Genome-wide lentiviral shRNA screen identifies serine/arginine-rich splicing factor 2 as a determinant of oncolytic virus activity in breast cancer cells.

Oncolytic human herpes simplex virus type 1 (HSV-1) shows promising treatment efficacy in late-stage clinical trials. The anticancer activity of oncolytic viruses relies on deregulated pathways in cancer cells, which make them permissive to oncolysis. To identify pathways that restrict HSV-1 KM100-mediated oncolysis, this study used a pooled genome-wide short hairpin RNA library and found that depletion of the splicing factor arginine-rich splicing factor 2 (SRSF2) leads to enhanced cytotoxicity of breast cancer cells by KM100. Serine/arginine-rich (SR) proteins are a family of RNA-binding phosphoproteins that control both constitutive and alternative pre-mRNA splicing. Further characterization showed that KM100 infection of HS578T cells under conditions of low SRSF2 leads to pronounced apoptosis without a corresponding increase in virus replication. As DNA topoisomerase I inhibitors can limit the phosphorylation of SRSF2, we combined a topoisomerase I inhibitor chemotherapeutic with KM100 and observed synergistic anticancer effect in vitro and prolonged survival of tumor-bearing mice in vivo.

Comprehensive essential gene identification as a platform for novel anti-infective drug discovery.

In large part, antimicrobial drug discovery is driven by the breadth and quality of both potential drug targets and available chemical libraries to screen. Traditionally, targets have been few in number and have been limited to those with known function, from which biochemical assays could be implemented into drug screens. Iterations of this same basic approach, applied to a few biochemically-defined targets have identified a limited set of novel antibiotics and even fewer antifungal agents. Indeed, in the last 50 years less than 30 antimicrobial targets have been exploited commercially. Within infectious disease, the industry was driven largely by chemistry-based approaches, simply making new analogs to existing drugs to overcome the growing problem of drug resistance. Elitra Pharmaceutical s approach has been to enable true functional genomics on a genome-wide scale. Elitra s vision has been to identify all of the essential genes directly in the key pathogenic organisms. Having moved rapidly towards the completion of this goal, we are now faced with the enviable challenge of prioritizing enormous target sets and developing novel sensitive screens for those best suited as definitive drug targets. These highly sensitive, cell-based screening paradigms enable re-screening of even well screened chemical libraries to reveal new chemical entities displaying novel modes of action against new targets. In parallel, we have also begun to shift the paradigm from screening targets singly, towards genome-wide approaches to drug screening.

Neutrophil free oxygen radical production and blood total antioxidant capacity in patients with coronary heart disease using various medications.

Despite convincing results of studies in vitro, less is known about the effects of antioxidants on in vivo redox balance in humans. We developed a novel parameter of in vivo redox balance, and studied it and its relation to dental infections in 51 patients on medication for coronary heart disease (CHD) and 39 random controls matched for age group, sex, social class and locality. In vivo redox balance was the ratio of plasma antioxidant capacity, as measured with radical-trapping assay, to neutrophil respiratory burst capacity, as measured with whole blood chemiluminescence assay. Dental infections were quantitated with four rating scales. CHD patients had higher values than controls. Patients on acetosalicylic acid (ASA), diuretics or beta blockers, but not the ones on calcium channel blocker, had significantly higher redox balance than non-users. Combination of calcium channel blockers and ASA was associated with redox balance similar to taking beta blockers or diuretics. Diuretics and ASA were independent determinants of redox balance in multivariate analyses. Redox balance did not correlate with severity of dental infections (Spearman's r 0.06 to 0.11). The results contrast experimental data indicating that calcium channel blockers are as antioxidants superior to other cardiovascular drugs. Total antioxidant capacity in parallel with oxygen species production capacity should be considered in attempts to solve the antioxidant paradox.

Saccharomyces cerevisiae mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway.

The MID2 gene of Saccharomyces cerevisiae encodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor. MID2 was isolated as a multicopy activator of the Skn7p transcription factor. Deletion of MID2 causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression of MID2 causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. alpha-Factor hypersensitivity of mid2Delta mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, including PKC1, RHO1, WSC1, and WSC2. Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since mid2Delta wsc1Delta mutants are inviable on medium that does not contain osmotic support. A role for MID2 in the cell integrity pathway is further supported by the finding that MID2 is required for induction of Mpk1p tyrosine phosphorylation during exposure to alpha-factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame, MTL1, which encodes a protein with both structural and functional similarity to Mid2p.

Modulation of arachidonic acid metabolism by phenols: relation to their structure and antioxidant/prooxidant properties.

The effects of substituted catechols (3-methylcatechol, 4-methylcatechol, 4-nitrocatechol, and guaiacol) and trihydroxybenzenes (pyrogallol, propyl gallate, 1,2,4-trihydroxybenzene, and 1,3,5-trihydroxybenzene) on the synthesis of prostaglandin (PG)E2 and leukotriene (LT)B4 were tested in human A23187-stimulated polymorphonuclear leukocytes. The effects were related to their peroxyl-radical-scavenging (antioxidant), superoxide-scavenging (antioxidant), and superoxide-generating (prooxidant) properties. In general, compounds with hydroxyl groups in the ortho position increased PGE2/LTB4 ratio, and compounds with hydroxyl groups in the meta position decreased PGE2/LTB4 ratio. Catechols, which have hydroxyl groups in the ortho position, were the most potent peroxyl radical and superoxide anion scavengers. Trihydroxybenzenes (pyrogallol, 1,2,4-trihydroxybenzene, and 1,3,5-trihydroxybenzene) generated superoxide, whereas dihydroxybenzenes did not. Thus, the positions and number of hydroxyl groups seem to be the most important properties determining the action of phenolic compounds on PGE2/LTB4 ratio and their antioxidant/prooxidant activities.

Plasma chain-breaking antioxidants in preterm infants with good and poor short-term outcome.

Many complications of prematurity have been suggested to result from free radical generation and an inadequacy of antioxidative capacity. We measured the plasma total peroxyl radical-trapping capability (TRAP) and concentrations of the main chain-breaking antioxidants contributing to it, i.e. uric acid, ascorbic acid, alpha-tocopherol, protein sulfhydryl groups and bilirubin, in 21 preterm infants with a mean birth weight of 1440 g and gestational age of 30 wk. The infants were divided into two groups according to their short-term outcome; the good outcome group (GOG) (N = 11) with no signs of morbidity and the poor outcome group (POG) (N = 10) with intraventricular haemorrhage and/or bronchopulmonary dysplasia and/or retinopathy. Arterial blood samples were obtained 3 and 10 days postpartum. TRAP was measured with a chemiluminescent method. As a comparison, venous blood samples from 13 adults (aged from 18 to 34) were used. At day 3 the poor outcome group had significantly higher TRAP than the good outcome or control group, mainly because of elevated uric acid concentration. Also the concentration of unidentified antioxidants was significantly lower in GOG. By day 10 the TRAP decreased substantially in both groups. However, from the components of TRAP, both ascorbate and the unidentified fraction decreased more in POG (p = 0.017 and 0.021, respectively). Furthermore in POG on day 10 urate concentration did not significantly differ from day 3 values. In conclusion, in preterm infants high TRAP was associated with high plasma uric acid concentration and a poor short-term prognosis.

Yeast Skn7p activity is modulated by the Sln1p-Ypd1p osmosensor and contributes to regulation of the HOG pathway.

Activation and control of the yeast HOG (High Osmolarity Glycerol) MAP kinase cascade is accomplished, in part, by a two-component sensory-response circuit comprised of the osmosensing histidine protein kinase Sln1p, the phospho-relay protein Ypd1p, and the response regulator protein Ssk1p. We found that deletion of SLN1 and/or YPD1 reduces reporter gene transcription driven by a second two-component response regulator -- Skn7p. The effect of sln1delta and ypd1delta mutations upon Skn7p activity is dependent on a functional two-component phosphorylation site (D427) in Skn7p, suggesting that Sln1p and Ypd1p may act as phosphodonors for Skn7p. We also observed that loss of PTC1 (a protein serine/threonine phosphatase implicated in negative control of the HOG pathway) in a skn7delta background results in severely retarded growth and in morphological defects. Deletion of either PBS2 or HOG1 alleviates the slow growth phenotype of ptc1delta skn7delta cells, suggesting that Skn7p may participate, in concert with known regulatory components, in modulating HOG pathway activity. The contribution of Skn7p to HOG pathway regulation appears to be modulated by the receiver domain, since non-phosphorylatable Skn7pD427N is unable to fully restore growth to ptc1/skn7 cells.

Effects of surgical removal of lung cancer on total plasma antioxidant capacity in lung cancer patients.

Previous evidence suggests that malignant tumors cause an oxidative burden to human antioxidative defense systems. We followed the plasma total radical-trapping antioxidant parameters (TRAP) and their main antioxidant components (alpha-tocopherol, uric acid, protein sulfhydryl groups, and unidentified antioxidant proportions) in 13 lung cancer patients and 7 control patients scheduled for thoracotomy. Plasma samples were collected 9 times during a 5 month follow-up period in the cancer patients. The objective of the study was to evaluate the effects of surgical removal of lung cancer on human plasma total antioxidant capacity. A significant reduction of plasma TRAP (period effect of ANOVA, p = 0.0006) and its components appeared in both groups during the first postoperative day. This decrease was due to reduction of ascorbate (p = 0.002) alpha-tocopherol (p = 0.0001) and urate (p = 0.05) concentrations. At 3 and 5 months after the surgical removal of the tumor there was an augmentation in plasma TRAP concentrations (p = 0.02, 3 months; p = 0.07, 5 months). This was mainly due to the increases in plasma yet as unidentified antioxidant components (UNID) and protein SH-groups. The data indicates that, first, thoracotomy itself causes a reduction in plasma TRAP during the early hours after operation, and secondly surgical removal of lung cancer increases plasma TRAP concentrations compared to the baseline values possibly reflecting the relief of oxidative stress caused by malignant tumors.

Radical releasing properties of nitric oxide donors GEA 3162, SIN-1 and S-nitroso-N-acetylpenicillamine.

The nitric oxide (NO)-, superoxide anion (O2.-)- and peroxynitrite (ONOO-)-releasing properties of 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162) were characterized and compared with the known NO-donors 3-morpholino-sydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine. All the three compounds released NO in aqueous solutions in a dose-dependent manner as measured by ozone-chemiluminescence. GEA 3162 produced more NO than SIN-1, but less than S-nitroso-N-acetylpenicillamine during a 45 min incubation time. SIN-1 reduced nitro blue tetrazolium and the effect was inhibitable by superoxide dismutase. Reduction of nitro blue tetrazolium was not detected in the solutions of GEA 3162 and S-nitroso-N-acetylpenicillamine suggesting that SIN-1 but not GEA 3162 and S-nitroso-N-acetylpenicillamine release O2.- in their decomposition process. Formation of ONOO- in solutions of GEA 3162, SIN-1 and S-nitroso-N-acetylpenicillamine was estimated indirectly by measuring the formation of nitrotyrosine. The data indicate that ONOO- was produced in the presence of SIN-1 but not in solutions of GEA 3162 and S-nitroso-N-acetylpenicillamine. The results suggest that GEA 3162 produces negligible amounts of O2.- and ONOO- as compared to SIN-1. This adds the value of GEA 3162 as an useful tool in NO research and could well explain the earlier findings on the superior NO-like biological activity of oxatriazole derivatives as compared to SIN-1.

Human plasma antioxidant capacity during radiotherapy for lung cancer: a clinical study.

Even though it is well established that oxygen-free radicals are the main mechanism responsible for the cytotoxicity produced during radiotherapy, the role of the human antioxidant defense system in clinical radiation oncology is still to be clarified. Changes in the human plasma total peroxyl radical trapping capacity (TRAP) and its individual components were followed during clinical radiotherapy for lung cancer. Sixteen patients receiving radical-aimed radiotherapy provided blood samples nine times during the treatment. Our hypothesis was that oxygen-free radical production increased by irradiation should decrease the plasma TRAP as a consequence of oxidative stress. Only a moderate reduction of the plasma TRAP was found during the therapy in the study group taken as a whole, but the development pattern of TRAP and its unidentified components were clearly different in those patients showing complete or partial response to the treatment and those in which the disease progressed unabated. Plasma ascorbate levels showed no significant changes during radiotherapy. A decrease in vitamin E concentrations was seen after 6 Gy (p=0.05). Uric acid concentrations increased towards the end of the radiotherapy in both response groups (p=0.02 at 50 Gy). In this study, 26.6% of the plasma TRAP was due to unidentified antioxidants (UNID).

Effect of a new nitric oxide donor on the biomechanical performance of the isolated ischaemic rat heart.

The effect of a new nitric oxide (NO) donor, a meso-ionic 3-aryl substituted oxatriazole-5-imine derivative, GEA 3162 was studied on constant flow-perfused ischaemic Langendorff rat heart. The perfusion was kept constant at a rate of 16 mL min-1. Ischaemia was induced by a low flow rate of 0.8 mL min-1 for 30 min, and was followed by a 40-minute reperfusion. In the first set of experiments the effects of GEA 3162-infusion were examined on perfusion pressure, left ventricular pressure, heart rate and left ventricular dP/dt. GEA 3162 infusion did not affect the pre-ischaemic maximum of left ventricular pressure. During reperfusion, maximal left ventricular pressure, maximal and minimal dP/dt values in the GEA 3162-treated group significantly exceeded those of the untreated controls (by 19.3, 36.0 and 18.0%, respectively). During reperfusion, perfusion pressure increased continuously in the control group indicating an increasing coronary resistance, but it was kept at a continuous low level with GEA 3162 treatment. In a second set of experiments bradykinin was infused in order to test the endothelial function before ischaemia and during late reperfusion. Bradykinin elicited significant vasodilation in the control group during reperfusion, meanwhile it did not cause further change in coronary resistance in the GEA 3162-infused group. We suggest, that GEA 3162 may have a protective effect on isolated rat heart in ischaemia and reperfusion, that results in an improved cardiac performance compared with untreated hearts.

Effects of dietary fluoride and magnesium supplements on cyclic adenosine monophosphate (cAMP), calcium and magnesium levels in aorta of genetically hypercholesterolaemic RICO rats.

Previous observations have suggested that low intakes of fluoride prevent pathological calcifications of internal organs, including the aortic wall, in experimental animals, fed a basically low magnesium diet. Our group found recently that fluoride has some potentially preventive effect against atherosclerotic serum lipid profiles in genetically hypercholesterolaemic rats. To study whether the apparently positive potential of fluoride against atherosclerosis is also reflected in aortic tissue, through its well known activation of adenylate cyclase, the aortic cAMP content of the rats used in our recent study was determined. Out of a total of 56 male RICO rats, mean weight 160 g, the control group C was fed an adequate diet, with 44% sucrose, a magnesium content of 883 p.p.m. and with 0.5% cholesterol. Group D had the same diet as group C except that the magnesium content was reduced to 200 p.p.m. Group E had the same diet as group D but with the fluoride content elevated from 1.9 to 12 p.p.m. Group G had the same diet as group E but with the magnesium content elevated from 200 to 300 p.p.m. After a feeding period of 6 weeks, the aortas of the animals were removed, cleaned and kept at -70 degrees C until analysed. The mean cAMP content of the aortas, measured by radioimmunoassay, in groups C, D E and G was 439, 546, 681, and 1394 mumol mg-1 protein, respectively. In group G only, the cAMP content was significantly higher than that of the other groups (p < 0.001). The mean calcium and magnesium contents of the aortas of different groups did not significantly differ from each other. Thus in RICO rats, fed a high-sugar low-magnesium diet with cholesterol, supplementation of the diet with a small amount of fluoride elevates the cAMP content of the aorta, provided that the intake of Mg is not very low.

Enhanced oxidizability of ubiquinol and alpha-tocopherol during lovastatin treatment.

A double-blinded, placebo-controlled cross-over trial was carried out with 27 hypercholesterolemic men with coronary heart disease. During the 6-week treatment period lovastatin (60 mg/day) decreased fasting serum LDL cholesterol by 45%, LDL phosphorus by 38% and apoB by 33%. Ubiquinol content diminished by 13% as measured per LDL phosphorus. When LDL was oxidized ex vivo with AMVN both LDL ubiquinol and alpha-tocopherol were exhausted faster after lovastatin treatment compared to placebo, by 24% (P < 0.005) and 36% (P < 0.0001), respectively. Lag time in copper-induced oxidation of LDL decreased by 7% (P < 0.01). This suggests diminished antioxidant-dependent resistance of LDL to the early phase of oxidative stress.

Plasma peroxyl radical trapping capacity in lung cancer patients: a case-control study.

Increasing evidence suggests that cancer patients express oxidative disturbances. The main objective of this cross-sectional case-control study (n = 57 + 76) was to explore whether lung cancer patients, when compared to healthy controls, have alterations in their plasma peroxyl radical trapping capacity (TRAP). Group matching was used with respect to age, sex and smoking history. A secondary objective was to observe the effects of life-long cigarette consumption on plasma TRAP and its components. Mean TRAP values were significantly lower in the cancer patients than in the control group (1143 vs 1273 mumol/l, p = 0.0002). Moreover, all the components of TRAP (except uric acid) were significantly lower in the cancer group: protein SH-groups 442 vs 571 mumol/l, ascorbic acid 34.0 vs 46.5 mumol/l and vitamin E 25.0 vs 33.8 mumol/l. The as yet unidentified antioxidant compounds in plasma contributed 26.5% of plasma TRAP in the cancer group and 30.2% in the control group. There was no correlation between cigarette consumption in pack-years and plasma TRAP; however, plasma concentrations of uric acid and ascorbic acid were negatively correlated with cigarette consumption.

Unidentified antioxidant defences of human plasma in immobilized patients: a possible relation to basic metabolic rate.

Plasma total peroxyl radical scavenging capacity was studied in terminal patients who were chronically immobilized because of an acute (stroke) or chronic neurodegenerative disease (Alzheimer's disease). A luminometric assay was used to measure total antioxidant capacity (TRAP). The immobilized patients showed significant decrease in TRAP primarily because of a decrease in the concentration of unknown antioxidants. Our results suggest that human plasma may contain unknown antioxidants, the regulation of which could be related to the basic metabolic rate.

Mode of cytostatic action of mesoionic oxatriazole nitric oxide donors in proliferating human hematopoietic cells.

The cytopathological effects of the novel nitric oxide (NO)-releasing, mesoionic 3-aryl-substituted oxatriazole-5-imine derivatives GEA 3162 and GEA 3175, and a reference NO donor SIN-1 were investigated in proliferating human hematopoietic cells. The GEA compounds (10-50 microM) induced rapid surface changes, which progressed as peculiar deep indentations and strictures in human leukemic T cells (MOLT-3) in 30 min. An excess of red cells partially prevented these surface changes. GEA 3162-treated MOLT-3 cells became permeable to ethidium bromide and lost their ability to be stained by acridine orange after 5 h of exposure. GEA 3162 and GEA 3175 suppressed thymidine and uridine incorporation in a dose-dependent manner, reflecting the inhibition of DNA and RNA synthesis respectively. In addition, the GEA compounds inhibited the growth of human bone marrow stem cells, CFU-GM colonies being more susceptible to the cytostatic action than BFU-E. The reference compound SIN-1 had comparative cytostatic effects at ten times greater concentrations (500 microM). We conclude that NO-releasing mesoionic oxatriazole derivatives have cytostatic action against human malignant and non-malignant hematopoietic cells, supporting the value of NO-releasing and NO-inducing compounds as anti-cancer agents.

Age-related changes in the peroxyl radical scavenging capacity of human plasma.

Aging and the diseases that typically follow with increasing age, notably atherosclerosis and cancer, are often proposed to be involved in increased oxidative stress. Animal studies, on the other hand, show no clear-cut pattern of age-related changes in enzymatic antioxidant defences. In this study we have demonstrated that total peroxyl radical scavenging antioxidant capacity (TRAP) in human plasma changes with age. We also found that among the antioxidants in human plasma there exists a major fraction of so far unidentified antioxidant(s). A chemiluminescent TRAP assay was used to determine the presence of peroxyl radical scavenging antioxidants in human plasma. The material consisted of 87 healthy volunteers, aged 20-96 years, who used no regular medication, vitamins, or trace elements. In females, total antioxidant capacity increased significantly during the life span. The increase in TRAP was mainly due to unidentified antioxidants. In males, TRAP increased until age 51-74, and then significantly decreased. The decrease observed among males was also due to the sharp decline in the concentration of unidentified antioxidants.

Effect of oral coenzyme Q10 supplementation on the oxidation resistance of human VLDL+LDL fraction: absorption and antioxidative properties of oil and granule-based preparations.

Coenzyme Q10 (Q10) is supposed to be an important endogenous lipid-soluble antioxidant. We studied 60 healthy 46 +/- 7 (mean +/- SD)-year-old smoking men. They were randomized into three groups to receive oil-based or granular Q10 (90 mg/d) or placebo for 2 months. Oil-based capsule elevated Q10 in plasma by 178% and in VLDL+LDL fraction by 160%. The granular preparation increased Q10 in plasma by 168% and in VLDL+LDL by 127%. However, the 2-month Q10 supplementation did not increase the oxidation resistance of VLDL+LDL fraction, as assessed by copper induced VLDL+LDL oxidation, haemin+H(2)O(2)-induced VLDL+LDL oxidation, total antioxidative capacity of LDL, and plasma malondialdehyde measurements. The first and the last dose was used to carry out a 12 h pharmacokinetic study (five subjects per group), which indicated that only a small part of supplemented Q10 was absorbed to the circulation in 12 h and that the absorption varied extensively between subjects. Our results suggest that at least among smoking men, 90 mg of orally supplemented Q10 daily does not increase the oxidation resistance of VLDL+LDL. Bioavailability of both the granular and the oil-based Q10 preparation was similar during the long-term supplementation, but one dose of 30 mg had only a marginal effect on the plasma levels of Q10.

Ubiquinol-10 and total peroxyl radical trapping capacity of LDL lipoproteins during aging: the effects of Q-10 supplementation.

Evidence is rapidly accumulating that oxidative modification of low density lipoprotein (LDL) may play an important role in the pathogenesis of atherosclerosis. In this study we measured the total peroxyl radical trapping capacity of human plasma LDL phospholipids (TRAPLDL) with a luminescent method. The study was carried out with 70 healthy volunteers, aged 28-77. In males an age-related decrease in TRAPLDL was observed. In the age group under 50 years the mean TRAPLDL was 31.36 +/- 1.45 pmol peroxyl radicals/nmol Pi; among those over 50 years it was significantly lower at 26.67 0.94 pmol/nmol Pi. As regards the components of TRAPLDL, the concentration of LDL-ubiquinol did not change and a non-significant decrease in the LDL-tocopherol concentration was detected with age. In females, the mean TRAPLDL, LDL-ubiquinol-10 and tocopherol concentrations did not differ between the age groups. When 17 of the participants were given coenzyme Q10 (Q10) supplementation, 100 mg/day, a highly significant increase in LDL-ubiquinol concentration was detected. Our results indicate that LDL antioxidant defenses tend to decrease with age in the Finnish male population. The decline is most significant in males under 50 years; in older age groups the values remain stable at a low level. Q10 supplementation doubles the number of ubiquinol-10-containing LDL molecules and may therefore have an inhibitory effect on LDL oxidation.