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 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

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

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

Placental extract protects bone marrow-derived stem/progenitor cells against radiation injury through anti-inflammatory activity

Placental extracts have been reported to have anti-oxidative and anti-inflammatory activities. Because there is increasing evidence that ionizing radiation induces the release of reactive oxygen species (ROS) and inflammatory cytokines, we examined the protective effects of a placental extract against radiation injury. C57BL/6 mice were exposed to 1 Gy of γ-ray radiation every day for 5 days, and placental extract (1 mg/day) was administrated orally soon after each exposure. At 2 days after the last irradiation, mice were euthanized to examine the numbers, colony-forming capacity, and DNA damage of stem/progenitor cells in the peripheral blood and bone marrow. To understand the related mechanisms, we also measured the levels of intracellular and mitochondrial ROS, and 8-OHdG in the plasma and urine, and IL-6 and TNF-α in the plasma. Compared with the placebo treatment, oral administration of placental extract significantly increased the number and colony-forming capacity, but decreased the DNA damage of bone marrow stem/progenitor cells. However, neither the levels of intracellular and mitochondrial ROS in bone marrow cells, nor the levels of 8-OHdG in the urine and plasma significantly differed between groups. Interestingly, in comparison with the placebo treatment, placental extract significantly decreased the levels of the inflammatory cytokines IL-6 and TNF-α in the plasma. Placental extract significantly attenuated the acute radiation injury to bone marrow-derived stem/progenitor cells, and this protection is likely to be related to the anti-inflammatory activity of the placental extract

Nicaraven Attenuates Radiation-Induced Injury in Hematopoietic Stem/Progenitor Cells in Mice

Nicaraven, a chemically synthesized hydroxyl radical-specific scavenger, has been demonstrated to protect against ischemia-reperfusion injury in various organs. We investigated whether nicaraven can attenuate radiation-induced injury in hematopoietic stem/progenitor cells, which is the conmen complication of radiotherapy and one of the major causes of death in sub-acute phase after accidental exposure to high dose radiation. C57BL/6 mice were exposed to 1 Gy γ-ray radiation daily for 5 days in succession (a total of 5 Gy), and given nicaraven or a placebo after each exposure. The mice were sacrificed 2 days after the last radiation treatment, and the protective effects and relevant mechanisms of nicaraven in hematopoietic stem/progenitor cells with radiation-induced damage were investigated by ex vivo examination. We found that post-radiation administration of nicaraven significantly increased the number, improved the colony-forming capacity, and decreased the DNA damage of hematopoietic stem/progenitor cells. The urinary levels of 8-oxo-2′-deoxyguanosine, a marker of DNA oxidation, were significantly lower in mice that were given nicaraven compared with those that received a placebo treatment, although the levels of intracellular and mitochondrial reactive oxygen species in the bone marrow cells did not differ significantly between the two groups. Interestingly, compared with the placebo treatment, the administration of nicaraven significantly decreased the levels of the inflammatory cytokines IL-6 and TNF-α in the plasma of mice. Our data suggest that nicaraven effectively diminished the effects of radiation-induced injury in hematopoietic stem/progenitor cells, which is likely associated with the anti-oxidative and anti-inflammatory properties of this compound

Nicaraven Attenuates Radiation-Induced Injury in Hematopoietic Stem/Progenitor Cells in Mice

Nicaraven, a chemically synthesized hydroxyl radical-specific scavenger, has been demonstrated to protect against ischemia-reperfusion injury in various organs. We investigated whether nicaraven can attenuate radiation-induced injury in hematopoietic stem/progenitor cells, which is the conmen complication of radiotherapy and one of the major causes of death in sub-acute phase after accidental exposure to high dose radiation. C57BL/6 mice were exposed to 1 Gy γ-ray radiation daily for 5 days in succession (a total of 5 Gy), and given nicaraven or a placebo after each exposure. The mice were sacrificed 2 days after the last radiation treatment, and the protective effects and relevant mechanisms of nicaraven in hematopoietic stem/progenitor cells with radiation-induced damage were investigated by ex vivo examination. We found that post-radiation administration of nicaraven significantly increased the number, improved the colony-forming capacity, and decreased the DNA damage of hematopoietic stem/progenitor cells. The urinary levels of 8-oxo-2′-deoxyguanosine, a marker of DNA oxidation, were significantly lower in mice that were given nicaraven compared with those that received a placebo treatment, although the levels of intracellular and mitochondrial reactive oxygen species in the bone marrow cells did not differ significantly between the two groups. Interestingly, compared with the placebo treatment, the administration of nicaraven significantly decreased the levels of the inflammatory cytokines IL-6 and TNF-α in the plasma of mice. Our data suggest that nicaraven effectively diminished the effects of radiation-induced injury in hematopoietic stem/progenitor cells, which is likely associated with the anti-oxidative and anti-inflammatory properties of this compound