Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Cellular defense against heat shock-induced oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of IDPm against heat shock in HEK293 cells, an embryonic kidney cell line, was investigated in control and cells transfected with the cDNA for IDPm, where IDPm activity was 6–7 fold higher than that in the control cells carrying the vector alone. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in control cells as compared to HEK293 cells in which IDPm was over-expressed. We also observed the significant difference in the cellular redox status reflected by the endogenous production of reactive oxygen species, NADPH pool and GSH recycling between two cells. The results suggest that IDPm plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of reactive oxygen species.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Carbonic Anhydrase Inhibitors: Synthesis of Sulfonamides Incorporating 2, 4, 6–Trisubstituted-Pyridinium-Ethylcarboxamido Moieties Possessing Membrane-Impermeability and in Vivo Selectivity for the Membrane-Bound (CA IV) Versus the Cytosolic (CA I and CA II) Isozymes

A new approach is proposed for the selective in vivo inhibition of membrane-bound versus cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes with a class of positively-charged, membrane-impermeant sulfonamides. Aromatic/heterocyclic sulfonamides acting as strong (but unselective) inhibitors of this zinc enzyme were derivatized by the attachment of trisub-stituted-pyridinium-ethylcarboxy moieties (obtained from 2, 4, 6–trisubstituted-pyrylium salts and β-alanine) to the amino, imino, hydrazino or hydroxyl groups present in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes, i.e., hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound isozyme; h = human; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, ethoxzolamide), are unable to penetrate biological membranes, as shown by CJ vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors suggest that: (i) when using the new type of positively-charged sulfonamides. only the membrane-bound enzyme (CA IV) was inhibited. whereas the cytosolic isozymes (CA I and II) were not affected, (ii) in the experiments in which the classical compounds (acetazolamide, bcn-zolamíde. etc.) were used. unselective inhibition of all CA isozymes (I. II and IV) occurred.     

PHOTOBIOTREATMENT: INFLUENCE OF NITROGEN AND PHOSPHORUS RATIO IN WASTEWATER ON GROWTH KINETICS OF SCENEDESMUS OBLIQUUS

Nitrogen and phosphorus concentration in the effluent of a wastewater treatment plant can vary significantly, which could affect the growth kinetic and chemical composition of microalgae when cultivated in this medium. The aim of this work was to study the rate of growth, nutrient removal and carbon dioxide biofixation as well as biomass composition of Scenedesmus obliquus (S. obliquus) when it is cultivated in wastewater at different nitrogen and phosphorus ratio, from 1:1 to 35:1. A more homogeneous method for calculating productivities in batch reactors was proposed. The proper N:P ratio for achieving optimum batch biomass productivity ranged between 9 and 13 (263 and 322 mg L^?1 d^?1 respectively). This was also the ratio range for achieving a total N and P removal. Above and below this range (9–13) the maximum biomass concentration changed, instead of the specific growth rate.The maximum carbon dioxide biofixation rate was achieved at N:P ratio between 13 and 22 (553 and 557 mg CO2 L^?1 d^?1 respectively). Lipid and crude protein content, both depend on the aging culture, reaching the maximum lipid content (34%) at the lowest N:P (1:1) and the maximum crude protein content (34.2%) at the highest N:P (35:1).     

Synthesis of D‐Altritol Nucleosides with a 3′‐O‐Tert‐Butyldimethylsilyl Protecting Group

Four D‐altritol nucleosides with a 3′‐O‐tert‐butyldimethylsilyl protecting group are synthesized (base moieties are adenine, guanine, thymine and 5‐methylcytosine). The nucleosides are obtained by ring opening reaction of 1,5:2,3‐dianhydro‐4,6‐O‐benzylidene‐D‐allitol. Optimal reaction circumstances (NaH, LiH, DBU, phase transfer, microwave irridation) for the introduction of the heterocycles are base‐specific. For the introduction of the 3′‐O‐silyl protecting group, long reaction times and several equivalents of tert‐butyldimethylsilyl chloride are needed.     

Mechanism of Inhibition of Xanthine Oxidoreductase by Allopurinol: Crystal Structure of Reduced Bovine Milk Xanthine Oxidoreductase Bound with Oxipurinol

Inhibitors of xanthine oxidoreductase block conversion of xanthine to uric acid and are therefore potentially useful for treatment of hyperuricemia or gout. We determined the crystal structure of reduced bovine milk xanthine oxidoreductase complexed with oxipurinol at 2.0 Å resolution. Clear electron density was observed between the N2 nitrogen of oxipurinol and the molybdenum atom of the molybdopterin cofactor, indicating that oxipurinol coordinated directly to molybdenum. Oxipurinol forms hydrogen bonds with glutamate802, arginine880, and glutamate1261, which have previously been shown to be essential for the enzyme reaction. We discuss possible differences in the hypouricemic effect of inhibitors, including allopurinol and newly developed inhibitors, based on their mode of binding in the crystal structures.     

CYP1A1 genetic polymorphisms,tobacco smoking and lung cancer risk in a French Caucasian population

The CYP1A1 gene encoding for an enzyme involved in the metabolic activation of important tobacco carcinogens could be implicated in smoking-induced lung cancer. Given the strong association between tobacco smoking and lung cancer, the effect of tobacco smoke exposure has to be taken into account when studying the potential association between lung cancer and CYP1A1 genotypes. The effect of two CYP1A1 genetic polymorphisms (Mspl and IIe-Val) on lung cancer risk were evaluated using peripheral blood DNA from 150 lung cancer patients and 171 controls. The Mspl sitepresent allele was found among 19.3% of both cases and controls and the variant allele Val among 6.7% of cases and 8.8% of controls. Lung cancer risks associated with the Mspl site-present allele (OR= 0.9; 95%Cl: 0.5-1.8) or with the Val allele (OR= 0.8; 95%Cl: 0.3-1.9) were not increased after adjustment for tobacco and asbestos exposures. These results persisted when analyses were stratified on smoking status, daily consumption of tobacco or duration of smoking. Similar findings were obtained when squamous cell or small cell carcinomas were studied separately. This study thus suggests a minor role for the known CYP1A1 gene polymorphisms in predisposition to lung cancer among Caucasian populations.