DHEA attenuates PDGF-induced phenotypic proliferation of vascular smooth muscle A7r5 cells through redox regulation.

It is known that dehydroepiandrosterone (DHEA) inhibits a phenotypic switch in vascular smooth muscle cells (VSMC) induced by platelet-derived growth factor (PDGF)-BB. However, the mechanism behind the effect of DHEA on VSMC is not clear. Previously we reported that low molecular weight-protein tyrosine phosphatase (LMW-PTP) dephosphorylates PDGF receptor (PDGFR)-beta via a redox-dependent mechanism involving glutathione (GSH)/glutaredoxin (GRX)1. Here we demonstrate that the redox regulation of PDGFR-beta is involved in the effect of DHEA on VSMC. DHEA suppressed the PDGF-BB-dependent phosphorylation of PDGFR-beta. As expected, DHEA increased the levels of GSH and GRX1, and the GSH/GRX1 system maintained the redox state of LMW-PTP. Down-regulation of the expression of LMW-PTP using siRNA restored the suppression of PDGFR-beta-phosphorylation by DHEA. A promoter analysis of GRX1 and gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme of GSH synthesis, showed that DHEA up-regulated the transcriptional activity at the peroxisome proliferator-activated receptor (PPAR) response element, suggesting PPARalpha plays a role in the induction of GRX1 and gamma-GCS expression by DHEA. In conclusion, the redox regulation of PDGFR-beta is involved in the suppressive effect of DHEA on VSMC proliferation through the up-regulation of GSH/GRX system

Glutathione Related Enzyme Activities in Spontaneous Hypertensive Rat Heart

It has been reported that oxygen radicals are involved in the development of tissue injury in hypertension. To prevent o xidative stress, there are antioxidant systems inside the cells such as superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reducatase (GR), catalase (CAT) and glutathione S-transferase (GST). In this study changes in these antioxidant activities were estimated in the outer wall of the left ventricles from spontaneously hypertensive rats (SHR), stroke prone SHR (SHRSP) and normal Wister Kyoto rats (WKY). The activities of manganese-superoxide dismutase (Mn-SOD), which localizes in mitochondria and GST were lower in the left ventricles of SHR and SHRSP compared to those in WKY. Slight decrease in the GPX activity was observed in the left ventricles from SHR and SHRSP. On the other hand, the activity of GR and catalase was not different in them. The effect of Nicardipine, a calcium channel blocker, on these antioxidant activities was also esimated. Treatment of these rats with nicardipine (150 mg/kg/day) for 4 weeks improved blood pressure, from 176ﾂｱ10 mmHg to 140ﾂｱ8 mmHg in SHR (n = 5), from 201ﾂｱ11 mmHg to 167ﾂｱ5 in SHRSP (n = 5), respectively, and restored the activities of Mn-SOD, GST and GPX. Collectively, these results suggest that oxidative stress in hypertensive rat heart causes supression of antioxidant activities, which may contribute to myocardical injury, and nicardipine plays a cardioprotective role to reduce the oxidative stress in hypertensive heart

Expression of Superoxide Dismutase in Basal Cell Carcinoma

There have been no studies of the expression of superoxide dismutase(SOD) at the mRNA and protein level in skin cancers. Northern blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed in order to analyze the expression of Cu, Zn-SOD and Mn-SOD in basal cell carcinomas (BCC) and normal skin (NS). The expression of Mn-SOD mRNA and protein was significantly higher in BCC than in NS. The expression of Cu,Zn-SOD, however, was high in BCC at the mRNA level, but not at the protein level. These results suggest that an increase in the expression of Mn-SOD relates to the development of BCCs

Glutathione S-transferase pi localizes in mitochondria and protects against oxidative stress.

Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the protection of cellular components against anti-cancer drugs or peroxidative stress. Previously we found that GST pi, an isoform of the GSTs, is transported into the nucleus. In the present study, we found that GST pi is present in mitochondria as well as in the cytosol and nucleus in mammalian cell lines. A construct comprising the 84 amino acid residues in the amino-terminal region of GST pi and green fluorescent protein was detected in the mitochondria. The mutation of arginine to alanine at positions 12, 14, 19, 71, and 75 in full-length GST pi completely abrogated the ability to distribute in the mitochondria, suggesting that arginine, a positively charged residue, is required for the mitochondrial transport of GST pi. Chemicals generating reactive oxygen species, such as rotenone and antimycin A, decreased cell viability and reduced mitochondrial membrane potential. The overexpression of GST pi diminished these changes. GST pi-targeting siRNA abolished the protective effect of GST pi on the mitochondria under oxidative stress. The findings indicate that the peptide signal is conducive to the mitochondrial localization of GST pi under steady-state conditions without alternative splicing or posttranslational modifications such as proteolysis, suggesting that GST pi protects mitochondria against oxidative stress

Enhanced Nox1 expression and oxidative stress resistance in c-kit-positive hematopoietic stem/progenitor cells

Although stem cells are generally thought to be resistant to oxidative stress, the fact and in detail molecular mechanism are still to be clearly identified. We herein tried to understand the overall characterization of redox regulatory signaling in hematopoietic stem cells. We purified c-kit-positive hematopoietic stem/progenitor cells from the bone marrow of healthy mice, and then evaluated their redox regulatory property. Compared to the c-kit-negative matured mononuclear cells, c-kit-positive stem/progenitor cells showed lower basic levels of intracellular reactive oxygen species, faster clearance of the accumulated intracellular reactive oxygen species, and higher resistant to oxidative stress. An overall view on the gene expression profile associated with redox regulation showed to be widely differed between cell types. We confirmed that the c-kit-positive stem/progenitor cells expressed significantly higher of Nox1 and catalase, but less of lactoperoxidase than these matured mononuclear cells. Our data suggests that stem cells keep specific redox regulatory property for defensing against oxidative stress

Significance of Impairment of Antioxidants in Colonic Epithelial Cells Isolated From TNBS-Iuduced Colitis Rats

The functional status of glutathione (GSH), its related enzymes and Cu, Zn-superoxide dismutase (SOD) in colonocytes isolated from trinitrobenzene sulphonic (TNBS)-induced colitis rats was studied. Colitis (T group) was induced in Wistar rats with 42 mg TNBS dissolved in 0.35 ml of 40% (v/v) ethanol instilled into the colon. The animals were sacrificed on day 14 and compared with saline-instilled rats (S group). The GSH concentration and the enzymatic activities of glutathione peroxidase (GPx), glutathione S-transferase (GST), and SOD were spectrophotometrically estimated. The severity of colitis was assessed histologically and by myeloperoxidase activity (MPO) in whole colonic tissue. The body weight loss of the rats in the T group was marked. In colonocytes isolated from rats in the T group, the concentration of GSH (7.9 ±1.4 vs.11.3 ± 0.4 nmol/mg protein, p < 0.05) and the activities of GST (104.4 ± 10.3 vs. 146.2 ± 18.5 mU/mg protein, p < 0.05) and SOD (74.4 ± 8.9 vs. 99.8 ± 7.5U/mg protein, p < 0.05) were lower, but the activity of GPx (430.0 -±14.1 vs. 283.9 ± 10.0 mU/mg protein, p < 0.05) was higher than in the S group. As expected, the activity of MPO in the T group was higher than in the S group (371.2 ± 14.7 vs. 158.9 ± 8.4 mU/mg tissue, p<0.05) and histologically, colitis was only observed in rats in the T group. In conclusion, the functional status of antioxidants in the colonic epithelial cells of rats challenged with TNBS solution is impaired. This impariment may make them more susceptible to oxidative damage that may contribute to the development of the lesions observed in this model. Further studies at the molecular level are necessary to investigate these novel findings in this model and their potential application for testing new therapeutic approaches in inflammatory diseases of the intestinal tract

Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability

Nuclear translocation of glutathione S-transferase π is mediated by a non-classical localization signal.

Glutathione S-transferase π (GSTπ), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GSTπ is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GSTπ appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GSTπ was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GSTπ195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GSTπ depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GSTπ, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs

The potential benefits of nicaraven to protect against radiation-induced injury in hematopoietic stem/progenitor cells with relative low dose exposures

Nicaraven, a hydroxyl radical-specific scavenger has been demonstrated to attenuate radiation injury in hematopoietic stem cells with 5 Gy γ-ray exposures. We explored the effect and related mechanisms of nicaraven for protecting radiation injury induced by sequential exposures to a relatively lower dose γ-ray. C57BL/6 mice were given nicaraven or placebo within 30 min before exposure to 50 mGy γ-ray daily for 30 days in sequences (cumulative dose of 1.5 Gy). Mice were victimized 24 h after the last radiation exposure, and the number, function and oxidative stress of hematopoietic stem cells were quantitatively estimated. We also compared the gene expression in these purified stem cells from mice received nicaraven and placebo treatment. Nicaraven increased the number of c-kit+ stem/progenitor cells in bone marrow and peripheral blood, with a recovery rate around 60-90% of age-matched non-irradiated healthy mice. The potency of colony forming from hematopoietic stem/progenitor cells as indicator of function was completely protected with nicaraven treatment. Furthermore, nicaraven treatment changed the expression of many genes associated to DNA repair, inflammatory response, and immunomodulation in c-kit+ stem/progenitor cells. Nicaraven effectively protected against damages of hematopoietic stem/progenitor cells induced by sequential exposures to a relatively low dose radiation, via complex mechanisms

Increased expression of PHD3 represses the HIF-1 signaling pathway and contributes to poor neovascularization in pancreatic ductal adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) is known as one of the most malignant potential diseases with poor neovascularization. By comparing PDAC to hepatocellular carcinoma (HCC), which is well vascularized, we investigated the mechanisms and tumor biological significance of the poor neovascularization in PDAC. Methods: Surgical specimens from primary PDAC and HCC patients were immunohistologically stained to detect the expressions of CD105, CD44, HIF-1α, PHD3, and Siah2. We also used two PDAC and two HCC cell lines to compare the expressions of HIF-1α, PHD3, and CD44, as well as the production of VEGF in hypoxic condition. The role of PHD3 in regulating HIF-1α expression was further confirmed by siRNA knockdown in a PDAC cell line that highly expressed PHD3. Results: There were significantly fewer microvessels but more cancer stem cells in PDAC specimens compared to HCC specimens. The expression of CD105 was reversely related to the expression of CD44 in PDAC and HCC specimens. PDAC specimens also showed higher expressions of PHD3 but lower expressions of HIF-1α. Similarly, the expression of PHD3 was observed clearly in PDAC cell lines, but was almost completely negative in HCC cell lines. Hypoxic stimulation clearly enhanced HIF-1α expression and VEGF secretion in both HCC cell lines, but did not significantly change in PDAC cell lines. The knockdown of PHD3 in PDAC cells restored the hypoxic-induced HIF-1α expression, which accordingly stimulated the cells’ VEGF secretion. Conclusions: The enhanced expression of PHD3 might likely contribute to the poor neovascularization and affect the biological characterization in PDAC cancer cells