Impact of peroxiredoxin-6 expression on colon adenocarcinoma.

PURPOSE: Peroxiredoxins (Prdxs) represent a family of proteins that act as antioxidant enzymes and are involved in a variety of metabolic functions including mainly the intracellular hydrogen peroxide (H2O2) levels reduction. Especially, Prdx-6 protein encoded by the PRDX6 gene (1q25.1) regulates also phospholipid modifications and induces response to oxidative stress and injuries. Our aim was to investigate the expression of Prdx-6 in colon adenocarcinoma (CA). METHODS: A series of 30 formalin-fixed, paraffin-embedded primary CAs tissue sections were used and analyzed. Immunohistochemistry was performed using an anti-Prdx-6 antibody. Digital image analysis was also implemented for evaluating objectively the protein expression levels on the corresponding stained cells. RESULTS: Prdx-6 protein overexpression (increased immunostaining levels) was observed in 12/30 (40%) cases, whereas 18/30 (60%) CA tissues demonstrated low to moderate protein levels, respectively. Prdx-6 overall expression was strongly associated with the stage of the examined tumors (p=0.011), whereas other statistical significances were not assessed (inflammatory infiltration: p=0.364; carcinoma location: p=0.93; differentiation grade: p=0.517; tumor diameter: p=0.983; ulceration: p=0.622). CONCLUSIONS: Prdx-6 overexpression is observed in a significant subset of CAs correlating with aggressive biological behavior (advanced stage). Prdx-6 is a crucial enzyme for oxidative stress/injury endogenous cell response and should be an interesting agent as a biomarker and potential therapeutic target.

KLF9 regulates PRDX6 expression in hyperglycemia-aggravated bupivacaine neurotoxicity.

BACKGROUND: Neurotoxicity induced by local anesthetics (LAs) is potentially life threatening, especially for patients with underlying diseases like diabetes. The anesthetic bupivacaine (Bup) has been reported to induce neurotoxicity mediated by reactive oxygen species (ROS), which is aggravated by hyperglycemia. Krüppel-like factor 9 (KLF9), an axon growth-suppressing transcription factor, plays a key role in neuronal maturation and promotes oxidative stress. This study was designed to investigate whether and how KLF9 regulates ROS levels related to LA neurotoxicity under hyperglycemic conditions. METHODS: Klf9/GFP ShRNA (LV Sh-Klf9) was used to achieve stable Klf9 knockdown in the SH-SY5Y cell line. KLF9-deficient and normal cells were cultured under normal or high-glucose (HG) culture conditions and then exposed to Bup. Cell viability, intracellular and mitochondrial ROS, and mitochondrial membrane potential (ΔΨm) were detected to examine the role of KLF9. Thereafter, KLF9-deficient and normal cells were pretreated with small-interfering RNA targeting peroxiredoxin 6 (siRNA-Prdx6) to determine if PRDX6 was the target protein in HG-aggravated Bup neurotoxicity. RESULTS: The mRNA and protein levels of KLF9 were increased after Bup and hyperglycemia treatment. In addition, cell survival and mitochondrial function were significantly improved, and ROS production was decreased after Sh-Klf9 treatment compared with Sh-Ctrl. Furthermore, the expression of PRDX6 was suppressed by Bup in hyperglycemic cultures and was upregulated in the Sh-Klf9 group. Moreover, the protection provided by KLF9 deficiency for cell survival, the increase in ROS production in cells and mitochondria, and the disruption of mitochondrial function were abolished by Prdx6 knockdown. CONCLUSIONS: The results of this study demonstrated that hyperglycemia aggravated Bup neurotoxicity by upregulating KLF9 expression, which repressed the antioxidant PRDX6 and led to mitochondrial dysfunction, ROS burst, and cell death. Understanding this mechanism may, thus, offer valuable insights for the prevention and treatment of neurotoxicity induced by LAs, especially in diabetic patients.

Presenilin Mutation Suppresses Lung Tumorigenesis via Inhibition of Peroxiredoxin 6 Activity and Expression.

Some epidemiological studies suggest an inverse correlation between cancer incidence and Alzheimer's disease (AD). In this study, we demonstrated experimental evidences for this inverse relationship. In the co-expression network analysis using the microarray data and GEO profile of gene expression omnibus data analysis, we showed that the expression of peroxiredoxin 6 (PRDX6), a tumor promoting protein was significantly increased in human squamous lung cancer, but decreased in mutant presenilin 2 (PS2) containing AD patient. We also found in animal model that mutant PS2 transgenic mice displayed a reduced incidence of spontaneous and carcinogen-induced lung tumor development compared to wildtype transgenic mice. Agreed with network and GEO profile study, we also revealed that significantly reduced expression of PRDX6 and activity of iPLA2 in these animal models. PS2 mutations increased their interaction with PRDX6, thereby increasing iPLA2 cleavage via increased γ-secretase leading to loss of PRDX6 activity. However, knockdown or inhibition of γ-secretase abolished the inhibitory effect of mutant PSs. Moreover, PS2 mutant skin fibroblasts derived from patients with AD showed diminished iPLA2 activity by the elevated γ-secretase activity. Thus, the present data suggest that PS2 mutations suppress lung tumor development by inhibiting the iPLA2 activity of PRDX6 via a γ-secretase cleavage mechanism and may explain the inverse relationship between cancer and AD incidence.

Prdx6 Upregulation by Curcumin Attenuates Ischemic Oxidative Damage via SP1 in Rats after Stroke.

BACKGROUND: The role of Peroxiredoxin 6 (Prdx6) in brain ischemia remains unclear. Curcumin (Cur) treatment elicits neuroprotective effects against cerebral ischemic injury, and the associated mechanisms may involve Prdx6. In this study, we investigated whether Prdx6 and the transcription factor specific protein 1 (SP1) were involved in the antioxidant effect of Cur after stoke. METHODS: Focal cerebral ischemic injury was induced by transient middle cerebral artery occlusion for 2 hours in male Sprague-Dawley rats treated with or without Prdx6 siRNA. Expression of Prdx6 in the penumbra was assessed by Real-Time PCR (RT-PCR), Western blot analysis, and immunoflourescent staining. In addition, infarct volume, neurological deficit score, and oxidative stress were evaluated. Prdx6 levels were also determined in the presence and absence of SP1 antagonist mithramycin A (MTM-A). RESULTS: Cur treatment upregulated Prdx6 protein expression and the number of Prdx6-positive neuronal cells 24 hours after reperfusion. Cur treatment also attenuated oxidative stress and induced neuroprotective effects against ischemic damage, whereas the beneficial effects of Cur treatment were lost in animals treated with Prdx6-siRNA. Prdx6 upregulation by Cur treatment was abolished by SP1 antagonists MTM. CONCLUSIONS: Prdx6 upregulation by Cur treatment attenuates ischemic oxidative damage through SP1 induction in rats after stroke. This represents a novel mechanism of Cur-induced neuroprotection against cerebral ischemia.

The role of Prdx6 in the protection of cells of the crystalline lens from oxidative stress induced by UV exposure.

PURPOSE: The immediate aim of this study was to investigate alterations in peroxiredoxin (Prdx) 6 at posttranslational levels, and the levels of protein oxidation, lipid peroxidation, and reactive oxygen species (ROS) in lens epithelial cells (LECs) after exposure to severe oxidative stress, such as ultraviolet-B (UV-B). Our ultimate aim was to provide new information on antioxidant defenses in the lens and their regulation, thereby broadening existing knowledge of the role of Prdx6 in lens physiology and pathophysiology. METHODS: The expression of the hyperoxidized form of Prdx6 and oxidation of protein were analyzed by western blotting and the OxyBlot assay in human LECs (hLECs). ROS levels were quantified using DCFH-DA dye, and cell viability was quantified by the MTS and TUNEL assays. To evaluate the protective effect of Prdx6, we cultured lenses with or without the TAT transduction domain (TAT-HA-Prdx6) and observed (and photographed) the cultures at specified time-points after the exposure to UV-B for the development of opacity. RESULTS: Prdx6 in hLECs was hyperoxidized after exposure to high amounts of UV-B. UV-B treatment of hLECs increased the levels of cell death, protein oxidation, and ROS. hLECs exposed to UV-B showed higher levels of ROS, which could be reduced by the application of extrinsic TAT-HA-Prdx6, attenuating UV-B-induced lens opacity and apoptotic cell death. CONCLUSION: Excessive oxidative stress induces the hyperoxidation of Prdx6 and may reduce the ability of Prdx6 to protect LECs against ROS or stresses. Because extrinsic Prdx6 could attenuate UV-B-induced abuse, this molecule may have a potential in preventing cataractogenesis.

Overexpression of Peroxiredoxin 6 Protects Neoplastic Cells against Apoptosis in Canine Haemangiosarcoma.

Canine haemangiosarcoma (HSA), like human angiosarcoma, is an uncommon malignant vascular endothelial cell tumour associated with a poor prognosis. The peroxiredoxin (PRDX) family of peroxidases, which comprises six members in mammals (PRDX1-6), might contribute to cancer cell survival in the face of oxidative stress as these proteins exhibit frequent upregulation in cancer cells. In this study, we investigated the expression levels of PRDX6 in spontaneously arising primary canine HSAs by immunohistochemical analysis, identifying marked expression of this protein. Both PRDX6 mRNA and protein were overexpressed in HSA cell lines compared with normal canine endothelial cells, although some variation was observed between the different HSA cell lines. Small interfering RNA-induced downregulation of PRDX6 promoted apoptosis in the HSA cell lines. The observation that PRDX6 suppression increased the cytotoxicity of these cells suggests that PRDX6 might play an important cytoprotective role.

Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression.

Snake venom toxin (SVT) from Vipera lebetina turanica contains a mixture of different enzymes and proteins. Peroxiredoxin 6 (PRDX6) is known to be a stimulator of lung cancer cell growth. PRDX6 is a member of peroxidases, and has calcium-independent phospholipase A2 (iPLA2) activities. PRDX6 has an AP-1 binding site in its promoter region of the gene. Since AP-1 is implicated in tumor growth and PRDX6 expression, in the present study, we investigated whether SVT inhibits PRDX6, thereby preventing human lung cancer cell growth (A549 and NCI-H460) through inactivation of AP-1. A docking model study and pull down assay showed that SVT completely fits on the basic leucine zipper (bZIP) region of c-Fos of AP-1. SVT (0-10 µg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decreased cIAP and Bcl2 expression via inactivation of AP-1. In an xenograft in vivo model, SVT (0.5 mg/kg and 1 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression, but increased expression of proapoptotic proteins. These data indicate that SVT inhibits tumor growth via inhibition of PRDX6 activity through interaction with its transcription factor AP-1.

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.

Protein and miRNA profiling of radiation-induced skin injury in rats: the protective role of peroxiredoxin-6 against ionizing radiation.

Radiation-induced skin injury is a serious concern during radiotherapy. However, the molecular mechanism underlying the pathogenesis of radiation-induced skin injury has not been extensively reported. Most biological functions are performed and regulated by proteins and noncoding RNAs, including microRNAs (miRNAs). The interplay between mRNA and miRNA has been implicated in disease initiation and progression. Technical advances in genomics and proteomics have enabled the exploration of the etiology of diseases and have the potential to broaden our understanding of the molecular pathogenesis of radiation-induced skin injury. In this study, we compared the protein and miRNA expression in rat skin irradiated with a 45-Gy electron beam with expression from adjacent normal tissues. We found 24 preferentially expressed proteins and 12 dysregulated miRNAs in irradiated skin. By analyzing the protein and miRNA profiles using bioinformatics tools, we identified a possible interaction between miR-214 and peroxiredoxin-6 (PRDX-6). Next, we investigated the expression of PRDX-6 and the consequences of its dysregulation. PRDX-6 is suppressed by radiation-inducible miR-214 and is involved in the pathogenesis of radiation-induced skin injury. Overexpression of PRDX-6 conferred radioresistance on cells, decreased cell apoptosis, and preserved mitochondrial integrity after radiation exposure. In addition, in vivo transfection with PRDX-6 reduced radiation-induced reactive oxygen species and the malondialdehyde concentration and ameliorated radiation-induced skin damage in rats. Our present findings illustrate the molecular changes during radiation-induced skin injury and the important role of PRDX-6 in ameliorating this damage in rats.

Keratinocyte growth factor and glucocorticoid induction of human peroxiredoxin 6 gene expression occur by independent mechanisms that are synergistic.

AIMS: 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. RESULTS: 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. INNOVATION: 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. CONCLUSION: 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.

The impact of antioxidant supplements and endurance exercise on genes of the carbohydrate and lipid metabolism in skeletal muscle of mice.

To ascertain whether reactive oxygen species (ROS) contribute to training-induced adaptation of skeletal muscle, we administered ROS-scavenging antioxidants (AOX; 140 mg/l of ascorbic acid, 12 mg/l of coenzyme Q10 and 1% N-acetyl-cysteine) via drinking water to 16 C57BL/6 mice. Sixteen other mice received unadulterated tap water (CON). One cohort of both groups (CON(EXE) and AOX(EXE) ) was subjected to treadmill exercise for 4 weeks (16-26 m/min, incline of 5°-10°). The other two cohorts (CON(SED) and AOX(SED) ) remained sedentary. In skeletal muscles of the AOX(EXE) mice, GSSG and the expression levels of SOD-1 and PRDX-6 were significantly lower than those in the CON(EXE) mice after training, suggesting disturbance of ROS levels. The peak power related to the body weight and citrate synthase activity was not significantly influenced in mice receiving AOX. Supplementation with AOX significantly altered the mRNA levels of the exercise-sensitive genes HK-II, GLUT-4 and SREBF-1c and the regulator gene PGC-1alpha but not G6PDH, glycogenin, FABP-3, MCAD and CD36 in skeletal muscle. Although the administration of AOX during endurance exercise alters the expression of particular genes of the ROS metabolism, it does not influence peak power or generally shift the metabolism, but it modulates the expression of specific genes of the carbohydrate and lipid metabolism and PGC-1alpha within murine skeletal muscle.

Classical swine fever virus NS5A protein localizes to endoplasmic reticulum and induces oxidative stress in vascular endothelial cells.

Classical swine fever virus (CSFV) causes a severe disease of pigs that is characterized by hemorrhage, disseminated intravascular coagulation, and leucopenia. Until now, the role of the nonstructural protein 5A (NS5A) produced by CSFV in the pathogenesis of CSF is not well known. In this study, we investigated the function of CSFV NS5A by examining its role in the induction of oxidative stress and related intracellular events. Stable swine umbilical vein endothelial cell lines expressing CSFV NS5A were established and showed that CSFV NS5A is localized to endoplasmic reticulum and induces oxidative stress associated with enhanced reactive oxygen species production. The expression of NS5A protein exerts different effects on the three major antioxidants. Particularly, it exhibits a significant increase in transcriptional activities of antioxidant proteins thioredoxin and peroxiredoxin-6, but accompanied by a concomitant decrease of antioxidant protein heme oxygenase-1. Further studies showed that cyclooxygenase-2, a pro-inflammatory protein related to oxidative stress, is up-regulated while anti-inflammatory protein peroxisome proliferator-activated receptor-γ, an important mediator in vascular functional regulation, is down-regulated in CSFV NS5A expressing cells. This study suggested that CSFV NS5A plays important roles in the induction of oxidative stress and inflammatory response in vascular endothelial cells. These findings provide novel information on the function of the poorly characterized CSFV NS5A and provide an insight into the mechanism by which CSFV NS5A can alter intracellular events associated with the viral infection.

Differential expression and function of peroxiredoxin 1 and peroxiredoxin 6 in cancerous MCF-7 and noncancerous MCF-10A breast epithelial cells.

Peroxiredoxins are thiol-specific antioxidant proteins whose expression is elevated in several cancers. We compared the expression and function of Prdx1 and Prdx6 between the MCF-7 mammary adenocarcinoma cell line and the noncancerous MCF-10A cell line. We found elevated Prdx1 expression in MCF-7 cells and comparable expression of Prdx6. Suppression of Prdx1 and/or Prdx6 resulted in a modest increase in peroxide-induced cytotoxicity of MCF-7 cells, and a dramatic increase in MCF-10A cytotoxicity with and without hydrogen peroxide treatment. Our data confirm a cytoprotective role for peroxiredoxins and suggest a synergistic role for Prdx1 and Prdx6 in MCF-10A cells.

Expression of peroxiredoxin 1, 2, and 6 in the rat brain during perinatal development and in response to dexamethasone.

Peroxiredoxins (Prdx), a family of antioxidant proteins, have important defensive roles in the degenerative brain diseases and neuronal cell death in adult subjects. However, little is known in the neonatal brain. Here, we studied the developmental expression of Prdxs and their response to dexamethasone in the perinatal rat brain. Prdx 1 expression increased during late gestations and peaked at postnatal-day 1, when its expression gradually decreased. Prdx 2 expression remained largely unchanged. Prdx 6 expression continually increased as growing. Using immunohistochemistry, each Prdx showed a strong expression in the cerebral cortex and hippocampus. Prdx 1 was strongly expressed in the corpus callosum. The dexamethasone injection increased the expression of Prdx 6. In conclusion, we reveal for the first time that Prdx 1, 2 and 6 are found in abundance in the perinatal rat brain and are differentially expressed during development. The expression of Prdx 6 was affected by dexamethasone treatment.

Age-related cataracts and Prdx6: correlation between severity of lens opacity, age and the level of Prdx 6 expression.

BACKGROUND: Peroxiredoxin 6 (Prdx6), a new family of antioxidants, regulates gene expression and function by controlling reactive oxygen species, delays hereditary cataracts in rats and protects epithelial cells in the lens against oxidative stresses. AIM: To investigate the correlation between Prdx6 expression, age and the severity of lens opacity at the time of cataract surgery. METHODS: 88 cataractous eyes were examined at Fukui University Hospital, Fukui, Japan, between March 2007 and October 2007. The patient age at the time of surgery, and the subtype and severity of cataract as classified according to the modified version of the Lens Opacities Classification System version III (LOCSIII) were recorded, as well as the expression level of Prdx6 mRNA in their lenses. RESULTS: The expression of Prdx6 was found to be significantly negatively associated with age at the time of cataract surgery (p<0.047). A significant correlation was also found between a higher nuclear or cortical cataract score and lower expression of Prdx6 in patients under 70 years old. CONCLUSION: These findings suggest that oxidative stress contributes to nuclear cataract formation and that a local decrease in Prdx6 in cataractous lenses may indicate the initiation of age-related cataract formation.

TAT-mediated peroxiredoxin 5 and 6 protein transduction protects against high-glucose-induced cytotoxicity in retinal pericytes.

AIMS: Hyperglycemia-induced oxidative stress is implicated in pericyte apoptosis seen in diabetic retinopathy. The six mammalian Peroxiredoxins (PRDXs) comprise a novel family of antioxidative proteins that negatively regulate oxidative stress-induced apoptosis by controlling reactive oxygen species (ROS) levels. MAIN METHODS: Sprague-Dawley rats were used to detect the retinal expressions of PRDXs1-6. Pig pericytes cultured in high-glucose medium were used to monitor the protective effect of PRDX5 and 6 against high-glucose-associated change. Recombinant PRDX5 and 6 proteins were linked to the Trans-Activating Transduction (TAT) domain from HIV-1 TAT protein for their efficient delivery into cells/tissues. KEY FINDINGS: We found higher expression of PRDX5 and 6 mRNAs and PRDX5 and 6 proteins in retina than the other Prdxs (Prdx1-4). Western blotting affirmed the intracellular presence of TAT-linked proteins and revealed the efficient transduction of TAT-HA-PRDX5 and 6 in these cells. Extrinsic supply of TAT-HA-PRDX5 and 6 proteins inhibited the oxidative stress-induced DNA damage after high-glucose exposure in pig pericytes. The cell survival and apoptosis assay revealed that extrinsic supply of TAT-HA-PRDX5 and 6 proteins was responsible for inhibiting hyperglycemia-induced pericyte apoptosis. SIGNIFICANCE: Results suggest that delivery of PRDX5 and 6 might protect hyperglycemia-induced pericyte loss to inhibit oxidative stress.

Overexpression of peroxiredoxin 6 does not prevent ethanol-mediated oxidative stress and may play a role in hepatic lipid accumulation.

Oxidative stress is implicated in the etiology of many diseases, including alcoholic liver disease (ALD). Peroxiredoxin 6 is a cytosolic peroxidase that has been demonstrated to protect various tissues, such as skin, lung, and cardiac muscle, against acute oxidative insults. Consequently, peroxiredoxin 6 was hypothesized to also protect the liver from oxidative stress generated during the process of chronic ethanol ingestion. To test this, wild-type peroxiredoxin 6 knockout mice (KO), and transgenic peroxiredoxin 6 overexpressing mice (TG) were fed an ethanol-containing diet. Various biomarkers of ALD were assessed, along with the effects of chronic ethanol consumption on the antioxidant defenses. After 9 weeks of ethanol consumption, all backgrounds exhibited elevations of plasma alanine aminotransferase activity, hepatosteatosis, CYP2E1 induction, and lipid peroxidation; however, hepatic triglyceride accumulation seemed to be exacerbated in ethanol-fed TG mice. Differences in antioxidant protein expression and activity in response to chronic ethanol consumption were also observed. Examples include significant inductions of catalase and glutathione transferase activity in ethanol-fed KO and TG mice, along with elevated levels of glutathione peroxidase activity. These alterations in antioxidant defenses could be attributed to either compensatory responses due to the genetic manipulations or ethanol-mediated responses. In conclusion, both ethanol-fed KO and ethanol-fed TG mice developed early stage ALD and peroxiredoxin 6 may play a role in ethanol-mediated hepatic lipid accumulation.

Quantitative mass spectrometry identifies drug targets in cancer stem cell-containing side population.

A multifaceted approach is presented as a general strategy to identify new drug targets in a breast cancer stem cell-containing side population. The approach we have utilized combines side population cell sorting and stable isotope labeling by amino acids in cell culture with mass spectrometry to compare and identify proteins with differential expression profiles between side population cells, know to be enriched in cancer stem cells, and nonside population cells, which are depleted in cancer stem cells, for two breast cancer cell lines, MCF7 and MDA-MB231. Almost 900 proteins were quantified, and several important proteins in cell cycle control and differentiation were found to be upregulated in the cancer stem cell-containing side population. Most interestingly, a splice isoform of pyruvate kinase M2 as well as peroxiredoxin 6 were found to be downregulated. The differential levels of three of these proteins, thymosin beta4 (TB4), proliferation-associated protein 2G4, and SIAH-interacting protein, were validated using Western blot. Furthermore, functional validation provided clear evidence that elevated TB4 expression contributes to drug resistance in the stem cell population. Small interfering RNA silencing of TB4 led to a loss of chemoresistance in two separate breast cancer populations. These proteins likely contribute to resistance in the cancer stem cell-containing side population, and their altered expression in a tumor causes clinical resistance to chemotherapy. The ability to perform quantitative mass spectrometry has enabled the identification of a series of proteins that could serve as future therapeutic targets.

Regulation of peroxiredoxins by nitric oxide in immunostimulated macrophages.

Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.

Differential expression of peroxiredoxin 6 in fetal rat testis following in utero exposure to di(n-butyl) phthalate.

OBJECTIVES: To isolate and identify differentially expressed proteins in fetal rat testis following in utero exposure to di(nbutyl) phthalate (DBP). METHODS: 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. RESULTS: We found significant differences in protein spot intensities. Subsequently several of these variant protein spots were identified by mass spectrometry. Peroxiredoxin 6 (Prdx6) was one of them. Prdx6, which expressed a higher level in DBP-treated fetal rat testes compared with normal ones, is a member of the peroxiredoxins family (Prdxs). Recently, Prdx6 had been shown to be important in protecting cells from oxidative injury. Further, immunohistochemical analyses of fetal rat testes sections were made to determine the cellular distribution of this protein, consequently a strong Prdx6 staining was found out primarily in Leydig cells. CONCLUSIONS: The present study had found several differentially regulated proteins and demonstrated the differential expression of Prdx6 in fetal rat testis following in utero exposure to DBP, when compared with controls. Combining the cellular location of Prdx6 and its function in other tissues, the results of this study could provide us with a possibility to interfere the reproductive toxicity of DBP for its specific antioxidant properties in testis tissues.