N‐acetyl‐L‐cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors

Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N‐acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA‐2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase‐3, ‐8, ‐9 activities and Bcl‐2, Bax, Cyt‐c, Annexin V‐FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD₅₀) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD₅₀) and H₂O₂ for 24 h increased Caspase‐3, ‐8, ‐9 activities, Cyt‐c and Bax levels and decreased Bcl‐2 levels. The concurrent incubation of NT2 cells with bleomycin/H₂O₂ and NAC (5 mM) for 24 h abolished bleomycin/H₂O₂‐dependent increases in Caspase‐3, ‐8, ‐9 activities, Bax and Cyt‐c levels and bleomycin/H₂O₂‐dependent decrease in Bcl‐2 level. Our results indicate that bleomycin/H₂O₂ induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H₂O₂ induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin‐induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells. J. Cell. Biochem. 114: 1685–1694, 2013. © 2013 Wiley Periodicals, Inc.     

Up‐regulation of glycolysis promotes the stemness and EMT phenotypes in gemcitabine‐resistant pancreatic cancer cells

Cancer stem cells (CSCs) and epithelial–mesenchymal transition (EMT)‐type cells are considered as underlying causes of chemoresistance, tumour recurrence and metastasis in pancreatic cancer. We aimed to describe the mechanisms – particularly glycolysis – involved in the regulation of the CSC and EMT phenotypes. We used a gemcitabine‐resistant (GR) Patu8988 cell line, which exhibited clear CSC and EMT phenotypes and showed reliance on glycolysis. Inhibition of glycolysis using 2‐deoxy‐D‐glucose (2‐DG) significantly enhanced the cytotoxicity of gemcitabine and inhibited the CSC and EMT phenotypes in GR cells both in vitro and in vivo. Intriguingly, the use of the reactive oxygen species (ROS) scavenger N‐acetylcysteine (NAC) restored the CSC and EMT phenotypes. H₂O₂ produced changes similar to those of 2‐DG, indicating that ROS were involved in the acquired cancer stemness and EMT phenotypes of GR cells. Moreover, doublecortin‐like kinase 1 (DCLK1), a pancreatic CSC marker, was highly expressed and regulated the stemness and EMT phenotypes in GR cell. Both 2‐DG and H₂O₂ treatment suppressed DCLK1 expression, which was also rescued by NAC. Together, these findings revealed that glycolysis promotes the expression of DCLK1 and maintains the CSC and EMT phenotypes via maintenance of low ROS levels in chemoresistant GR cells. The glycolysis‐ROS‐DCLK1 pathway may be potential targets for reversing the malignant behaviour of pancreatic cancer.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Role of HemF and HemN in the heme biosynthesis of Vibrio vulnificus under S‐adenosylmethionine‐limiting conditions

Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O₂‐dependent HemF and O₂‐independent HemN. The growth of the hemF mutant HF1 was similar to wild‐type cells at pH 7.5 under 2% O₂ conditions where HemN was active and had a half‐life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S‐adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM‐limiting conditions, a mutation arose in hemN, encoding HemN_Y74F. Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemN_Y74F for the cofactor SAM1, which coordinates the 4Fe‐4S cluster, was approximately sixfold higher than that of HemN. The K_m of HemN_Y74F for the co‐substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.     

Fabricating PLGA microparticles with high loads of the small molecule antioxidant N‐acetylcysteine that rescue oligodendrocyte progenitor cells from oxidative stress

Reactive oxygen species (ROS), encompassing all oxygen radical or non‐radical oxidizing agents, play key roles in disease progression. Controlled delivery of antioxidants is therapeutically relevant in such oxidant‐stressed environments. Encapsulating small hydrophilic molecules into hydrophobic polymer microparticles via traditional emulsion methods has long been a challenge due to rapid mass transport of small molecules out of particle pores. We have developed a simple alteration to the existing water‐in‐oil‐in‐water (W/O/W) drug encapsulation method that dramatically improves loading efficiency: doping external water phases with drug to mitigate drug diffusion out of the particle during fabrication. PLGA microparticles with diameters ranging from 0.6 to 0.9 micrometers were fabricated, encapsulating high loads of 0.6–0.9 µm diameter PLGA microparticles were fabricated, encapsulating high loads of the antioxidant N‐acetylcysteine (NAC), and released active, ROS‐scavenging NAC for up to 5 weeks. Encapsulation efficiencies, normalized to the theoretical load of traditional encapsulation without doping, ranged from 96% to 400%, indicating that NAC‐loaded external water phases not only prevented drug loss due to diffusion, but also doped the particles with additional drug. Antioxidant‐doped particles positively affected the metabolism of oligodendrocyte progenitor cells (OPCs) under H₂O₂‐mediated oxidative stress when administered both before (protection) or after (rescue) injury. Antioxidant doped particles improved outcomes of OPCs experiencing multiple doses of H₂O₂ by increasing the intracellular glutathione content and preserving cellular viability relative to the injury control. Furthermore, antioxidant‐doped particles preserve cell number, number of process extensions, cytoskeletal morphology, and nuclear size of H₂O₂‐stressed OPCs relative to the injury control. These NAC‐doped particles have the potential to provide temporally‐controlled antioxidant therapy in neurodegenerative disorders such as multiple sclerosis (MS) that are characterized by continuous oxidative stress.     

Investigation of the effect of naringenin on oxidative stress‐related alterations in testis of hydrogen peroxide‐administered rats

Testis tissue is prone to oxidation because its plasma membrane contains many polyunsaturated fatty acids. Naringenin is a plant‐derived natural flavonoid. We investigated the possible ameliorative role of naringenin on the hydrogen peroxide (H₂O₂)‐induced testicular damage in Wistar rats. Animals received 12 mg/kg H₂O₂ by intraperitoneal injection, and 50 mg/kg naringenin via orogastric gavage for 4 weeks. In the H₂O₂ group, the testis malondialdehyde level increased, while the amount of reduced glutathione, glutathione transferase activities, and the testis weight decreased. There were severe testicular damages in the H₂O₂ group otherwise their grade were less in the naringenin + H₂O₂ group. However, the serum testosterone concentrations decreased in both the H₂O₂ and the naringenin + H₂O₂ groups. The testicular zinc and calcium levels reduced in the H₂O₂‐treated rats. In conclusion, the administration of H₂O₂ caused oxidative stress in the testes and naringenin supplementation decreased the H₂O₂‐induced effects, except for changes in testosterone levels.     

Redox‐regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9 induces H‐site flexibility

Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X‐ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH‐binding site (G‐site) and a hydrophobic co‐substrate‐binding site (H‐site). At elevated H₂O₂ concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H‐site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox‐regulated enzyme.     

Trace analysis of N‐acetyl‐L‐cysteine using luminol–H2O2 chemiluminescence system catalyzed by silver nanoparticles

N‐Acetyl‐L‐cysteine (NAC) can inhibit the luminol–H₂O₂, reaction, which is catalyzed by silver nanoparticles. Based on this phenomenon a new method was developed for NAC determination. Under optimum conditions, a linear relationship between chemiluminescence intensity and NAC concentration was found in the range 0.034–0.98 µg/mL. The detection limit was 0.010 µg/mL (S/N =3), and the relative standard deviation (RSD) was <5% for 0.480 µg/mL NAC (n =5). This simple, sensitive and inexpensive method has been applied to measure the concentration of NAC in pharmaceutical tablets. Copyright © 2014 John Wiley & Sons, Ltd.     

N‐acetylcysteine counteracts oxidative stress and prevents hCG‐induced apoptosis in rat Leydig cells through down regulation of caspase‐8 and JNK

We have earlier reported that following persistent stimulation with hCG, oxidative stress‐induced apoptosis in rat Leydig cells was mainly achieved through the extrinsic pathway. In the present study, the role of N‐acetylcysteine (NAC) in counteracting the oxidative stress and the mechanisms of inhibition of apoptosis under such conditions were investigated. NAC (1 mM) intervention with repeated hCG stimulation (50 ng/ml, four times, each with 30 min challenge) prevented the decline in Leydig cell viability and the rise in lipid peroxidation and reactive oxygen species. Simultaneously, the activities of the enzymes glutathione‐S‐transferase, catalase, superoxide dismutase and the intracellular glutathione and antioxidant capacity of the treated cells improved significantly. Apoptotic markers Fas, FasL, and caspase‐8, up‐regulated following repeated hCG exposure, were significantly down‐regulated following NAC co‐incubation. While Bcl‐2 expression was fully restored, Bax and caspase‐9 remained unchanged. NAC treatment induced down‐regulation of upstream JNK/pJNK and down‐stream caspase‐3 in the target cells. Taken together, the above findings indicate that NAC counteracted the oxidative stress in Leydig cells induced as a result of repeated hCG stimulation, and inhibited apoptosis by mainly regulating the extrinsic and JNK pathways of metazoan apoptosis. Mol. Reprod. Dev. 77:900–909, 2010. © 2010 Wiley‐Liss, Inc.     

O‐Methyltransferases involved in biphenyl and dibenzofuran biosynthesis

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O‐methylation reactions. cDNAs encoding the O‐methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab‐causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5‐dihydroxybiphenyl, supplied by the first pathway‐specific enzyme, biphenyl synthase (BIS). 3,5‐Dihydroxybiphenyl underwent a single methylation reaction in the presence of S‐adenosyl‐l ‐methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5‐hydroxyferulic acid. Both substrates were only methylated at the meta‐positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor‐treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N‐ and C‐terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is associated with altered pancreatic islets redox status

In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H₂O₂), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H₂O₂ and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H₂O₂ production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H₂O₂ (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine.     

Characterisation of antioxidants in photosynthetic and non‐photosynthetic leaf tissues of variegated Pelargonium zonale plants

Hydrogen peroxide is an important signalling molecule, involved in regulation of numerous metabolic processes in plants. The most important sources of H₂O₂ in photosynthetically active cells are chloroplasts and peroxisomes. Here we employed variegated Pelargonium zonale to characterise and compare enzymatic and non‐enzymatic components of the antioxidative system in autotrophic and heterotrophic leaf tissues at (sub)cellular level under optimal growth conditions. The results revealed that both leaf tissues had specific strategies to regulate H₂O₂ levels. In photosynthetic cells, the redox regulatory system was based on ascorbate, and on the activities of thylakoid‐bound ascorbate peroxidase (tAPX) and catalase. In this leaf tissue, ascorbate was predominantly localised in the nucleus, peroxisomes, plastids and mitochondria. On the other hand, non‐photosynthetic cells contained higher glutathione content, mostly located in mitochondria. The enzymatic antioxidative system in non‐photosynthetic cells relied on the ascorbate–glutathione cycle and both Mn and Cu/Zn superoxide dismutase. Interestingly, higher content of ascorbate and glutathione, and higher activities of APX in the cytosol of non‐photosynthetic leaf cells compared to the photosynthetic ones, suggest the importance of this compartment in H₂O₂ regulation. Together, these results imply different regulation of processes linked with H₂O₂ signalling at subcellular level. Thus, we propose green‐white variegated leaves as an excellent system for examination of redox signal transduction and redox communication between two cell types, autotrophic and heterotrophic, within the same organ.     

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

Neuregulin‐1 protects myocardial cells against H2O2‐induced apoptosis by regulating endoplasmic reticulum stress

Neuregulin‐1 (NRG‐1) is a stress‐mediated growth factor secreted by cardiovascular endothelial cells and provides the protection to myocardial cells, but the underlying mechanisms are not fully understood. This study aimed to demonstrate that NRG‐1 protects myocardial cells exposed to oxidative damage by regulating endoplasmic reticulum (ER) stress. Neonatal rat cardiac myocytes (NRCMs) were isolated and treated with H₂O₂ as a cellular model of ER stress. NRCMs were pretreated with different concentrations of NRG‐1. We found that NRG‐1 increased the viability and reduced the apoptosis of NRCMs treated by H₂O₂. Moreover, NRG‐1 reduced lactate dehydrogenase level, increased superoxide dismutase activity and decreased malondialdehyde content in NRCMs treated by H₂O₂. Finally, we demonstrated that NRG‐1 alleviated ER stress and decreased CHOP and GRP78 protein levels in NRCMs treated by H₂O₂. Taken together, these data indicate that NRG‐1 relieves oxidative and ER stress in NRCMs and suggest that NRG‐1 is a promising agent for cardioprotection. Copyright © 2014 John Wiley & Sons, Ltd.