S-Glutathionylation of Protein Disulfide Isomerase Regulates Estrogen Receptor α Stability and Function

S-Glutathionylation of cysteine residues within target proteins is a posttranslational modification that alters structure and function. We have shown that S-glutathionylation of protein disulfide isomerase (PDI) disrupts protein folding and leads to the activation of the unfolded protein response (UPR). PDI is a molecular chaperone for estrogen receptor alpha (ERα). Our present data show in breast cancer cells that S-glutathionylation of PDI interferes with its chaperone activity and abolishes its capacity to form a complex with ERα. Such drug treatment also reverses estradiol-induced upregulation of c-Myc, cyclinD1, and P21Cip, gene products involved in cell proliferation. Expression of an S-glutathionylation refractory PDI mutant diminishes the toxic effects of PABA/NO. Thus, redox regulation of PDI causes its S-glutathionylation, thereby mediating cell death through activation of the UPR and abrogation of ERα stability and signaling

Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

Background: PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood. Methodology/Principal Findings: We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2+-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca 2+ release and causing auto-regulation of eNOS through S-glutathionylation. Conclusions/Significance: The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca 2+ flux and calmodulin (CaM) activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNO

Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS-induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras-induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation-induced inactivation. Along those lines, Prdx1 tumour suppression of Ras- or ErbB-2-induced transformation was mediated mainly via PTEN

Transgenic Mice Overexpressing Peroxiredoxin 6 Show Increased Resistance to Lung Injury in Hyperoxia

Peroxiredoxin 6 (Prdx6) is a novel peroxidase enzyme that is expressed at a high level in the lung. We tested the hypothesis that transgenic (Tg) mice overexpressing Prdx6 would exhibit increased resistance to hyperoxia-induced lung injury. Wild-type and Tg mice were exposed to 100% O2 and evaluated for survival, lung histopathology, total protein, and nucleated cells in bronchoalveolar lavage fluid (BALF), and oxidation of lung protein and lipids. Prdx6 protein expression and enzyme activity were ∼ 3-fold higher in Tg lungs compared with wild-type. Tg mice survived longer during exposure to 100% O2 (LT50 104 ± 2.8 h in Tg versus 88.9 ± 1.1 h for wild-type). Lung wet/dry weight ratio and total protein and nucleated cell count in lung lavage fluid were significantly greater in wild-type mice at 72 and 96 h of hyperoxia compared with Tg mice. At 96 h of hyperoxia, Tg mice had less epithelial cell necrosis, perivascular edema, and inflammatory cell recruitment by light microscopy, and lower TBARS and protein carbonyls in lung homogenate (P < 0.05). These results show that Tg mice have increased defense against lung injury in hyperoxia, providing evidence that Prdx6 functions as a lung antioxidant enzyme

Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344

ABSTRACT ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function. In a dose-dependent manner the drug caused instantaneous and pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells (quantitatively significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by ME-344, with specific effects on the respiratory chain (resistance correlated with higher glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased extracellular acidification rates in drugresistant cells (significantly less in drug-sensitive cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug-sensitive cells did ME-344 dose-dependently increase the intracellular generation of reactive oxygen species and cause oxidation of total (mainly glutathione) and protein thiols and the concomitant immediate increases in NADPH levels. We conclude that ME-344 causes complex, redox-specific, and mitochondria-targeted effects in lung cancer cells, which differ in extent from normal cells, correlate with drug sensitivity, and provide indications of a beneficial in vitro therapeutic index

PABA/NO effect on surface protein thiols and plasma trans-membrane potential in HL60 cells and controls for PABA, DETA/NO and DEA NONOate effects.

<p>A) Fluorescent detection (TG-1) of dose-dependent HL60 surface protein sulfhydryls level after PABA/NO, PABA and ThG addition. B). PABA (50 µM) addition to HL60 cells (with or without extracellular Ca²⁺) did not initiate generation of fluorescent metabolite (MW 622). C) Plasma trans-membrane potential (V_m) recorded as Bis-Oxanol emission at 520 nm after addition of 20 nM ThG, 25 µM PABA or 25 µM PABA/NO in HL60 cells. D) DETA/NO and DEA NONOate (25 µM) addition to HL60 cells did not initiate Ca²⁺ fluxes. Data represent: mean ± SE (panel A), representative traces averaged and smoothed with Sigma Plot 10.0 software (panel C), and original representative traces (panel B and D) of 3 independent experiments.</p