Upregulation of Peroxiredoxin 3 Protects Afg3l2-KO Cortical Neurons In Vitro from Oxidative Stress: A Paradigm for Neuronal Cell Survival under Neurodegenerative Conditions.

Several neurodegenerative disorders exhibit selective vulnerability, with subsets of neurons more affected than others, possibly because of the high expression of an altered gene or the presence of particular features that make them more susceptible to insults. On the other hand, resilient neurons may display the ability to develop antioxidant defenses, particularly in diseases of mitochondrial origin, where oxidative stress might contribute to the neurodegenerative process. In this work, we investigated the oxidative stress response of embryonic fibroblasts and cortical neurons obtained from Afg3l2-KO mice. AFG3L2 encodes a subunit of a protease complex that is expressed in mitochondria and acts as both quality control and regulatory enzyme affecting respiration and mitochondrial dynamics. When cells were subjected to an acute oxidative stress protocol, the survival of AFG3L2-KO MEFs was not significantly influenced and was comparable to that of WT; however, the basal level of the antioxidant molecule glutathione was higher. Indeed, glutathione depletion strongly affected the viability of KO, but not of WT MEF, thereby indicating that oxidative stress is more elevated in KO MEF even though well controlled by glutathione. On the other hand, when cortical KO neurons were put in culture, they immediately appeared more vulnerable than WT to the acute oxidative stress condition, but after few days in vitro, the situation was reversed with KO neurons being more resistant than WT to acute stress. This compensatory, protective competence was not due to the upregulation of glutathione, rather of two mitochondrial antioxidant proteins: superoxide dismutase 2 and, at an even higher level, peroxiredoxin 3. This body of evidence sheds light on the capability of neurons to activate neuroprotective pathways and points the attention to peroxiredoxin 3, an antioxidant enzyme that might be critical for neuronal survival also in other disorders affecting mitochondria.

Peroxiredoxin-3 attenuates traumatic neuronal injury through preservation of mitochondrial function.

Peroxiredoxins (PRDXs) are a highly conserved family of thiol peroxidases that scavenge peroxides in cells. PRDX3 is one member of PRDXs localized in the mitochondria, and has been shown to be involved in antioxidant defense and redox signaling. In this study, we investigated the role of PRDX3 in neuronal trauma using a traumatic neuronal injury (TNI) model in primary cultured cortical neurons. We found that TNI significantly decreased the expression of PRDX3 at both mRNA and protein levels. Overexpression of PRDX3 by lentivirus (LV-PRDX3) transfection attenuated lactate dehydrogenase (LDH) release and neuronal apoptosis after TNI. The results of immunostaining showed that LV-PRDX3 transfection markedly reduced TNI-induced intracellular ROS production, protein radical formation and lipid peroxidation. In addition, overexpression of PRDX3 preserved mitochondrial membrane potential (MMP) levels and ATP generation, and inhibited mitochondrial cytochrome c release in TNI-injured neurons. The results of polymerase chain reaction (PCR) showed that PRDX3 overexpression also increased mitochondrial DNA (mtDNA) content and upregulated the expression of mitochondrial biogenesis-related factors. Taken together, our data demonstrate that PRDX3 protects against TNI insult by preserving mitochondrial function and mitochondrial biogenesis, and may have potential therapeutic value for traumatic brain injury (TBI).

Downregulation of peroxiredoxin-3 by hydrophobic bile acid induces mitochondrial dysfunction and cellular senescence in human trophoblasts.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder characterised by raised bile acids in foetal-maternal circulation, which threatens perinatal health. During the progression of ICP, the effect of oxidative stress is underscored. Peroxiredoxin-3 (PRDX3) is a mitochondrial antioxidant enzyme that is crucial to balance intracellular oxidative stress. However, the role of PRDX3 in placental trophoblast cells under ICP is not fully understood. We demonstrated that the level of PRDX3 was downregulated in ICP placentas as well as bile acids-treated trophoblast cells and villous explant in vitro. Toxic levels of bile acids and PRDX3 knockdown induced oxidative stress and mitochondrial dysfunction in trophoblast cells. Moreover, silencing of PRDX3 in trophoblast cell line HTR8/SVneo induced growth arrest and cellular senescence via activation of p38-mitogen-activated protein kinase (MAPK) and induction of p21WAF1/CIP and p16INK4A. Additionally, enhanced cellular senescence, determined by senescence-associated beta-galactosidase staining, was obviously attenuated by p38-MAPK inhibitor SB203580. Our data determined that exposure to bile acid decreased PRDX3 level in human trophoblasts. PRDX3 protected trophoblast cells against mitochondrial dysfunction and cellular senescence induced by oxidative stress. Our results suggest that decreased PRDX3 by excessive bile acids in trophoblasts plays a critical role in the pathogenesis and progression of ICP.

Accelerated hepatocellular carcinoma development in CUL4B transgenic mice.

Cullin 4B (CUL4B) is a component of the Cullin 4B-Ring E3 ligase (CRL4B) complex that functions in proteolysis and in epigenetic regulation. CUL4B possesses tumor-promoting properties and is markedly upregulated in many types of human cancers. To determine the role of CUL4B in liver tumorigenesis, we generated transgenic mice that expressed human CUL4B in livers and other tissues and evaluated the development of spontaneous and chemically-induced hepatocellular carcinomas. We observed that CUL4B transgenic mice spontaneously developed liver tumors at a high incidence at old ages and exhibited enhanced DEN-induced hepatocarcinogenesis. There was a high proliferation rate in the livers of CUL4B transgenic mice that was accompanied by increased levels of Cdk1, Cdk4 and cyclin D1 and decreased level of p16. The transgenic mice also exhibited increased compensatory proliferation after DEN-induced liver injury, which was accompanied by activation of Akt, Erk, p38 and NF-κB. We also found that Prdx3 was downregulated and that DEN induced a higher level of reactive oxygen species in the livers of transgenic mice. Together, our results demonstrate a critical role of CUL4B in hepatocarcinogenesis in mice.

HSP70, Peroxiredoxin-3 and -6 are upregulated during renal warm ischaemia in a donation after circulatory death model.

BACKGROUND: The use of donation after circulatory death (DCD) kidneys for transplantation is increasing. Subsequent delayed graft function is related to ischaemia/reperfusion injury (I/R), warm ischaemia (WI) being one of the main contributing factors. This proteomics study aimed to identify candidate biomarkers of WI. METHODS: Termination biopsies were obtained over 180min in 6 pigs. Proteins were subjected to differential in-gel electrophoresis (DIGE) and identified using LC MS/MS. RESULTS: Thirty nine protein spots showed significant changes in expression (ANOVA, p<0.05). Peroxiredoxin-3 and -6 (PRX3 and PRX6) were expressed with a fold change (FD) of +1.8 (p=0.03 and 0.02 respectively). A significant upregulation of Alpha-2-HS-glycoprotein (A2HSG, FD+1.9, p=0.047) and heat-shock protein 70-1b (HSP70-1b, FD+2.1 p=0.002) was recorded. CONCLUSIONS: The expression of PRX3, PRX6 and HSP70-1b during the first 30min of WI may be critical in measuring cellular responses. This is the first large animal model to describe the novel candidate biomarker, structural protein A2HSG. A2HSG upregulation during WI alone in this study is encouraging and further assessment in a DCD auto-transplant model is warranted. BIOLOGICAL SIGNIFICANCE: Warm ischaemia (WI) during donation after circulatory death (DCD) organ retrieval is associated with higher rates of post transplant organ dysfunction. The cellular and molecular mechanism of this paradigm is poorly reported. The work carried out in this large animal study has been performed to enable better understanding of protein expression during DCD WI at the time of retrieval. We have identified differential increased expression of PRX3, PRX6 and HSP70 during the first 30min of WI. Observation of this behaviour has not been reported before. Application of these results in a reperfusion model or autograft animal study would further help study of the named proteins as clinical biomarkers of WI. Alpha 2-HS Glycoprotein (A2HSG) species were also differentially expressed during the WI period. This remains a novel finding. Assessment of A2HSG is also recommended for further study in a reperfusion context. Previous reports of A2HSG have suggested an association in chronic kidney disease and diabetes, but no association with WI has previously been noted in either small or large animals.

Peroxiredoxin III protein expression is associated with platinum resistance in epithelial ovarian cancer.

We investigated the association between peroxiredoxin III (PRX III) and chemotherapy resistance in ovarian cancer. Patient's specimens were taken at the time of surgery. Determination of resistance is based on whether first diagnosis of relapse occurred within 6 months after the cessation of chemotherapy. PRX III expression was immunohistochemically determined in paraffin-embedded specimens from platinum-resistant (Pt-resistant) and platinum-sensitive (Pt-sensitive) patients. The Pt-resistant group had significantly higher PRX III protein compared to the Pt-sensitive group. The two groups showed no significant differences in pathological classification and age, although they differed significantly in tissue differentiation and stage. PRX III protein was significantly higher in the Pt-resistant serous carcinomas, in moderately and poorly differentiated, and in stage III and IV ovarian cancer tissues compared to the Pt-sensitive group. PRX III may be associated with drug resistance in ovarian cancer.

Mitochondrial-targeted nitroxides disrupt mitochondrial architecture and inhibit expression of peroxiredoxin 3 and FOXM1 in malignant mesothelioma cells.

Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently, we described an interplay between mitochondrial-derived oxidants and expression of FOXM1, a redox-responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here we have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito-carboxy-proxyl (MCP) and Mito-TEMPOL (MT) caused dose-dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP, and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox-responsive green fluorescent protein targeted to mitochondria (mito-roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi-1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM.

MicroRNA-23b downregulates peroxiredoxin III in human prostate cancer.

To investigate the mechanism by which peroxiredoxin III (PRDX3) is altered in human prostate cancer (PCa), we used microRNA (miRNA) target prediction program and miRNA microarray to predict and identify miR-23b as a candidate miRNA that targets PRDX3. We showed that miR-23b suppresses PRDX3 protein expression in human DU145 cells under normal and hypoxic conditions. Additionally, the clinical significance of miR-23b and PRDX3 expression in PCa patients was also confirmed. In conclusion, our data suggest that the effects of PRDX3 in PCa progression may be caused by the regulation function of miR-23b, and consequently, miR-23b may be involved in the response of PCa cells to hypoxia stress.

The most negatively charged low-density lipoprotein L5 induces stress pathways in vascular endothelial cells.

BACKGROUND/AIMS: L5, the most negatively charged species of low-density lipoprotein (LDL), has been implicated in atherogenesis by inducing apoptosis of endothelial cells (ECs) and inhibiting the differentiation of endothelial progenitor cells. In this study, we compared the effects of LDL charge on cellular stress pathways leading to atherogenesis. METHODS: We isolated L5 and L1 (the least negatively charged LDL) from the plasma of patients with familial hypercholesterolemia and used JC-1 staining to examine the effects of L5 and L1 on the mitochondrial membrane potential (DCm) in human umbilical vein ECs (HUVECs). Additionally, we characterized the gene expression profiles of 7 proteins involved in various types of cellular stress. RESULTS: The DCm was severely compromised in HUVECs treated with L5. Furthermore, compared with L1, L5 induced a decrease in mRNA and protein expression of the endoplasmic reticulum (ER) chaperone proteins ORP150, Grp94, and Grp58, mitochondrial proteins Prdx3 and ATP synthase, and an increase in the expression of the pro-inflammatory protein hnRNP C1/C2. CONCLUSIONS: Our work suggests that L5, but not L1, may promote the destruction of ECs that occurs during atherogenesis by causing mitochondrial dysfunction and modulating the expression of key proteins to promote inflammation, ER dysfunction, oxidative stress, and apoptosis.

Peroxiredoxins 3 and 4 are overexpressed in prostate cancer tissue and affect the proliferation of prostate cancer cells in vitro.

The present study aimed to investigate the proteome profiling of surgically treated prostate cancers. Hereto, 2D-DIGE and mass spectrometry were performed for protein identification, and data validation for peroxiredoxin 3 and 4 (PRDX3 and PRDX4) was accomplished by reverse phase protein arrays (RPPA). The Formal Concept Analysis (FCA) method was applied to assess whether the TMPRSS2-ERG gene fusion could influence the degree of overexpression of PRDX3 and PRDX4 in prostate cancer. Lastly, we performed an in vitro functional characterization of both PRDX3 and PRDX4 using the classical human prostate cancer cell lines DU145 and LNCaP. Reverse phase protein arrays verified that the overexpression of both PRDX3 and PRDX4 in tumor samples is negatively correlated with the presence of the TMPRSS2-ERG gene fusion. Functional characterization of PRDX3 and PRDX4 activity in PCa cell lines suggests a role of these members of the peroxiredoxin family in the pathophysiology of this tumor entity.

Identification of protein targets underlying dietary nitrate-induced protection against doxorubicin cardiotoxicity.

We recently demonstrated protective effect of chronic oral nitrate supplementation against cardiomyopathy caused by doxorubicin (DOX), a highly effective anticancer drug. The present study was designed to identify novel protein targets related to nitrate-induced cardioprotection. Adult male CF-1 mice received cardioprotective regimen of nitrate (1 g NaNO(3) per litre of drinking water) for 7 days before DOX injection (15 mg/kg, i.p.) and continued for 5 days after DOX treatment. Subsequently the heart samples were collected for proteomic analysis with two-dimensional differential in-gel electrophoresis with 3 CyDye labelling. Using 1.5 cut-off ratio, we identified 36 proteins that were up-regulated by DOX in which 32 were completely reversed by nitrate supplementation (89%). Among 19 proteins down-regulated by DOX, 9 were fully normalized by nitrate (47%). The protein spots were further identified with Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF)/TOF tandem mass spectrometry. Three mitochondrial antioxidant enzymes were altered by DOX, i.e. up-regulation of manganese superoxide dismutase and peroxiredoxin 3 (Prx3), and down-regulation of Prx5, which were reversed by nitrate. These results were further confirmed by Western blots. Nitrate supplementation also significantly improved animal survival rate from 80% in DOX alone group to 93% in Nitrate + DOX group 5 days after the DOX treatment. In conclusion, the proteomic analysis has identified novel protein targets underlying nitrate-induced cardioprotection. Up-regulation of Prx5 by nitrate may explain the observed enhancement of cardiac antioxidant defence by nitrate supplementation.