Redox regulation of tumor suppressor PTEN in cell signaling.

Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) is a potent tumor suppressor and often dysregulated in cancers. Cellular PTEN activity is restrained by the oxidation of active-site cysteine by reactive oxygen species (ROS). Recovery of its enzymatic activity predominantly depends on the availability of cellular thioredoxin (Trx) and peroxiredoxins (Prx), both are important players in cell signaling. Trx and Prx undergo redox-dependent conformational changes through the oxidation of cysteine residues at their active sites. Their dynamics are essential for protein functionality and regulation. In this review, we summarized the recent advances regarding the redox regulation of PTEN, with a specific focus on our current state-of-the-art understanding of the redox regulation of PTEN. We also proposed a tight association of the redox regulation of PTEN with Trx dimerization and Prx hyperoxidation, providing guidance for the identification of novel therapeutic targets.

Native low density lipoprotein increases the production of both nitric oxide and reactive oxygen species in the human umbilical vein endothelial cells.

BACKGROUND: Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells. OBJECTIVE: The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2',7'-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader. RESULTS: NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400 W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs. CONCLUSION: These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.

Erratum to "Long-Term Treatment of Native LDL Induces Senescence of Cultured Human Endothelial Cells".

[This corrects the article DOI: 10.1155/2017/6487825.].

Role of Recepteur D'origine Nantais on Gastric Cancer Development and Progression.

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase belonging to the subfamily of which c-MET is the prototype. Large epidemiologic studies have confirmed the strong association between RON and gastric cancer development. Constitutive activation of RON signaling directly correlates with tumorigenic phenotypes of gastric cancer and a poor survival rate in advanced gastric cancer patients. In this review, we focus on recent evidence of the aberrant expression and activation of RON in gastric cancer tumors and provide insights into the mechanism of RON signaling associated with gastric cancer progression and metastasis. Current therapeutics against RON in gastric cancer are summarized.

Redox regulation of the tumor suppressor PTEN by the thioredoxin system and cumene hydroperoxide.

Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.

Long-Term Treatment of Native LDL Induces Senescence of Cultured Human Endothelial Cells.

The study aimed to evaluate whether the treatment of primary cultured human endothelial cells with native low-density lipoprotein (nLDL) could induce their senescence and to uncover some of the putative mechanisms involved. For this purpose, human umbilical vein endothelial cells (HUVECs) were subcultured and/or continuously cultured with nLDL (0, 2, 5, and 10 µg protein/mL), for up to 9 days. The results indicated that nLDL inhibited the proliferation of HUVECs by arresting the cell cycle at G1 phase. The G1-arrested cells showed increase in cytosolic senescence-associated-ß-galactosidase (SA-ß-Gal) activity, a biomarker of cellular senescence. The causative factor of the cellular senescence was nLDL itself and not oxidized LDL (oxLDL), since blocking LDL receptor (LDLR) with the anti-LDLR antibody opposed the nLDL-induced increase of SA-ß-Gal activity and decrease of cellular proliferation. In addition, nLDL-induced cellular senescence by inhibiting the phosphorylation of pRb (G1 arrest) via p53 as well as p16 signal transduction pathways. G1 phase arrest of the senescent cells was not overcome by nLDL removal from the culture medium. Moreover, the nLDL-treated cells produced reactive oxygen species (ROS) dose- and time-dependently. These results suggested, for the first time, that long-term treatment of nLDL could induce the premature senescence of endothelial cells.

Assay of the redox state of the tumor suppressor PTEN by mobility shift.

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.

Gastroprotective Effects of Glutinous Rice Extract against Ethanol-, Indomethacin-, and Stress-induced Ulcers in Rats.

This study was designed to evaluate the efficacy of an orally administered aqueous extract of glutinous rice (GRE) to protect against acute gastric mucosal lesions induced by ethanol, indomethacin, and water immersion restraint stress in rats and to characterize the active substances responsible for the protection. GRE was shown to dose-dependently prevent the gastric lesions induced by the above ulcerogenic treatments at doses of 30 to 300 mg/kg. GRE treatment increased the gastric mucin content and partially blocked the ethanol-induced depletion of the gastric mucus layer. Also, it increased the nonprotein sulfhydryl concentration in the gastric mucosa. The gastroprotective action of GRE was markedly enhanced by co-treatment with 4-8 mg/kg tea extracts. The activity of GRE was completely lost by heat treatment at 80℃ for 3 min or treatment with 0.01% pepsin at 37℃ for 1 h. Protein extraction studies indicated that prolamins are involved in the gastroprotective activity of GRE. Our results suggest that glutinous rice proteins are useful for the prevention and treatment of gastritis and peptic ulcer.

Redox regulation of the tumor suppressor PTEN by glutathione.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Delta and gsh2Delta mutants. Expression of gamma-glutamylcysteine synthetase Gsh1 in the gsh1Delta mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration- and time-dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.

Alterations in the activity and expression of endothelial NO synthase in aged human endothelial cells.

This study was to investigate factors underlying the age-related decrease in NO production in vascular endothelial cells. The age-related changes in NO production, the activity and expression level of eNOS, and eNOS binding proteins, were studied in HUVECs. NO production in HUVECs significantly decreased in an age-dependent manner. The potentiation of NO production by L-Arg was significantly suppressed by L-NIO (eNOS-specific inhibitor) in young HUVECs and was suppressed by 1400W (iNOS-specific inhibitor) in aged HUVECs. The aged HUVECs had lower eNOS protein levels than young cells. eNOS phosphorylation at Ser-1177 (active) decreased gradually from PDL 23 through 40, and eNOS phosphorylation at Thr-495 (inactive) increased in aged cells. Changes of intracellular eNOS binding proteins, such as caveolin-1, pAkt, and Hsp90, as well as interaction between eNOS and eNOS binding proteins, indicated decreasing enzyme activity in aged HUVECs. Aging might decrease the activity as well as expression level of eNOS in HUVECs. And the decrease in eNOS activity probably implicated to the alterations in the regulatory binding proteins. For further study, it needs to be confirmed that the age-related change in the intracellular distribution of eNOS and the relative contribution of eNOS and iNOS on vascular dysfunction in aged endothelial cells.

Therapeutic effect of intracameral amphotericin B injection in the treatment of fungal keratitis.

PURPOSE: To study the therapeutic efficacy of intracameral amphotericin B (ICAMB) injection in the treatment of fungal keratitis. METHODS: Fourteen patients with fungal keratitis received ICAMB, 10 microg/0.1 mL (group A), and 17 patients received conventional treatment only (group B). Visual acuity, time to hypopyon disappearance, time to epithelial defect closure, time to final improvement, and final outcome were analyzed and compared between the 2 groups. The concentration of amphotericin B in the aqueous humor after injection was measured using high-performance liquid chromatography. RESULTS: The mean final visual acuity (log MAR) was 1.6 +/- 1.1 in group A and 1.3 +/- 1.4 in group B (P = 0.24). The mean time to disappearance of hypopyon, epithelial defect closure, and final improvement was 9.4 +/- 9.4, 19.8 +/- 10.4, and 26.6 +/- 9.2 days in group A and 26.7 +/- 21.3 (P = 0.03), 32.6 +/- 22.8 (P = 0.08), and 52.8 +/- 38.2 days (P = 0.04) in group B, respectively. At the last follow-up, treatment success was achieved in 92.9% of group A and 82.4% of group B (P = 0.38). The mean concentration of intracameral amphotericin B was 601.6 +/- 51.3 ng/mL at 6 hours, 98.8 +/- 43.1 ng/mL at 1 day, 57.0 +/- 11.6 ng/mL at 3 days, and 52.3 +/- 8.3 ng/mL at 7 days after injection. CONCLUSIONS: ICAMB seems to be effective in reducing time to disappearance of hypopyon and final improvement in the treatment of fungal keratitis.

Interleukin-6 and tumor necrosis factor-alpha levels in tears of patients with dry eye syndrome.

PURPOSE: To determine the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in tears of patients with dry eye syndrome. METHODS: IL-6 and TNF-alpha levels were measured by enzyme-linked immunosorbent assay in tear samples obtained from 18 patients with dry eye (8 patients with Sjögren syndrome and 10 patients with non-Sjögren syndrome) and 14 control subjects. The correlation between IL-6 and TNF-alpha levels and tear film and ocular surface parameters was analyzed. The relative expression of these cytokines was evaluated in conjunctival impression cytology and conjunctival biopsy specimens by using immunohistochemical staining. RESULTS: The mean levels of IL-6 and TNF-alpha were, respectively, 18.57 +/- 8.92 and 3.68 +/- 3.45 pg/mL in patients with dry eye and 3.59 +/- 3.38 (P < 0.01) and < 0.5 (P < 0.01) pg/mL in control subjects. IL-6 level was significantly increased in tears of patients with Sjögren syndrome compared with those with non-Sjögren syndrome (P < 0.01). IL-6 level correlated significantly with tear film breakup time (P = 0.04), Schirmer test (P < 0.01), tear clearance (P = 0.02), keratoepithelioplasty score (P < 0.01), and goblet cell density (P = 0.03), but not with corneal sensitivity (P = 0.08). There was no significant difference in TNF-alpha level between patients with non-Sjögren and Sjögren syndrome. TNF-alpha levels did not correlate with tear film and ocular surface parameters. Immunohistochemical staining showed positive staining for IL-6 in specimens from patients with dry eye, especially in specimens from patients with Sjögren syndrome. CONCLUSION: IL-6 and TNF-alpha levels are elevated in tears of patients with dry eye syndrome. IL-6 level, but not TNF-alpha level, is associated with the severity of the disease and correlates with various tear film and ocular surface parameters.

Total peroxyl radical-trapping ability and anti-oxidant vitamins of the umbilical venous plasma and the placenta in pre-eclampsia.

AIM: Our purpose was to investigate lipid peroxide levels, total peroxyl radical-trapping anti-oxidative parameter (TRAP) values, and anti-oxidant vitamin levels in umbilical venous plasma and placental tissues, and to evaluate their roles in the pathophysiology of pre-eclampsia. METHODS: Samples of umbilical venous plasma and placental tissue homogenates were obtained from 23 normal and 18 pre-eclamptic women at between 33 and 40 weeks' gestation. The enzyme-linked immunosorbent assay method was used to assay alpha-tumor necrosis factor (TNF-alpha), and lipid peroxide levels were measured by thiobarbituric acid reaction. The TRAP values were measured using the modified Wayner's method. Ascorbic acid, retinol alpha-tocopherol and gamma-tocopherol were measured by high performance liquid chromatography. RESULTS: Levels of TNF-alpha in placental tissue homogenates of women with pre-eclampsia were significantly higher than those of women with normal pregnancy (21.4 +/- 3.39 v. 10.3 +/- 1.06 pg/mL, P < 0.05). Lipid peroxide levels in umbilical venous plasma and placental tissue homogenates of women with pre-eclampsia were significantly higher than those of women with normal pregnancy (10.3 +/- 1.1 v. 5.85 +/- 0.53, P < 0.01, 5.14 +/- 0.40 v. 3.99 +/- 0.33 nmol/mg protein, P < 0.05, respectively). The TRAP values in umbilical venous plasma and placental tissue homogenates of women with pre-eclampsia were significantly lower than those of women with normal pregnancy (0.39 +/- 0.02 v. 0.45 +/- 0.02, P < 0.05, 0.27 +/- 0.02 v. 0.34 +/- 0.03 mM, P < 0.05, respectively). Ascorbic acid levels in umbilical venous plasma and placental tissue homogenates of women with pre-eclampsia were significantly lower than those of women with normal pregnancy (325.4 +/- 50.4 v. 543 +/- 73.8, P < 0.05, 219.0 +/- 21.0 v. 333.3 +/- 32.6 nmol/mL, P < 0.05, respectively). CONCLUSIONS: The above results suggest that increased oxidative stress in the placenta is involved in the pathophysiology of pre-eclampsia, and ascorbic acid may act as an important preventative factor in the development of pre-eclampsia.

Antioxidant responsiveness in BALB/c mice exposed to ozone.

BACKGROUND: A single, acute exposure to ozone has been shown to modify the antioxidant defense mechanism in the respiratory tract. OBJECTIVE: The aim of this study was to evaluate the effects of ozone exposure on antioxidant response in BALB/c mice. METHODS: We measured enhanced pause of breathing (Penh) as a marker of airway obstruction using barometric whole-body plethysmography before and after ozone exposure [groups (n = 6): filtered air, 0.12 ppm, 0.5 ppm, 1 ppm, 2 ppm] for 3 h. Antioxidant levels were measured using high-performance liquid chromatography with electrochemical detection in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates. RESULTS: Malondialdehyde concentrations in lung tissue homogenates were significantly increased in the group exposed to 2-ppm ozone compared to the filtered air group. Uric acid and gamma-tocopherol concentrations in BAL fluid were significantly increased in the ozone exposure group compared to the filtered air group (p < 0.01). Uric acid concentrations were increased in a concentration-dependent manner according to ozone concentration to which the animals were exposed. Increases in Penh after ozone exposure were significantly higher in an ozone concentration-dependent manner. The proportion of neutrophils in BAL fluid was significantly higher in the group exposed to 2 ppm than in the filtered air and the group exposed to 0.12 ppm (p < 0.01, respectively). The level of ascorbate correlated with the level of gamma-tocopherol. CONCLUSION: These findings suggest that antioxidant responses may serve as a protective mechanism against a range of oxidants in BALB/c mice exposed to ozone.

A novel murine long-chain acyl-CoA synthetase expressed in brain participates in neuronal cell proliferation.

Refsum disease (RfD) is an autosomal recessive neurologic disorder of the lipid metabolism. We have identified a novel murine long-chain acyl-CoA synthetase (mLACS) associated with the RfD gene using yeast two-hybrid assay. Northern blot analyses revealed that mLACS was expressed mainly in the brain and testis. mLACS was highly expressed in the brain at 2 weeks after birth and maintained through adult life. Expressions of the brain-specific LACS family increased in the PC12 cells undergoing neurite outgrowth by nerve growth factor. mLACS preferentially catalyzed the formation of arachidonoyl-CoA more than palmitoyl-CoA or oleoyl-CoA in PC12 cells. Triacsin C, an inhibitor of LACS, suppressed the cell proliferation and decreased mLACS expression in parent PC12 cells, but not in stably anti-sense mLACS cDNA-transfected cells. Our results indicate that mLACS participates in neuronal cell proliferation and differentiation, and interaction of the RfD gene with brain-selective mLACS may be involved in the pathogenesis of RfD.

Effect of drinking green tea on age-associated accumulation of Maillard-type fluorescence and carbonyl groups in rat aortic and skin collagen.

Tea catechins and other flavonoids have been shown to potentially protect against chronic cardiovascular diseases such as coronary heart disease and atherosclerosis. In this study, 6-month-old female Sprague-Dawley rats were fed green tea extract (50 mg/100 ml in drinking water) up to the age of 22 months, and the age-associated changes in Maillard-type fluorescence and carbonyl groups in the aortic and skin collagen were compared with those occurring in the water-fed control animals. Collagen-linked Maillard-type fluorescence was found to increase in both the aortic and skin tissues as animals aged. The age-associated increase in the fluorescence in the aortic collagen was remarkably inhibited by the green tea extract treatment, while that occurring in the skin collagen was not significantly inhibited by the treatment. The collagen carbonyl content also increased in both the aortic and skin tissues as animals aged. In contrast with the case of Maillard-type fluorescence, however, the age-associated increase in the carbonyl content was not inhibited by the green tea extract treatment either in the aortic or skin collagen. These results suggest that the inhibition of AGE formation in collagen is an important mechanism for the protective effects of tea catechins against cardiovascular diseases.