Noxa couples lysosomal membrane permeabilization and apoptosis during oxidative stress

The exact roles of lysosomal membrane permeabilization (LMP) in oxidative stress-triggered apoptosis are not completely understood. Here, we first studied the temporal relation between LMP and mitochondrial outer membrane permeabilization (MOMP) during the initial stage of apoptosis caused by the oxidative stress inducer H₂O₂. Despite its essential role in mediating apoptosis, the expression of the BH3-only Bcl-2 protein Noxa was dispensable for LMP. In contrast, MOMP was dependent on Noxa expression and occurred downstream of LMP. When lysosomal membranes were stabilized by the iron-chelating agent desferrioxamine, H₂O₂-induced increase in DNA damage, Noxa expression, and subsequent apoptosis were abolished by the inhibition of LMP. Importantly, LMP-induced Noxa expression increase was mediated by p53 and seems to be a unique feature of apoptosis caused by oxidative stress. Finally, exogenous iron loading recapitulated the effects of H₂O₂ on the expression of BH3-only Bcl-2 proteins. Overall, these data reveal a Noxa-mediated signaling pathway that couples LMP with MOMP and ultimate apoptosis during oxidative stress.     

Role of labile iron in the toxicity of pharmacological ascorbate

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H₂O₂ via the formation of ascorbate radical (Asc^•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H₂O₂ generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H₂O₂ generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe³⁺/Fe²⁺, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.     

Manganoporphyrins and ascorbate enhance gemcitabine cytotoxicity in pancreatic cancer

Pharmacological ascorbate (AscH⁻) selectively induces cytotoxicity in pancreatic cancer cells vs normal cells via the generation of extracellular hydrogen peroxide (H₂O₂), producing double-stranded DNA breaks and ultimately cell death. Catalytic manganoporphyrins (MnPs) can enhance ascorbate-induced cytotoxicity by increasing the rate of AscH⁻ oxidation and therefore the rate of generation of H₂O₂. We hypothesized that combining MnPs and AscH⁻ with the chemotherapeutic agent gemcitabine would further enhance pancreatic cancer cell cytotoxicity without increasing toxicity in normal pancreatic cells or other organs. Redox-active MnPs were combined with AscH⁻ and administered with or without gemcitabine to human pancreatic cancer cell lines, as well as immortalized normal pancreatic ductal epithelial cells. The MnPs MnT2EPyP (Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin pentachloride) and MnT4MPyP (Mn(III)tetrakis(N-methylpyridinium-4-yl) porphyrin pentachloride) were investigated. Clonogenic survival was significantly decreased in all pancreatic cancer cell lines studied when treated with MnP + AscH⁻ + gemcitabine, whereas nontumorigenic cells were resistant. The concentration of ascorbate radical (Asc^•−, an indicator of oxidative flux) was significantly increased in treatment groups containing MnP and AscH⁻. Furthermore, MnP + AscH⁻ increased double-stranded DNA breaks in gemcitabine-treated cells. These results were abrogated by extracellular catalase, further supporting the role of the flux of H₂O₂. In vivo growth was inhibited and survival increased in mice treated with MnT2EPyP, AscH⁻, and gemcitabine without a concomitant increase in systemic oxidative stress. These data suggest a promising role for the use of MnPs in combination with pharmacologic AscH⁻ and chemotherapeutics in pancreatic cancer.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

Lipid peroxidation product 4-hydroxy-2-nonenal modulates base excision repair in human cells

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway.One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H₂O₂ and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N⁶-ethenoadenine (ɛA) and 3,N⁴-ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H₂O₂ or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway.     

The antioxidant machinery of the endoplasmic reticulum: Protection and signaling

Cellular metabolism is inherently linked to the production of oxidizing by-products, including reactive oxygen species (ROS) hydrogen peroxide (H₂O₂). When present in excess, H₂O₂ can damage cellular biomolecules, but when produced in coordinated fashion, it typically serves as a mobile signaling messenger. It is therefore not surprising that cell health critically relies on both low-molecular-weight and enzymatic antioxidant components, which protect from ROS-mediated damage and shape the propagation and duration of ROS signals. This review focuses on H₂O₂–antioxidant cross talk in the endoplasmic reticulum (ER), which is intimately linked to the process of oxidative protein folding. ER-resident or ER-regulated sources of H₂O₂ and other ROS, which are subgrouped into constitutive and stimulated sources, are discussed and set into context with the diverse antioxidant mechanisms in the organelle. These include two types of peroxide-reducing enzymes, a high concentration of glutathione derived from the cytosol, and feedback-regulated thiol–disulfide switches, which negatively control the major ER oxidase ER oxidoreductin-1. Finally, new evidence highlighting emerging principles of H₂O₂-based cues at the ER will likely set a basis for establishing ER redox processes as a major line of future signaling research. A fundamental problem that remains to be solved is the specific, quantitative, time resolved, and targeted detection of H₂O₂ in the ER and in specialized ER subdomains.     

Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement, and administration of DHEA produces a number of beneficial effects in the elderly. Many researchers have suggested that DHEA exerts it function after conversion into more biologically active hormones in peripheral target cells. The actions of DHEA in Leydig cells, a major target cell of DHEA biotransformation in males, are not clear. The present study found that DHEA increased cell viability and decreased reactive oxygen species (ROS) and malondialdehyde contents in H₂O₂-induced Leydig cells. DHEA significantly increased the activities of superoxide dismutase, catalase and peroxidase, and decreased the DNA damage in H₂O₂-induced Leydig cells. Apoptosis was significant decreased in H₂O₂-induced Leydig cells after DHEA treatment. DHEA inhibited the loss of mitochondrial membrane potential (ΔΨm) and the upregulation of the caspase-3 protein level induced by H₂O₂ in Leydig cells. DHEA also reversed the decrease in PI3K and p-Akt protein levels induced by H₂O₂. These data showed that DHEA could ameliorate H₂O₂-induced oxidative damage by increasing anti-oxidative enzyme activities, which resulted in reduced ROS content, and decreased apoptosis, mainly by preventing the loss of ΔΨm and inhibiting caspase-3 protein levels via activation of PI3K/Akt signaling pathways. These results increase our understanding of the molecular mechanism of the anti-ageing effect of DHEA.     

Targeting the upregulation of reactive oxygen species subsequent to hyperglycemia prevents type 1 diabetic cardiomyopathy in mice

Cardiac oxidative stress is an early event associated with diabetic cardiomyopathy, triggered by hyperglycemia. We tested the hypothesis that targeting left-ventricular (LV) reactive oxygen species (ROS) upregulation subsequent to hyperglycemia attenuates type 1 diabetes-induced LV remodeling and dysfunction, accompanied by attenuated proinflammatory markers and cardiomyocyte apoptosis. Male 6-week-old mice received either streptozotocin (55 mg/kg/day for 5 days), to induce type 1 diabetes, or citrate buffer vehicle. After 4 weeks of hyperglycemia, the mice were allocated to coenzyme Q₁₀ supplementation (10 mg/kg/day), treatment with the angiotensin-converting-enzyme inhibitor (ACE-I) ramipril (3 mg/kg/day), treatment with olive oil vehicle, or no treatment for 8 weeks. Type 1 diabetes upregulated LV NADPH oxidase (Nox2, p22^phox, p47^phox and superoxide production), LV uncoupling protein UCP3 expression, and both LV and systemic oxidative stress (LV 3-nitrotyrosine and plasma lipid peroxidation). All of these were significantly attenuated by coenzyme Q₁₀. Coenzyme Q₁₀ substantially limited type 1 diabetes-induced impairments in LV diastolic function (E:A ratio and deceleration time by echocardiography, LV end-diastolic pressure, and LV −dP/dt by micromanometry), LV remodeling (cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis), and LV expression of proinflammatory mediators (tumor necrosis factor-α, with a similar trend for interleukin IL-1β). Coenzyme Q_10's actions were independent of glycemic control, body mass, and blood pressure. Coenzyme Q₁₀ compared favorably to improvements observed with ramipril. In summary, these data suggest that coenzyme Q₁₀ effectively targets LV ROS upregulation to limit type 1 diabetic cardiomyopathy. Coenzyme Q₁₀ supplementation may thus represent an effective alternative to ACE-Is for the treatment of cardiac complications in type 1 diabetic patients.     

On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: A reassessment

Recently, D.J. Hall et al. reported that ethidium (E⁺) is formed as a major product of hydroethidine (HE) or dihydroethidium reaction with superoxide (O₂⁻) in intact animals with low tissue oxygen levels (J. Cereb. Blood Flow Metab. 32:23–32, 2012). The authors concluded that measurement of E⁺ is an indicator of O₂⁻ formation in intact brains of animals. This finding is in stark contrast to previous reports using in vitro systems showing that 2-hydroxyethidium, not ethidium, is formed from the reaction between O₂⁻ and HE. Published in vivo results support the in vitro findings. In this study, we performed additional experiments in which HE oxidation products were monitored under different fluxes of O₂⁻. Results from these experiments further reaffirm our earlier findings (H. Zhao et al., Free Radic. Biol. Med. 34:1359, 2003). We conclude that whether in vitro or in vivo, E⁺ measured by HPLC or by fluorescence lifetime imaging is not a diagnostic marker product for O₂⁻ reaction with HE.     

ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.     

Effect of cryopreservation on viability and growth efficiency of stromal-epithelial cells derived from neonatal human thymus

The thymus is the major site of T lymphocyte generation and so is critical for a functional adaptive immune system. Since, thymectomy is a component of neonatal surgery for congenital heart diseases, it provides great potential for collection and storage of thymic tissue for autologous transplantation. However, specific investigation into the optimum parameters for thymic tissue cryopreservation have not been conducted. In this research, we evaluated the effect of different cryoprotective media compositions, which included penetrating (Me₂SO, glycerol) and non-penetrating (dextran-40, sucrose, hydroxyethyl starch) components, on the viability and functionality of frozen-thawed human thymic samples to select an optimal cryoprotective medium suitable for long-term storage of thymic tissue and a stromal-epithelial enriched population. Our primary focus was on receiving, low-temperature storage, culturing and evaluation of thymic tissue samples from newborns and infants with congenital heart diseases, who had undergone thymectomy as a part of standard surgical procedure. Thus, this work builds the platform for autologous clinical intervention into the thymus-deficient patients with congenital heart diseases. From our data, we conclude that although there were no significant differences in efficiency of tested cryoprotective media compositions, the combination of Me₂SO and dextran-40 compounds was the most suitable for long-term storage both thymic cell suspensions and thymic fragments based on the viability of CD326⁺ epithelial cells and stromal-epithelial cell monolayer formation.     

Design,synthesis and biological evaluation of Helicobacter pylori inosine 5′-monophosphate dehydrogenase (HpIMPDH) inhibitors. Further optimization of selectivity towards HpIMPDH over human IMPDH2

Inosine 5′-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes a crucial step in guanine nucleotide biosynthesis, thereby governing cell proliferation. In contrast to mammalian IMPDHs, microbial IMPDHs are relatively less explored as potential targets for antimicrobial drug discovery. In continuation with our previous work, here we report the discovery of moderately potent and highly selective Helicobacter pylori IMPDH (HpIMPDH) inhibitors. The present study is mainly focused around our previously identified, modestly potent and relatively nonselective (for HpIMPDH over human IMPDH2) hit molecule IX (16i). In an attempt to optimize the selectivity for the bacterial enzyme, we screened a set of 48 redesigned new chemical entities (NCEs) belonging to 5-aminoisobenzofuran-1(3H)-one series for their in vitro HpIMPDH and human IMPDH2 inhibition. A total of 12 compounds (hits) demonstrated ≥70% HpIMPDH inhibition at 10 μM concentration; none of the hits were active against hIMPDH2. Compound 24 was found to be the most potent and selective molecule (HpIMPDH IC₅₀ = 2.21 µM) in the series. The study reaffirmed the utility of 5-aminoisobenzofuran-1(3H)-one as a promising scaffold with great potential for further development of potent and selective HpIMPDH inhibitors.     

Temperature-induced lipocalin (TIL) is translocated under salt stress and protects chloroplasts from ion toxicity

Temperature-induced lipocalins (TIL) have been invoked in the defense from heat, cold and oxidative stress. Here we document a function of TIL for basal protection from salinity stress. Heterologous expression of TIL from the salt resistant poplar Populus euphratica did not rescue growth but prevented chlorophyll b destruction in salt-exposed Arabidopsis thaliana. The protein was localized to the plasma membrane but was re-translocated to the symplast under salt stress. The A. thaliana knock out and knock down lines Attil1-1 and Attil1-2 showed stronger stress symptoms and stronger chlorophyll b degradation than the wildtype (WT) under excess salinity. They accumulated more chloride and sodium in chloroplasts than the WT. Chloroplast chloride accumulation was found even in the absence of salt stress. Since lipocalins are known to bind regulatory fatty acids of channel proteins as well as iron, we suggest that the salt-induced trafficking of TIL may be required for protection of chloroplasts by affecting ion homeostasis.     

The development of 2-acetylphenol-donepezil hybrids as multifunctional agents for the treatment of Alzheimer’s disease

A series of 2-acetylphenol-donepezil hybrids was designed and synthesized based on multi-target-directed ligands strategy. The biological activities were evaluated by AChE/BChE inhibition and MAO-A/MAO-B inhibition. The results revealed that the tertiary amines and methylene chain length significantly affected the eeAChE inhibitory potency, in particular, compound TM-14 showed the best eeAChE inhibitory activity with IC₅₀ value of 2.9 μM, in addition, both kinetic analysis of AChE inhibition and docking study displayed that TM-14 could simultaneously bind to the catalytic active site and peripheral anionic site of AChE. Moreover, compound TM-14 was a selective metal chelator and could form 1:1 TM-14-Cu²⁺ complex. The structure-active-relationship also indicated that the O-alkylamine fragment remarkably decreased hMAO-B inhibitory activity, compound TM-2 exhibited potent hMAO-B inhibitory activity (IC₅₀ = 6.8 μM), which was supported by the molecular docking study. More interestingly, compounds TM-14 and TM-2 could cross the blood-brain barrier in vitro. Therefore, the structure-active-relationship of 2-acetylphenol-donepezil hybrids could encourage the development of multifunction agents with selective AChE inhibition or selective MAO-B inhibition for the treatment of Alzheimer’s disease.