomal, calcium-independent phospholipase A2 (PLA2) activity [2]. Acting as PLA2 in lipid catabolism at low pH, Prdx6 hydrolyses the sn-2 ester linkage of phospholipids to release a fatty acid and a lysophospholipid. As a peroxidase, Prdx6 can reduce H2O2, short chain organic hydroperoxides and phospholipid hydroperoxides at cytosolic, neutral pH [2]. Due to these functions it was of particular interest to determine the role of Prdx6 in skin carcinogenesis. For this purpose we made use of transgenic mice overexpressing Prdx6 in the epidermis as well as of Prdx6 knockout mice. Interestingly, overexpression of Prdx6 protected keratinocytes of the skin from oxidative damage in vitro and in vivo [3]. Vice versa, mice lacking Prdx6 were more susceptible to oxidative damage in the skin [4]. Based on these data, we analyzed the function of Prdx6 in skin tumorigenesis using genetic and chemically-induced skin cancer models [5]. In the genetic model we used transgenic mice, which express the oncogenes of the human papilloma virus type 8 (HPV8) in keratinocytes and which develop spontaneous skin papillomas within a few weeks. These mice were mated with either Prdx6 transgenic mice or Prdx6 knockout mice. Interestingly, loss of Prdx6 enhanced tumor multiplicity in the HPV8 model, while overexpression of Prdx6 significantly reduced the tumor number. Results obtained with Prdx6 transgenic mice subjected to a model where tumors are induced with a mutagen (DMBA) and a tumor promoter (TPA) confirmed the tumor-preventive effect of this enzyme. In both tumor models, Prdx6 activation had no influence on keratinocyte proliferation, apoptosis or the inflammatory response of the mice. However, following TPA treatment of wild-type mice, we detected an increase in the levels of several epidermal phospholipid hydroperoxides. This increase was much less pronounced in Prdx6 transgenic mice, but more pronounced in Prdx6 knockout mice. This finding suggests that Prdx6 reduces oxidative stress and thus protects from lipid peroxidation and most likely also from oxidative DNA damage. Figure 1 The dual role of Prdx6 in skin carcinogenesis On the other hand, we found an increase in tumor progression in Prdx6 overexpressing mice, most likely due to protection of tumor cells from inflammation-induced ROS damage and subsequent cell death. Therefore, our data revealed a surprising dual function of Prdx6 in skin tumorigenesis depending on the stage of tumor development: While it protects normal cells from oxidative damage and thus reduces mutagenesis and tumorigenesis, it is also beneficial for cancer cells, thus promoting their survival under stress conditions and subsequent tumor progression. These results are likely to be of human relevance, since we detected strong PRDX6 expression in normal skin and also in cutaneous SCCs [5]. Surprisingly, however, the expression levels were highly variable between different patients. Therefore, it will be important to determine if individuals with strong PRDX6 expression in normal skin are less susceptible to tumor development and vice versa, and if PRDX6 overexpression in skin tumors correlates with tumor aggressiveness. These results will provide the basis for the potential use of PRDX6 activators for skin tumor prevention, whereas PRDX6 inhibitors might have therapeutic potential for the treatment of malignant epithelial skin cancers. Consistent with this assumption, thiacremonone, a compound isolated from garlic, was shown to bind to the catalytic site Cys47 of Prdx6 and it reduced the tumor volume of lung carcinoma cells allografted to wild-type mice and in particular of cells allografted to Prdx6 transgenic mice [6]. This promising result points to the usefulness of PRDX6 inhibitors in cancer therapy. These data combined with our results obtained in the skin also demonstrate that Prdx6 activation/ overexpression and ROS-detoxifying strategies in general are not always beneficial. This is also reflected by the recently described acceleration of lung cancer progression in mice by antioxidants [7]. Therefore, activation of ROS-detoxifying enzymes and reduction of ROS levels should be carefully evaluated, in particular in patients with diagnosed cancer.

PRDX6 promotes lung tumor progression via its GPx and iPLA2 activities

PRDX6 is a bifunctional protein with both glutathione peroxidase (GPx) and calcium-independent phospholipase A2 (iPLA2) activities, which are concomitantly increased with the expression of PRDX6. PRDX6 promoted lung tumor growth in an in vivo allograft model. Herein, we further studied the vital roles in tumor progression of PRDX6 in lung cancer using nude mice bearing PRDX6-overexpressing lung cancer cells. Nude mice xenografted with PRDX6 showed increases in tumor size and weight compared to control mice. Histopathological and Western blotting examination demonstrated that expression of proliferating cell nuclear antigen, vascular endothelial growth factor, metalloproteinases 2 and 9, and cyclin-dependent kinases accompanied by increased iPLA2 and GPx activities were increased in the tumor tissues of PRDX6-overexpressing nude mice. In tumor tissues of PRDX6-overexpressing mice, the activation of mitogen-activated protein kinases and AP-1 DNA binding were also increased. The growth of lung cancer cell lines (A549 and NCI-H460) was enhanced by the increase in iPLA2 and GPx activities of PRDX6. In addition, mutant PRDX6 (C47S) attenuated PRDX6-mediated p38, ERK1/2, and AP-1 activities as well as its enzyme activities in the A549 and NCI-H460 lines. Furthermore, tumor growth and p38, ERK1/2, and AP-1 activities were also inhibited in nude mice bearing mutant PRDX6 (C47S) compared to PRDX6. Therefore, our findings indicate that PRDX6 promotes lung tumor growth via increased glutathione peroxidase and iPLA2 activities.

Effects of dioscin on apoptosis in pancreatic cancer MiaPaCa-2 cells and its mechanism

The aim of this study was to observe the effects of dioscin on apoptosis and on expression of PRDX1 in pancreatic cancer MiaPaCa-2 cells in vitro. MTT assay was used to detect the growth rate among the medication groups treated with different concentrations of dioscin. The apoptosis rate was determined by annexin V-fluorescein isothiocyanate/propidium iodide double staining and flow cytometry. Western blot analysis was used to assay the expression of PRDX1 and apoptotic proteins in the cells. Reactive oxygen species (ROS) formation was measured by 2'7'-dichlorofluorescein diacetate (DCFH-DA). Dioscin considerably inhibited the proliferation of MiaPaCa-2 cells in vitro. The inhibitory action was enhanced in a dose-dependent manner. The levels of intracellular ROS detected with DCFH-DA were highly increased after dioscin treatment. The flow cytometry analysis using annexin V-PI staining showed that compared with the apoptotic rate of control group [(3.5 ± 0.7)%], 2.5 µmol/L and 5 µmol/L dioscin induced apoptosis in (28.4 ± 0.9)% and (49.6 ± 2.7)% MiaPaCa-2 cells, and Western blot analysis showed that apoptotic proteins Bax and cleaved caspase-3 expressions were increased and antiapoptotic protein Bcl-2 expression was decreased. In addition, these effects could be blocked by antioxidant N-acetylcysteine (NAC) administration, and the apoptotic rates decreased to (10.8 ± 2.3)% and (18.8 ± 3.0)%, respectively. We further observed the decrease of PRDX1 expression after dioscin treatment. Moreover, after PRDX1 overexpression, dioscin treatment no longer induced high levels of ROS and apoptosis, and the apoptotic rate was decreased to (21.3 ± 5.9)%. Dioscin can down-regulate the PRDX1 expression, and then induces ROS-mediated apoptosis in cancer cells.

PRDX1 and PRDX6 are repressed in papillary thyroid carcinomas via BRAF V600E-dependent and -independent mechanisms

Many clinical studies highlight the dichotomous role of PRDXs in human cancers, where they can exhibit strong tumor-suppressive or tumor-promoting functions. Recent evidence suggests that lower expression of PRDXs correlates with cancer progression in colorectal cancer (CRC) or in esophageal squamous carcinoma. In the thyroid, increased levels of PRDX1 has been described in follicular adenomas and carcinomas, as well as in thyroiditis, while reduced levels of PRDX6 has been found in follicular adenomas. We studied the expression of PRDX1 and PRDX6, in a series of thyroid tissue samples, covering different thyroid diseases, including 13 papillary thyroid carcinomas (PTCs). Our results show that PRDX1 and PRDX6 are significantly reduced in all PTCs compared to normal tissues, to non-neoplastic tissue (MNG) or follicular neoplasms. This reduction is strongly evident in PTCs harboring BRAF V600E (31% of our cases). Using TPC-1 and BCPAP and FRTL-5 cell lines, we demonstrate for the first time that the presence of BRAF V600E is responsible of the hypoexpression of PRDX1 and PRDX6 both at mRNA and protein levels. Finally, independently of BRAF status, we observe an interesting correlation between the tumor size, the presence of lymph node metastasis and the lowest PRDX1 and PRDX6 levels. Therefore, these data indicate that PRDX1 and PRDX6 expression not only may play a key role in papillary thyroid carcinogenesis via a BRAF V600E-dependent mechanism, but their determination could be considered as potential tumor marker for indicating tumor progression in PTCs, independently of BRAF status.

Keratinocyte Growth Factor and Glucocorticoid Induction of Human Peroxiredoxin 6 Gene Expression Occur by Independent Mechanisms That Are Synergistic

Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage.

Peroxiredoxin 1 is a tumor-associated antigen in esophageal squamous cell carcinoma

Peroxiredoxin 1 (Prdx1) is an antioxidant and plays an important role in H2O2-mediated cell signaling. We previously found that the expression level of Prdx1 was elevated in esophagus squamous cell carcinoma (ESCC) tissue using a proteomics approach. Since overexpressed protein can induce an autoimmune response, to further examine whether serum from ESCC patients exhibits immunoreactivity against Prdx1, autoantibody responses to Prdx1 were evaluated by ELISA, western blotting and indirect immunofluorescence assay in sera from patients with ESCC and normal individuals. Immunohistochemical study with tissue array slides and western blot analysis with cancer cell lines were also performed to analyze the protein expression profiles of Prdx1 in ESCC tissues and cancer cell lines. The results demonstrated that the positive rate of autoantibody against Prdx1 in ESCC sera was 13.2% (9/68), whereas this rate was 0% (0/89) in normal individuals. Data also showed that expression of Prdx1 was significantly increased in ESCC tissues when compared to expression in paired adjacent normal tissues (P<0.05). The data indicate that Prdx1 may contribute to malignant transformation of the esophagus, and may be used as a biomarker in the immunodiagnosis of ESCC.

Dual Role of the Antioxidant Enzyme Peroxiredoxin 6 in Skin Carcinogenesis

The antioxidant enzyme peroxiredoxin 6 (Prdx6) is a key regulator of the cellular redox balance, particularly under stress conditions. We identified Prdx6 as an important player in different phases of skin carcinogenesis. Loss of Prdx6 in mice enhanced the susceptibility to skin tumorigenesis, whereas overexpression of Prdx6 in keratinocytes of transgenic mice had the opposite effect. The tumor-preventive effect of Prdx6, which was observed in a human papilloma virus 8-induced and a chemically induced tumor model, was not due to alterations in keratinocyte proliferation, apoptosis, or in the inflammatory response. Rather, endogenous and overexpressed Prdx6 reduced oxidative stress as reflected by the lower levels of oxidized phospholipids in the protumorigenic skin of Prdx6 transgenic mice and the higher levels in Prdx6-knockout mice than in control animals. In contrast to its beneficial effect in tumor prevention, overexpression of Prdx6 led to an acceleration of malignant progression of existing tumors, revealing a dual function of this enzyme in the pathogenesis of skin cancer. Finally, we found strong expression of PRDX6 in keratinocytes of normal human skin and in the tumor cells of squamous cell carcinomas, indicating a role of Prdx6 in human skin carcinogenesis. Taken together, our data point to the potential usefulness of Prdx6 activators or inhibitors for controlling different stages of skin carcinogenesis.

Differential expression of peroxiredoxin 6, annexin A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1 in testis of rat fetuses after maternal exposure to di-n-butyl phthalate

ObjectivesTo isolate and identify differentially expressed proteins in testis of rat fetuses after maternal exposure to di-n-butyl phthalate (DBP). MethodsPregnant rats were daily treated by gavage with 1ml/kg corn oil or 750mg/kg DBP from GD14 to GD18. We used the technique of proteomic analysis to compare the testis protein patterns obtained by two-dimensional gel electrophoresis from fetal rats of gestation day 19. ResultsWe found significant differences in protein spot intensities compared to control. Subsequently several of these variant protein spots were identified by mass spectrometry. Peroxiredoxin 6 (Prdx6), annexin A5 (AnxA5) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UchL1) were three of them, the differential expression of which were confirmed by western blotting. Further, immunohistochemical analyses of fetal rat testes sections were made to determine the cellular distribution of these proteins, consequently strong Prdx6 and AnxA5 stainings were found primarily in Leydig cells, while a weak UchL1 staining was found primarily in spermatogonium. ConclusionsThe present study had found several differentially regulated proteins and demonstrated the differential expression of Prdx6, AnxA5 and UchL1 in fetal rat testis after maternal exposure to DBP, when compared with controls. Combining the cellular location of these proteins and their function in other tissues, the results of this study indicated that oxidative injury and abnormal apoptotic regulation might participate the formation of testicular dysgenesis in fetuses of dams exposed to DBP.

4-Hydroxybenzyl Alcohol Confers Neuroprotection Through Up-Regulation of Antioxidant Protein Expression

An herb-derived phenolic compound, 4-hydroxybenzyl alcohol (4-HBA), exhibits beneficial effects in cerebral ischemic injury. However, the molecular mechanisms underlying this observation remain unclear. Here we used an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD/R) and an in vivo ischemic model of middle cerebral artery occlusion to investigate the relevant neuroprotective mechanisms. We demonstrated that 4-HBA reduced the neuronal injury, LDH release, and up-regulation of 8-hydroxydeoxyguanosine (8-OHdG) induced by OGD/R. Furthermore, 4-HBA reduced the cerebral infarct size and improved the behavioral parameters after cerebral ischemia. These neuroprotective effects may be conferred by the 4-HBA mediated upregulation of the transcription factor nuclear factor E2-related factor 2 (Nrf2), peroxiredoxin 6 (Prdx6) and protein disulfide isomerase (PDI) by the use of 4-HBA. Interestingly, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the increase in phosphorylation of Akt and abolished the neuroprotection associated with 4-HBA. Our results suggested that 4-HBA protects neurons against cerebral ischemic injury, and this neuroprotection may occur through upregulation of Nrf2, Prdx6, and PDI expression via the PI3K/Akt pathway.

Curcumin abates hypoxia-induced oxidative stress based-ER stress-mediated cell death in mouse hippocampal cells (HT22) by controlling Prdx6 and NF-κB regulation

Oxidative stress and endoplasmic reticulum (ER) stress are emerging as crucial events in the etiopathology of many neurodegenerative diseases. While the neuroprotective contributions of the dietary compound curcumin has been recognized, the molecular mechanisms underlying curcumin's neuroprotection under oxidative and ER stresses remains elusive. Herein, we show that curcumin protects HT22 from oxidative and ER stresses evoked by the hypoxia (1% O(2) or CoCl(2) treatment) by enhancing peroxiredoxin 6 (Prdx6) expression. Cells exposed to CoCl(2) displayed reduced expression of Prdx6 with higher reactive oxygen species (ROS) expression and activation of NF-κB with IκB phosphorylation. When NF-κB activity was blocked by using SN50, an inhibitor of NF-κB, or cells treated with curcumin, the repression of Prdx6 expression was restored, suggesting the involvement of NF-κB in modulating Prdx6 expression. These cells were enriched with an accumulation of ER stress proteins, C/EBP homologous protein (CHOP), GRP/78, and calreticulin, and had activated states of caspases 12, 9, and 3. Reinforced expression of Prdx6 in HT22 cells by curcumin reestablished survival signaling by reducing propagation of ROS and blunting ER stress signaling. Intriguingly, knockdown of Prdx6 by antisense revealed that loss of Prdx6 contributed to cell death by sustaining enhanced levels of ER stress-responsive proapoptotic proteins, which was due to elevated ROS production, suggesting that Prdx6 deficiency is a cause of initiation of ROS-mediated ER stress-induced apoptosis. We propose that using curcumin to reinforce the naturally occurring Prdx6 expression and attenuate ROS-based ER stress and NF-κB-mediated aberrant signaling improves cell survival and may provide an avenue to treat and/or postpone diseases associated with ROS or ER stress.