A study of glutathione peroxidase 4 function in human intestinal epithelial cells

PhD ThesisIntake of the micronutrient selenium, which is incorporated into selenoproteins in humans, has been implicated in affecting risk of colorectal cancer. A genetic variant in the gene encoding the selenoprotein glutathione peroxidase 4 (GPx4) has been reported to influence colorectal cancer risk. In this study the role of GPx4 was investigated in the Caco-2 intestinal cell line using RNA silencing. GPX4 expression was knocked–down by ~60% and an unbiased gene microarray analysis of the total Caco-2 cell transcriptome was carried out using Illumina HumanHT-12v3 beadchips. The data were validated by real-time PCR. Ingenuity Pathway analysis showed that the major canonical pathways affected by GPX4 knock-down were oxidative phosphorylation, ubiquinone biosynthesis and mitochondrial dysfunction and the top two toxicological lists were mitochondrial dysfunction and oxidative stress. Western blotting and real-time PCR confirmed that knock-down affected target genes encoding components of respiratory complexes I, IV and V as well as the protein apoptosis-inducing factor (AIF). GPX4 knock-down increased levels of mitochondrial reactive oxygen species and oxidised lipid, and decreased mitochondrial adenosine triphosphate (ATP) levels and mitochondrial membrane potential. Time course experiments showed changes in AIF expression preceded those in the respiratory complexes. GPX4 knock-down increased apoptosis and changed protein expression of Caspase-9, Bax and Bcl-2. Treatment of cells with the antioxidant mitoquinone prevented the effects of GPX4 knockdown on mitochondrial reactive oxygen species, oxidised lipid and mitochondrial membrane potential but not the effect on AIF. These data suggest that in intestinal epithelial cells GPx4, through effects on lipid peroxidation and AIF, plays a complex role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage and apoptosis.EPSR

Obesity in a model of haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy

Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice (GPx4(+/-)) and in samples of human myocardium. METHODS: Wild-type (WT) and GPx4(+/-) mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and cardiovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from a cohort of 103 patients undergoing elective heart surgery. RESULTS: Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4(+/-)) mice had marked carbonyl stress in these organs accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with obesity, and cardiac fibrosis more pronounced in obese GPx4(+/-) mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity and increased reactive oxygen species was also present in obese GPx4(+/-) but not WT hearts, along with up-regulation of pro-inflammatory and pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts, compared with age-matched non-diabetic patients. CONCLUSION: These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4 serves a critical role as an adaptive countermeasure

Glutathione and mitochondria

Glutathione (GSH) is the main non-protein thiol in cells whose functions are dependent on the redox-active thiol of its cysteine moiety that serves as a cofactor for a number of antioxidant and detoxifying enzymes. While synthesized exclusively in the cytosol from its constituent amino acids, GSH is distributed in different compartments, including mitochondria where its concentration in the matrix equals that of the cytosol. This feature and its negative charge at physiological pH imply the existence of specific carriers to import GSH from the cytosol to the mitochondrial matrix, where it plays a key role in defense against respiration-induced reactive oxygen species and in the detoxification of lipid hydroperoxides and electrophiles. Moreover, as mitochondria play a central strategic role in the activation and mode of cell death, mitochondrial GSH has been shown to critically regulate the level of sensitization to secondary hits that induce mitochondrial membrane permeabilization and release of proteins confined in the intermembrane space that once in the cytosol engage the molecular machinery of cell death. In this review, we summarize recent data on the regulation of mitochondrial GSH and its role in cell death and prevalent human diseases, such as cancer, fatty liver disease, and Alzheimer's disease. © 2014 Ribas, Garcia-Ruiz and Fernandez-Checa.Vicent Ribas is recipient of an Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) Post-doctoral Fellowship-BIOTRACK, supported by the European Community’s Seventh Framework Programme (EC FP7/2007-2013) under the grant agreement number 229673 and the Spanish Ministry of Economy and Competitiveness (MINECO) through the grant COFUND2013-40261. The work was supported by Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Fundació la Marató de TV3 and grants PI11/0325 (META) from the Instituto Salud Carlos III and grants, SAF2011-23031, and SAF2012-34831 from Plan Nacional de I+D, Spain; Fundación Mutua Madrileña and the center grant P50-AA-11999 (Research Center for Liver and Pancreatic Diseases, NIAAA/NIH)Peer Reviewe

Selenium Compounds, Apoptosis and Other Types of Cell Death: An Overview for Cancer Therapy

Selenium (Se) is an essential trace element involved in different physiological functions of the human body and plays a role in cancer prevention and treatment. Induction of apoptosis is considered an important cellular event that can account for the cancer preventive effects of Se. The mechanisms of Se-induced apoptosis are associated with the chemical forms of Se and their metabolism as well as the type of cancer studied. So, some selenocompounds, such as SeO<sub>2</sub> involve the activation of caspase-3 while sodium selenite induces apoptosis in the absence of the activation of caspases. Modulation of mitochondrial functions has been reported to play a key role in the regulation of apoptosis and also to be one of the targets of Se compounds. Other mechanisms for apoptosis induction are the modulation of glutathione and reactive oxygen species levels, which may function as intracellular messengers to regulate signaling pathways, or the regulation of kinase, among others. Emerging evidence indicates the overlaps between the apoptosis and other types of cell death such as autophagy. In this review we report different processes of cell death induced by Se compounds in cancer treatment and prevention