Overexpression Of Glutathione Peroxidase Research Articles

A reciprocal relationship between mitochondria and lipid peroxidation determines the chondrocyte intracellular redox environment

In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism.

Antioxidants for Early Treatment of Type 2 Diabetes in Rodents and Humans: Lost in Translation?

Reactive oxygen species (ROS) are formed by virtually all tissues. In normal concentrations they facilitate many physiologic activities, but in excess they cause oxidative stress and tissue damage. Local antioxidant enzyme synthesis in cells is regulated by the cytoplasmic KEAP-1/ Nrf2 complex, which is stimulated by ROS, to release Nrf2 for entry into the nucleus where it upregulates antioxidant gene expression. Major antioxidant enzymes include glutathione peroxidase (GPx), catalase (CAT), superoxide dismutases (SOD), hemoxygenases (HO), and peroxiredoxins (Prdx). Notably, the pancreatic islet beta cell does not express GPx or CAT, which puts it at greater risk for ROS damage caused by postprandial hyperglycemia. Experimentally, overexpression of GPx in beta cell lines and isolated islets as well as in vivo studies using genetic models of type 2 diabetes, have demonstrated enhanced protection against hyperglycemia and oxidative stress. Oral treatment of diabetic rodents with Ebselen, a GPx mimetic that is approved for human clinical usereproduced these findings. Prdxs detoxify hydrogen peroxide and reduce lipid peroxides. This suggests that pharmacologic development of more potent, beta cell specific antioxidants could be valuable as a treatment for oxidative stress due to post-prandial hyperglycemia in early type 2 diabetes in humans.

Differential effects of saturated and unsaturated free fatty acids on ferroptosis in rat β-cells

Elevated plasma concentrations of saturated free fatty acids (SFAs) are involved in pancreatic β-cell dysfunction and apoptosis, referred to as lipotoxicity. However, in contrast to apoptosis, the involvement of ferroptosis, as a distinct type of oxidative regulated cell death in β-cell lipotoxicity remains elusive. Therefore, the aim of this study was to determine the effects of various free fatty acids on ferroptosis induction in rat insulin-producing β-cells. Herein, rat insulin-producing β-cells underwent lipid peroxidation in the presence of long-chain SFAs and ω-6-polyunsaturated fatty acids (PUFAs), but only the latter induced ferroptosis. On the other hand, the ω-3-PUFA α-linolenate did not induce ferroptosis but sensitized insulin-producing β-cells to SFA-mediated lipid peroxidation. While the monounsaturated fatty acid oleate, overexpression of glutathione peroxidase 4, and the specific ferroptosis inhibitor ferrostatin-1 significantly abrogated lipid peroxidation, neither glutathione peroxidase 4 nor ferrostatin-1 affected palmitate-mediated toxicity. Site-specific expression of catalase in cytosol, mitochondria, and ER attenuated lipid peroxidation, indicating the contribution of metabolically generated H2O2 from all three subcellular compartments. These observations suggest that only ω-6-PUFAs reach the thresholds of lipid peroxidation required for ferroptosis, whereas SFAs favour apoptosis in β-cells. Hence, avoiding an excessive dietary intake of ω-6-PUFAs might be a crucial prerequisite for prevention of reactive oxygen species-mediated ferroptosis in insulin-producing cells.

In vitro assessment of synergistic effects in combinations of a temoporfin-based photodynamic therapy with glutathione peroxidase 1 inhibitors

BackgroundDue to an increased elimination of reactive oxygen species (ROS), in particular hydrogen peroxide (H2O2), overexpression of glutathione peroxidase 1 (GPX1) can lead to an attenuation of apoptosis and development of resistance in cancer cells, thereby promoting tumor cell survival. Consequently, GPX1 inhibitors have the potential to be used in cancer therapy as they support oxidative stress in cancer cells. Similarly, photodynamic therapy (PDT) induces oxidative stress in cancer cells by the formation of ROS upon illumination. Thus, both methods of treatment might act in synergy when used in combination. MethodsTo investigate this hypothesis, combinations of the known GPX1 inhibitors 9-chloro-6-ethyl-6H-[1,2,3,4,5]pentathiepino[6,7-b]indole (CEPI) or mercaptosuccinic acid (MSA) with PDT induced by the photosensitizer (PS) temoporfin (5,10,15,20-tetra(m-hydroxyphenyl)chlorin, mTHPC) were studied in vitro. This new combinatory approach was intended to accumulate ROS formed during PDT via blockage of GPX1-catalyzed H2O2 degradation, and thus to enhance PDT-induced phototoxicity. Five human cancer cell lines from tumor origins treatable with PDT were utilized to investigate ROS generation, apoptosis induction, and cell cycle distribution. ResultsSynergy was identified with both GPX1 inhibitors, but not in all cell lines. ROS levels were increased after combined treatment with mTHPC and CEPI, but not MSA, in some cell lines, indicating that oxidative stress and ROS accumulation were enhanced by CEPI. Surprisingly, enhanced apoptosis induction was also observed with MSA afterwards, suggesting that other pathways contributed to the initiation of apoptosis. Cell cycle analysis confirmed apoptosis induction via the detection of DNA fragmentation. ConclusionA combination of GPX1 inhibitors with mTHPC-PDT has the potential to generate synergistic effects and to increase overall phototoxicity, but the success of this combination approach was dependent on cancer type, and even antagonistic effects can occur.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

TIM-3 blockade leads to increased DNA sensing by cGAS–STING (by Kissiovd via Wikimedia Commons)TIM-3 blocking antibodies, which are showing promise in cancer clinical trials, can reverse T-cell exhaustion, but their effects on other immune cells are less clear. de Mingo Pulido et al. show that blocking TIM-3 promotes HMGB1-dependent endocytosis of extracellular DNA by classical dendritic cells. This activates the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway, leading to type I IFN and subsequently CXCL9 production. This pathway is required for the antitumor effects of TIM-3 blockade and paclitaxel in a mouse breast cancer model. The study provides new understanding of the mechanisms underlying TIM-3–targeted therapy.de Mingo Pulido Á, …, Ruffell B. Immunity 2021 Jun 8;54:1154–67.e7.TRMs in the lungs provide a niche that promotes tumor progression (from Nephron via Wikimedia Commons)Increased understanding of the diversity of macrophages in tumors is needed to effectively target the cells therapeutically. Using single-cell RNA sequencing, Casanova-Acebes et al. find four myeloid populations in human non–small cell lung carcinoma and in a mouse model of lung adenocarcinoma. The tissue-resident macrophage (TRM) population is reduced in mouse and human tumors compared with normal lung tissues. TRMs accumulate close to tumor cells early in tumor formation, promoting epithelial–mesenchymal transition, tumor-cell invasiveness, and regulatory T-cell responses. The data identify TRMs as potential targets for preventing and treating early-stage lung cancer.Casanova-Acebes M, …, Merad M. Nature 2021 Jun 16. DOI: 10.1038/s41586-021-03651-8.CD8+ TIL function is impaired by uptake of oxidized lipids via CD36 (from Victor A. Drover via Wikipedia)Lipid accumulation is a common metabolic characteristic of the tumor microenvironment (TME). Xu et al. show that oxidized lipids are prevalent in the TME in two mouse tumor models. Oxidized lipids are taken up by CD8+ tumor-infiltrating lymphocytes (TIL) via CD36, which is expressed at elevated levels on terminally exhausted PD-1+TIM-3+CD8+ TILs. Uptake of oxidized lipids induces lipid peroxidation and p38 MAPK activation, which reduces IFNγ and TNF production. Ablation of Cd36 and overexpression of glutathione peroxidase 4 (GPX4), which suppresses lipid peroxidation, restore CD8+ TIL function, highlighting the potential for targeting this pathway for cancer immunotherapy.Xu S, …, Kaech SM. Immunity 2021 Jul 13;54:1561–77.e7.ILC2s are a new potential therapeutic target in melanoma (by Julio C. Valencia, NCI Center for Cancer Research)Group 2 innate lymphoid cells (ILC2) in the skin can have both protumorigenic and antitumorigenic properties, but their effects in the melanoma tumor microenvironment (TME) are not well-known. Jacquelot et al. show that ILC2s infiltrate melanomas and are associated with a favorable prognosis in patients. In mouse models, ILC2-derived GM-CSF recruits eosinophils into the TME, boosting antitumor responses. However, infiltrating ILC2s also express PD-1, contributing to immunosuppression. Blockade of PD-1 in combination with IL33, an activating cytokine, unleashes ILC2/eosinophil antitumor immunity in the TME. The data highlight the potential to target ILC2s in melanoma to improve antitumor responses.Jacquelot N, …, Belz GT. Nat Immunol 2021 Jul 1;22:851–64.The liver microenvironment can switch on and off dormant tumor cells (by Firstfreddy via Wikimedia Commons)How disseminated tumor cells “turn on” after dormancy to seed metastases remains unclear. Correia et al. demonstrate a key role for NK cells in preventing breast tumor cells from exiting dormancy and forming metastases in the liver. Administration of IL15 maintains IFNγ in the microenvironment and is required for keeping tumor cells in a dormant state. A reduction in NK cells in the liver, as well as a concurrent accumulation of activated hepatic stellate cells (HSC), precedes tumor-cell exit from dormancy. This process is mediated by HSC-derived CXCL12, which induces NK-cell quiescence through interactions with CXCR4. The study provides insight into how tissue environments can permit or prevent progression of metastases.Correia AL, …, Bentires-Alj M. Nature 2021 Jun 2;594:566–71.Tregs in the TME can self-regulate via a feedback loop (photo by A. Benavides)How regulatory T cells (Treg) are regulated in the tumor microenvironment (TME) is not fully understood. Marangoni et al. find that conventional dendritic cells (cDC) in the TME can present tumor-associated antigens to pre-enriched polyclonal Tregs. These Tregs are maintained in the TME via transient T-cell receptor signals received during short-lived, unstable interactions with cDCs, a process maintained by a CTLA-4–dependent feedback loop that regulates Treg activation and proliferation via CD28. Blockade of CTLA-4 stabilizes Treg–cDC interactions, promoting Treg expansion. The data highlight that further intervention may be needed during immune checkpoint blockade of CTLA-4 so that immune-suppressive cell types are not promoted.Marangoni F, …, Mempel TR. Cell 2021 Jun 21. DOI: 10.1016/j.cell.2021.05.027.

Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8+ T cells in tumors

A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive Tcell dysfunction. Cd36-/- Tcells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into Tcells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector Tcell functions invitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs invivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ Tcell dysfunction and serves as a therapeutic avenue for immunotherapies.

SECISBP2 is a novel prognostic predictor that regulates selenoproteins in diffuse large B-cell lymphoma

AbstractThe overexpression of glutathione peroxidase 4 (GPX4; an enzyme that suppresses peroxidation of membrane phospholipids) is considered a poor prognostic predictor of diffuse large B-cell lymphoma (DLBCL). However, the mechanisms employed in GPX4 overexpression remain unknown. GPX4 is translated as a complete protein upon the binding of SECISBP2 to the selenocysteine insertion sequence (SECIS) on the 3′UTR of GPX4 mRNA. In this study, we investigated the expression of SECISBP2 and its subsequent regulation of GPX4 and TXNRD1 in DLBCL patients. Moreover, we determined the significance of the expression of these selenoproteins in vitro using MD901 and Raji cells. SECISBP2 was positive in 45.5% (75/165 cases) of DLBCL samples. The SECISBP2-positive group was associated with low overall survival (OS) as compared to the SECISBP2-negative group (P = 0.006). Similarly, the SECISBP2 and GPX4 or TXNRD1 double-positive groups (P < 0.001), as well as the SECISBP2, GPX4, and TXNRD1 triple-positive group correlated with poor OS (P = 0.001), suggesting that SECISBP2 may serve as an independent prognostic predictor for DLBCL (hazard ratio (HR): 2.693, P = 0.008). In addition, western blotting showed a decrease in GPX4 and TXNRD1 levels in SECISBP2-knockout (KO) MD901 and Raji cells. Oxidative stress increased the accumulation of reactive oxygen species in SECISBP2-KO cells (MD901; P < 0.001, Raji; P = 0.020), and reduced cell proliferation (MD901; P = 0.001, Raji; P = 0.030), suggesting that SECISBP2-KO suppressed resistance to oxidative stress. Doxorubicin treatment increased the rate of cell death in SECISBP2-KO cells (MD901; P < 0.001, Raji; P = 0.048). Removal of oxidative stress inhibited the altered cell death rate. Taken together, our results suggest that SECISBP2 may be a novel therapeutic target in DLBCL.SECISBP2 overexpression is an independent negative prognostic predictor in diffuse large B-cell lymphoma. Additionally, SECISBP2 positively correlates with selenoprotein expression. In vitro, SECISBP2 knockout increases intracellular reactive oxygen species accumulation via the downregulation of selenoproteins, inhibiting cell growth and promoting cell death after doxorubicin treatment. Therefore, SECISBP2 is a potential therapeutic target for malignant lymphoma.

Influence of non-surgical periodontal therapy on insulin resistance in chronic periodontitis subjects with prediabetes

Prediabetes (PRD) has been closely associated with chronic periodontitis (CP). Lately, studies have proposed the role of glutathione peroxidase (GPx), a ubiquitous antioxidant, in potentiating insulin resistance (IR), a significant factor in PRD. As overexpression of GPx is associated with CP as well as IR, the current study was undertaken to evaluate the influence of nonsurgical periodontal therapy (NSPT) on GPx levels and hence the IR in CP subjects with PRD. Forty-seven subjects were divided into two major groups: healthy controls (n = 11) and CP (n = 36), according to probing depth (PD) and clinical attachment loss (CAL). The CP patients were subdivided into CP only (n = 18) and CP with PRD (n = 18) based on baseline FBS levels. Serum and crevicular GPx and malondialdehyde (MDA) were measured in all the subjects using spectrophotometry. The participants underwent NSPTat baseline and 3 months. At 6 months, all the parameters were re-recorded. At 6 months after NSPT, there was significant improvement in periodontal health (p < 0.05) in all the three groups which was associated with reduced GPx and MDA levels. Besides, a significant reduction in IR was noted in both CP and CP with PRD groups (p < 0.05). NSPT may be a vital tool in reducing IR in CP subjects with PRD. This was clearly evident as there was progressive decline in IR with GPx levels along with improvement in other parameters, specifically in the PRD group.

Overexpression of glutathione peroxidase 1 predicts poor prognosis in oral squamous cell carcinoma.

Intracellular antioxidant enzymes are commonly upregulated in various cancer types and are associated with treatment outcomes. Because the relationship has rarely been examined in oral squamous cell carcinoma (OSCC), we aimed to evaluate the association between the levels of glutathione peroxidase (GPX)1, GPX4, and thioredoxin reductase (TrxR)1 expression and prognosis in patients with OSCC who underwent curative surgical resection. This study included 233 patients who underwent curative surgery for previously untreated OSCC between 2000 and 2012. Tumour GPX1, GPX4, and TrxR1 expression was evaluated by immunohistochemistry and was dichotomised to low and high values according to defined expression levels. The association between GPX1, GPX4, and TrxR1 expression and clinicopathological results was analysed. Univariate and multivariate analyses using the Cox proportional hazards model were conducted to assess the significance of differences in recurrence or survival outcomes between variables. High GPX1, GPX4, and TrxR1 expression was observed in 99 (42.5%), 133 (57.1%), and 46 (19.7%) patients, respectively. GPX1 overexpression was significantly correlated with nodal metastasis, advanced overall stage, depth of invasion of >10mm, high grade and perineural invasion (P<0.05). High GPX4 expression was also related to nodal metastasis, overall advanced stage and high grade (P<0.05). Univariate and multivariate analyses showed that increased GPX1 expression was significantly associated with poor disease-free, cancer-specific and overall survival (all P<0.05), while increased GPX4 or TrxR1 expression was not (all P>0.1). Tumour GPX1 expression is a useful biomarker predictive of recurrence and survival in OSCC patients.

Therapeutic Efficacy of a Subunit Vaccine in Controlling Chronic Trypanosoma cruzi Infection and Chagas Disease Is Enhanced by Glutathione Peroxidase Over-Expression.

Trypanosoma cruzi-induced oxidative and inflammatory responses are implicated in chagasic cardiomyopathy. In this study, we examined the therapeutic utility of a subunit vaccine against T. cruzi and determined if glutathione peroxidase (GPx1, antioxidant) protects the heart from chagasic pathogenesis. C57BL/6 mice (wild-type (WT) and GPx1 transgenic (GPxtg) were infected with T. cruzi and at 45 days post-infection (dpi), immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach. The plasma and tissue-sections were analyzed on 150 dpi for parasite burden, inflammatory and oxidative stress markers, inflammatory infiltrate and fibrosis. WT mice infected with T. cruzi had significantly more blood and tissue parasite burden compared with infected/GPxtg mice (n = 5-8, p<0.01). Therapeutic vaccination provided >15-fold reduction in blood and tissue parasites in both WT and GPxtg mice. The increase in plasma levels of myeloperoxidase (MPO, 24.7%) and nitrite (iNOS activity, 45%) was associated with myocardial increase in oxidant levels (3-4-fold) and non-responsive antioxidant status in chagasic/WT mice; and these responses were not controlled after vaccination (n = 5-7). The GPxtg mice were better equipped than the WT mice in controlling T. cruzi-induced inflammatory and oxidative stress markers. Extensive myocardial and skeletal tissue inflammation noted in chagasic/WT mice, was significantly more compared with chagasic/GPxtg mice (n = 4-6, p<0.05). Vaccination was equally effective in reducing the chronic inflammatory infiltrate in the heart and skeletal tissue of infected WT and GPxtg mice (n = 6, p<0.05). Hypertrophy (increased BNP and ANP mRNA) and fibrosis (increased collagen) of the heart were extensively present in chronically-infected WT and GPxtg mice and notably decreased after therapeutic vaccination. We conclude the therapeutic delivery of D/P vaccine was effective in arresting the chronic parasite persistence and chagasic pathology; and GPx1 over-expression provided additive benefits in reducing the parasite burden, inflammatory/oxidative stress and cardiac remodeling in Chagas disease.

Alteration in Downstream Hypoxia Gene Signaling in Neonatal Glutathione Peroxidase Overexpressing Mouse Brain after Hypoxia-Ischemia

We have previously shown that glutathione peroxidase (GPx) overexpressing mice (hGPx-tg) have reduced brain injury after neonatal hypoxia-ischemia (HI) as a consequence of reduced hydrogen peroxide accumulation. However, this protection is reversed with hypoxia preconditioning, raising the question of the roles of the genes regulated by hypoxia-inducible factor-1α (HIF-1α) and their transcription products, such as erythropoietin (EPO), in both the initial protection and subsequent reversal of protection. hGPx-tg and their wild-type (WT) littermates underwent the Vannucci procedure of HI brain injury at postnatal day 9 - left carotid artery ligation followed by exposure to 10% oxygen for 50 min. Brain cortices and hippocampi were subsequently collected 0.5, 4 and 24 h later for the determination of protein expression by Western blot for GPx, HIF-1α, HIF-2α, EPO, EPO receptor, ERK1/2, phospho-ERK1/2, spectrin 145/150 (as a marker of calpain-specific necrotic cell death), and spectrin 120 (as a marker of apoptotic cell death mediated via caspase-3). As expected, the GPx overexpressing mouse cortex had approximately 3 times the GPx expression as WT naïve. Also, GPx expression remained higher in the GPx overexpressing brain than WT at all time points after HI (0.5, 4, 24 h). HIF-1α was not significantly changed in hGPx-tg as a consequence of HI but decreased in the WT cortex 4 h after HI. HIF-2α decreased in the WT hippocampus after HI. EPO was higher in the GPx overexpressing cortex and hippocampus 30 min after HI compared to WT, but the EPO receptor was unchanged by HI. ERK1/2 phosphorylation increased in the hippocampus at 4 h after HI and in the cortex at 24 h after HI in both WT and hGPx-tg. Spectrin 145/150 was increased in the WT cortex at 4 and 24 h after HI, and spectrin 120 increased 24 h after HI, perhaps reflecting greater injury in the WT brain, especially at 24 h when brain injury is more evident. The effect of GPx overexpression does not appear to upregulate the HIF pathway, yet EPO was upregulated, perhaps via ERK. This might explain, in part, why cell death takes a necrotic or apoptotic path. This may also be an explanation for why the GPx overexpressing brain cannot be preconditioned. This information may prove valuable in the development of therapies for neonatal HI brain injury.

'OMICS' AND CHEMICAL APPROACHES USED TO MONITOR IRON-DEFICIENCY IN CITRUS ROOTSTOCKS

Two different ‘omics’ approaches were performed to a better comprehension of biological mechanisms involved in citrus iron (Fe) deficiency. Tips roots from Swingle citrumelo and Carrizo citrange (sensitive and tolerant rootstocks, respectively), growing in pots with control and chlorotic soil, were used for transcriptomic and proteomic analysis. CombiMatrix array was performed to isolate differential genes, among which glutathione peroxidase (GPX), SAUR gene and glutamate dehydrogenase (GDH) showed to be the most involved ones. They were switched on Swingle grown on calcareous conditions compared to Carrizo (in the same soil) and to the same stock in the control soil. The over-expression of GPX could be the effort of plants to neutralize the oxidative environment produced by stress. The involvement of auxin (and as consequence of SAUR gene) in the regulation of Fe deficiency responses is also well known. Both genes were considered in association to peroxidase, ferric chelate reductase activities, iron and chlorophyll content, to monitor the degree of suffering of rootstocks. Among differentially expressed proteins, identified by means of 2D-PAGE and RP-HPLC/nESI-MSMS, a strong down-regulation of cytosolic pyrophosphate-dependent phosphofructokinase I²-subunit and NADPH-isocitrate dehydrogenase could produce plant inability to sustain the energetic request of cell roots.

Ebselen Treatment Prevents Islet Apoptosis, Maintains Intranuclear Pdx-1 and MafA Levels, and Preserves β-Cell Mass and Function in ZDF Rats

We reported earlier that β-cell–specific overexpression of glutathione peroxidase (GPx)-1 significantly ameliorated hyperglycemia in diabetic db/db mice and prevented glucotoxicity-induced deterioration of β-cell mass and function. We have now ascertained whether early treatment of Zucker diabetic fatty (ZDF) rats with ebselen, an oral GPx mimetic, will prevent β-cell deterioration. No other antihyperglycemic treatment was given. Ebselen ameliorated fasting hyperglycemia, sustained nonfasting insulin levels, lowered nonfasting glucose levels, and lowered HbA1c levels with no effects on body weight. Ebselen doubled β-cell mass, prevented apoptosis, prevented expression of oxidative stress markers, and enhanced intranuclear localization of pancreatic and duodenal homeobox (Pdx)-1 and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), two critical insulin transcription factors. Minimal β-cell replication was observed in both groups. These findings indicate that prevention of oxidative stress is the mechanism whereby ebselen prevents apoptosis and preserves intranuclear Pdx-1 and MafA, which, in turn, is a likely explanation for the beneficial effects of ebselen on β-cell mass and function. Since ebselen is an oral antioxidant currently used in clinical trials, it is a novel therapeutic candidate to ameliorate fasting hyperglycemia and further deterioration of β-cell mass and function in humans undergoing the onset of type 2 diabetes.

Expression of glutathione peroxidase 2 is associated with not only early hepatocarcinogenesis but also late stage metastasis

Understanding of mechanisms of cancer progression is very important for reduction of cancer mortality. Of six rat hepatocellular carcinoma (HCC) cell lines, differing in their metastatic potential to the lung after inoculation into the tail vein of nude mice, the most metastatic featured particular overexpression of glutathione peroxidase 2 (GPX2). Therefore, we analyzed the influence of interference in highly metastatic L2 cells by siRNA transfection. Gpx2 siRNA significantly inhibited cell proliferation at 24 and 48h time points with induction of apoptosis but not cell cycle arrest. High expression of mutated p53 was detected in all HCC cell lines, with reduction in Gpx2 siRNA-transfected cells. Migration and invasion in vitro were also suppressed as compared to control siRNA-transfected cells and secretion of matrix metalloproteinase 9 was reduced. In vivo, the numbers and areas of metastatic nodules per area in the lungs were significantly reduced in the mice inoculated with Gpx2 siRNA-transfected cells as compared to control siRNA-transfected cells. In conclusion, expression of GPX2 is associated with cancer metastasis from rat HCCs both in vitro and in vivo. Together with immunohistochemical findings of elevated expression in rat and also human liver lesions, the results point to important roles in hepatocarcinogenesis.

Overexpression of glutathione peroxidase 4 prevents β-cell dysfunction induced by prolonged elevation of lipids in vivo

We have shown that oxidative stress is a mechanism of free fatty acid (FFA)-induced β-cell dysfunction. Unsaturated fatty acids in membranes, including plasma and mitochondrial membranes, are substrates for lipid peroxidation, and lipid peroxidation products are known to cause impaired insulin secretion. Therefore, we hypothesized that mice overexpressing glutathione peroxidase-4 (GPx4), an enzyme that specifically reduces lipid peroxides, are protected from fat-induced β-cell dysfunction. GPx4-overexpressing mice and their wild-type littermate controls were infused intravenously with saline or oleate for 48 h, after which reactive oxygen species (ROS) were imaged, using dihydrodichlorofluorescein diacetate in isolated islets, and β-cell function was assessed ex vivo in isolated islets and in vivo during hyperglycemic clamps. Forty-eight-hour FFA elevation in wild-type mice increased ROS and the lipid peroxidation product malondialdehyde and impaired β-cell function ex vivo in isolated islets and in vivo, as assessed by decreased disposition index. Also, islets of wild-type mice exposed to oleate for 48 h had increased ROS and lipid peroxides and decreased β-cell function. In contrast, GPx4-overexpressing mice showed no FFA-induced increase in ROS and lipid peroxidation and were protected from the FFA-induced impairment of β-cell function assessed in vitro, ex vivo and in vivo. These results implicate lipid peroxidation in FFA-induced β-cell dysfunction.

Inhibition of Telomerase Activity by Oleanane Triterpenoid CDDO-Me in Pancreatic Cancer Cells is ROS-Dependent

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a synthetic derivative of oleanolic acid, a triterpene, with apoptosis-inducing activity in a wide range of cancer cells. Induction of apoptosis by CDDO-Me is associated with the generation of reactive oxygen species (ROS) and inhibition of telomerase activity. In the present study, we investigated the role of ROS in inhibition of telomerase by CDDO-me. Treatment of MiaPaCa-2 and Panc-1 pancreatic cancer cell lines with CDDO-Me induced the production of hydrogen peroxide and superoxide anions and inhibited the telomerase activity. Pretreatment of cells with N-acetylcycsteine, a general purpose antioxidant or overexpression of glutathione peroxidase (GPx) or superoxide dismutase-1 (SOD-1) blocked the telomerase inhibitory activity of CDDO-Me. Furthermore, blocking ROS generation also prevented the inhibition of hTERT gene expression, hTERT protein production and expression of a number of hTERT–regulatory proteins by CDDO-Me (e.g., c-Myc, Sp1, NF-κB and p-Akt). Data also showed that Akt plays an important role in the activation of telomerase activity. Together, these data suggest that inhibition of telomerase activity by CDDO-Me is mediated through a ROS-dependent mechanism; however, more work is needed to fully understand the role of ROS in down-regulation of hTERT gene and hTERT-regulatory proteins by CDDO-Me.

Elevated Glutathione Peroxidase in Newly Diagnosed Hypertension: Its Relation to Insulin Resistance

Several studies have reported that insulin resistance and compensatory hyperinsulinemia often coexist with hypertension. Increased activity of glutathione peroxidase has been reported in patients with essential hypertension. Recently, overexpression of glutathione peroxidase has also been linked to the etiology of insulin resistance. The aim of this study was to determine the possible relation between the activity of glutathione peroxidase and insulin resistance status in essential hypertensive patients. A case–control study was performed on 35 hypertensive patients and 30 control subjects. The levels of insulin and insulin resistance as measured by the homeostasis model assessment insulin resistance index (HOMA-IR) and glutathione peroxidase were significantly increased in hypertensive patients compared with control subjects. In this study, a direct association was observed between glutathione peroxidase with HOMA-IR and insulin levels in essential hypertensive subjects. In conclusion, our results suggest that increased glutathione peroxidase can be one of the detrimental factors in contributing to insulin resistance in essential hypertensive subjects.

The role of gap junction communication and oxidative stress in the propagation of toxic effects among high-dose α-particle-irradiated human cells.

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.

Transgenic tomatoes showing higher glutathione peroxydase antioxidant activity are more resistant to an abiotic stress but more susceptible to biotic stresses

The function of selenium independent glutathione peroxidase (GPx) in response to biotic and abiotic stresses was investigated in transgenic tomato plants overexpressing an exogenous GPx and exhibiting a 50% increase in total GPx activity. GPx-overexpressing and control plants were challenged either by a mechanical stress or by infection with the biotrophic parasite Oidium neolycopersici or the necrotrophic parasite Botrytis cinerea. In mechanically stressed plants, internode growth was significantly less modified in GPx-overexpressing plants compared to controls. This stress resistant phenotype was not accompanied with any change in the global antioxidant response of the plants other than their increased GPx activity. Following infection by O. neolycopersici or by B. cinerea, lesion extension was increased in GPx-overexpressing plants compared with controls. These results showed that GPx overexpression provoked opposite effects in situations of biotic and abiotic challenges, suggesting a key role for this scavenger enzyme in controlling biotic and abiotic stress responses.

Immunohistochemical evidence for the over-expression of Glutathione peroxidase 3 in clear cell type ovarian adenocarcinoma

Glutathione peroxidase 3 (GPX3) is a member of glutathione peroxidase family, exerting one of the most important cellular defense mechanisms against stress signals, including oxidative damage. In this study, the expression of GPX3 mRNA and protein was analyzed for ovarian cancer tissues to test its applicability as a biomarker that can distinguish the four major histologic types of epithelial ovarian cancer. A public microarray dataset containing 99 ovarian cancer and 4 normal ovary samples was downloaded, and GPX3 mRNA expression was analyzed. The expression of GPX3 protein was measured by immunohistochemical staining in 40 epithelial ovarian cancer tissues, 10 for each of the serous, endometrioid, mucinous, and clear cell type. Histoscores were made from the immunohistostaining, and analysis of variance (ANOVA) was performed to quantitate the differences in protein level. Analysis of genomic dataset confirms a GPX3 overexpression in clear cell type ovarian adenocarcinoma compared with normal ovary and 3 other subtypes of epithelial ovarian cancer at mRNA level. GPX3 also shows the highest average antibody staining intensities in clear cell type ovarian adenocarcinomas over the other 3 types in immunostaining on tissue arrays. This is the first validation of GPX3 as a clear cell type-specific biomarker in ovarian cancer patients' tissues by immunostaining. GPX3 may serve as an important molecular marker for the diagnosis and molecular understanding of clear cell carcinoma of the ovary.