Subcellular compartmentalization of glutathione peroxidase 1 allelic isoforms differentially impact parameters of energy metabolism

Specific genetic variations in the gene for the selenium-containing antioxidant protein glutathione peroxidase 1 (GPX1) are associated with the risk of a variety of common diseases, including cancer, diabetes, and cardiovascular disorders. Two common variations have been focused upon, one resulting in leucine or proline at codon 198 and another resulting in 5, 6, or 7 alanine repeats were previously shown to affect the distribution of GPX1 between the cytoplasm and mitochondria. Human MCF7 cells engineered to exclusively express GPX1 with five alanine repeats at amino terminus and proline at codon 198 (A5P) and seven alanine repeats at amino terminus and leucine at codon 198 (A7L), as well as derivatives targeted to the mitochondria by the addition of a mitochondrial localization sequence (mA5P and mA7L) were used to assess the consequences of the expression of these proteins on the cellular redox state and bioenergetics. Ectopic expression of A5P and A7L reduced the levels of reactive oxygen species, and the mitochondrially targeted derivatives exhibited better activity in these assays. Bioenergetics and mitochondrial integrity were assessed by measuring mitochondrial membrane potential, oxygen consumption, adenosine triphosphate (ATP) levels, and the levels of lactate dehydrogenase. The results of these assays indicated distinctively, and sometimes opposing, patterns with regard to differences between the consequences of the expression of A5P, A7L, mA5P, and mA7L. These data provide new information on the consequences of differences in the primary structure and cellular location of GPX1 proteins and contribute to the understanding of how these effects might contribute to human disease

Rapamycin increases levels of GPx-4, MnSOD and pS6 protein levels in cell lines.

<p>The effect of 1 ng/mL rapamycin on GPx-4 and MnSOD and pS6 protein levels in KU812a, MEG-01, GM10832 and MDA-MB-231 is shown. GPx-4 by 3-day 1 ng/mL rapamycin treatment in KU812a and MEG-01 (<i>P</i> > 0.2), but was increased 3-fold in GM10832 (<i>P</i> = 0.05) and 6-fold in MDA-MB-231 cells (<i>P</i> = 0.02). The disappearance of pS6 signal following rapamycin treatment indicates inhibition of mTOR. Data shown is representative of three independent experiments.</p

Treatment with LY294002 for 4 days inhibits PI3K phosphorylation activity and decreases GPx activity.

<p>The effect of 20 μM LY294002 treatment of KU812a cells on GPx activity levels alone or in combination with 150 nM imatinib is shown. Data presented is derived from three independent experiments. * =  <i>P</i><0.001, ** = <i>P</i><0.01.</p

The translational efficiency of UGA readthrough by the GPx-1 SECIS is not enhanced by rapamycin.

<p>Diagrammatic representation of the pLNCX-UGA-GPx-1 reporter construct used for quantification of UGA read-through, containing the GPx-1 SECIS element (A). The effect of rapamycin (1 ng/mL), selenium (100 nM), or both on the translational efficiency of UGA readthrough in MDA-MB-231 cells infected with the pLNCX-UGA-GPx1 SECIS reporter construct as assessed by determining luciferase activity; relative luciferase levels were normalized to β-galactosidase levels to calculate translational efficiency (B). SECIS-dependent translational efficiency was not increased following 1 ng/mL rapamycin treatment, but was increased 2.6-fold by incubation of cells containing the reporter construct with selenium. Data shown represents two independent experiments. * = <i>P</i><0.001. Error bars indicate the S.D. The effect of rapamycin on U49C GPx-1 levels was determined by immunoblotting for GPx-1 by western blot analysis (C).</p

Rapamycin enhances GPx-1 protein and enzyme activity levels in all cell lines investigated.

<p>The effect of rapamycin on GPx-1 activity (A) and protein levels (B) in KU812a, MEG-01 GM10832 or MDA-MB-231 is shown. The effect of rapamycin on steady-state GPx-1 transcript levels as determined by RT-qPCR and normalization of GPx-1 Ct values to Ct values for 18s RNA (C). Rapamycin significantly increased protein respectively 3-fold and 1.3-fold in KU812a and MEG-01 (<i>P</i> = 0.05). Steady state transcript levels for GPx-1 were unaffected by treatment with rapamycin in KU812a. Data shown is representative of three independent experiments. * = <i>P</i><0.001, ** = <i>P</i><0.01. † = <i>P</i><0.05. Error bars indicate S.D.</p

GPx-1 levels are enhanced in a dose- and time-dependent manner following imatinib treatment.

<p>The dose- and time-dependent increases in GPx activity following 100 nM and 150 nM imatinib treatment of KU812a cells for 7 days (A) or 150 nM imatinib treatment for 3 or 7 days (B). The effect of imatinib treatment on GPx1 protein levels. Data shown is representative of thee independent experiments were performed (C). * = <i>P</i><0.001, † = <i>P</i><0.05, compared to 100 nM or 3-day imatinib treatment. Error bars indicate S.D.</p

GPx-1 protein and activity levels are decreased by exogenous Bcr-Abl in MDA-MB-231.

<p>MDA-MB-231 cells transfected with a GFP-tagged Bcr-Abl vector and the transfection success was as assessed by fluorescence microscopy (A). The effect of ectopic Bcr-Abl expression on GPx enzyme activity in MDA-MB-231 cells. GPx activity was decreased 0.6-fold by ectopic expression of Bcr-Abl. Data shown is representative of one experiment. Two independent experiments were performed. * = <i>P</i><0.001. Error bars indicate S.D.</p

GPx-1 is increased in MEG-01, but not MDA-MB-231 and GM10832, following imatinib treatment.

<p>The effect of imatinib on GPx enzyme activity (A) and GPx-1 protein (B) in MEG-01, MDA-MB-231 and GM10832 is shown. GPx activity was increased 2-fold, and protein increased 4-fold (<i>P</i><0.01) by 300 nM imatinib treatment in MEG-01. GPx-1 activity and protein levels did not change following imatinib treatment of MDA-MB-231 (500 nM imatinib) and GM10832 (300 nM). Data presented in A is the result of three independent experiments * = <i>P</i><0.001. Error bars indicate S.D.</p

GPx-1 activity and protein, but not steady-state transcript levels are enhanced by treatment with imatinib.

<p>KU812a cells were treated for 7 days with 150 nM imatinib and the effects on GPx enzyme activity (A), GPx-1 protein (B) and GPx-1 transcript levels expressed as the GPx-1 Ct values normalized to the Ct values for TBP mRNA(C) is shown. GPx activity was increased 2.5-fold, and protein increased 7-fold (<i>P</i> = 0.02) by imatinib treatment, while steady-state transcript levels were unaltered. Data shown in A and C are the results of three independent experiments. * = <i>P</i><0.001. Error bars indicate the S.D.</p

Allele-specific interaction between glutathione peroxidase 1 and manganese superoxide dismutase affects the levels of Bcl-2, Sirt3 and E-cadherin

<p>Manganese superoxide dismutase (MnSOD) is a mitochondrial-resident enzyme that reduces superoxide to hydrogen peroxide (H₂O₂), which can be further reduced to water by glutathione peroxidase (GPX1). Data from human studies have indicated that common polymorphisms in both of these proteins are associated with the risk of several cancers, including breast cancer. Moreover, polymorphisms in MnSOD and GPX1 were shown to interact to increase the risk of breast cancer. To gain an understanding of the molecular mechanisms behind these observations, we engineered human MCF-7 breast cancer cells to exclusively express GPX1 and/or MnSOD alleles and investigated the consequences on the expression of several proteins associated with cancer aetiology. Little or no effect was observed on the ectopic expression of these genes on the phosphorylation of Akt, although allele-specific effects and interactions were observed for the impact on the levels of Bcl-2, E-cadherin and Sirt3. The patterns observed were not consistent with the steady-state levels of H₂O₂ determined in the transfected cells. These results indicate plausible contributing factors to the effects of allelic variations on cancer risk observed in human epidemiological studies.</p