Redox Regulation of the Tumor Suppressor PTEN by Hydrogen Peroxide and Tert-Butyl Hydroperoxide

Abstract

Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx) system. Here, the role of tert-butyl hydroperoxide (t-BHP) in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t-BHP led to oxidation of recombinant PTEN. In contrast to H2O2, PTEN oxidation by t-BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t-BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t-BHP in the promotion of tumorigenesis.

Peroxiredoxin III Protects Tumor Suppressor PTEN from Oxidation by 15-Hydroperoxy-eicosatetraenoic Acid

“…To substantiate whether an identical intramolecular disulfide bond of PTEN was formed after 15s-HpETE treatment, lysates from MEFs treated with H 2 O 2 or the mixture of 15s-HpETE and Lipofectamine were fractionated on nonreducing gels and probed with PTEN antibody. Faster migrating bands, similar to those seen in our previous studies [30, 34, 36, 39], were detected following H 2 O 2 and a mixture of 15s-HpETE and Lipofectamine treatment (Figure 3(b)).…”

“…We previously reported that exogenous organic peroxides and hydroperoxides can cause the irreversible oxidation of PTEN by impairing the cellular Trx system [34, 36]. The ability of lipid peroxides to oxidize PTPs has been reported previously [37], as well as the PTEN oxidation by unidentified arachidonic acid metabolites [38].…”

Upstream signaling events leading to elevated production of pro-survival nitric oxide in photodynamically-challenged glioblastoma cells

“…L-Histidine (3 mM) was added to some cells prior to irradiation to check for any 1 O 2 -mediated PTEN photooxidation; control cells received Lalanine at the same starting concentration. After irradiation, the cell medium was aspirated and replaced with 10% FBS-containing medium without or with L-His or L-Ala. At selected post-irradiation incubation times, non-oxidized thiol groups were blocked by treating cells with 20 mM NEM in lysing medium [20 mM Tris-Cl buffer (pH 7.4), 150 mM NaCl, 5% glycerol, 0.1% Nonidet-P40 detergent, and protease inhibitor mixture], followed by sonication and centrifugation [41]. Some cells served as positive controls by incubation with the known PTEN oxidant, H 2 O 2 (e.g.…”

“…Supernatant fractions recovered after centrifugation were analyzed for total protein content by BCA assay, and then subjected to Western blotting under non-reducing conditions. Other details were as described by Zhang et al [41].…”

“…2.4), were suspended in cold pH 7.4 lysis buffer containing protease inhibitors and homogenized as described previously [25][26][27][28][29]. After centrifugation, the supernatant fractions were analyzed for total protein content, after which samples were separated by SDS-PAGE using appropriate proportions of acrylamide/bis-acrylamide and reducing conditions, except for PTEN [41]. Separated proteins were transferred to a polyvinylidene difluoride membrane.…”