Quasi-elastic scattering measurements of the 28Si + 142Nd system at back-angle

409-414The barrier distribution of a system can be extracted from excitation function data obtained either through fusion reaction or through quasi-elastic scattering measurement. In the present work, the quasi-elastic excitation function has precisely been measured at back angle for the 28Si + 142Nd system at energies around the Coulomb barrier and the corresponding experimental barrier distribution has been extracted. The experimental data has been interpreted in the frame work of the coupled channel calculations which include couplings to different possible modes of excitations of the interacting target-projectile combination. The possible effect of the nature of projectile excitations on the derived barrier distribution has been presented

Quasi-elastic scattering measurements of the 28Si + 142Nd system at back-angle

The barrier distribution of a system can be extracted from excitation function data obtained either through fusion reaction or through quasi-elastic scattering measurement. In the present work, the quasi-elastic excitation function has precisely been measured at back angle for the 28Si + 142Nd system at energies around the Coulomb barrier and the corresponding experimental barrier distribution has been extracted. The experimental data has been interpreted in the frame work of the coupled channel calculations which include couplings to different possible modes of excitations of the interacting target-projectile combination. The possible effect of the nature of projectile excitations on the derived barrier distribution has been presented

Redox Manipulation: An Approach for Preferential Killing of Cancer Cells by Oxidative Stress Inducers.

The beginning of modern era of chemotherapy can be traced back to 1942 with the first use of nitrogen mustards drugs. Though some other modes of treatment like radiation therapy emerged successively, they didn’t bring complete cure. As a result chemotherapeutic approach have come under the focus worldwide. Several decades of intensive study in this area revealed a number of mechanisms through which they mediate cell death by apoptosis and/or necrosis. Reactive oxygen species (ROS) are normal metabolic byproducts that are generated continuously in cells. Redox manipulation is emerging as a therapeutic approach to treat cancer. Opposite effects of intracellular ROS on normal cells versus cancer cells have recently been hypothesized. As the basal level of intracellular ROS is low in normal cells, its increase up to certain extent is expected to be associated with cell proliferation. On the other hand, higher threshold of intracellular ROS in tumor cells, close to the threshold of cytotoxicity, is associated with higher cellular proliferation. Additional increase of intracellular ROS is therefore likely to inhibit tumor cell proliferation. Intrinsic higher threshold of ROS in cancer cells has been exploited in the present study for preferential killing of cancer cells of different origin by some ROS inducing agents. Cell death will be assessed in vitro on a number of cancer cell lines and normal human peripheral blood mononuclear cells and in vivo in nude mice model using human tumor xenografts. Apoptosis will be evaluated by flowcytometry after staining with annexin V staining and by evaluation of caspase activation. Redox- sensitive signal transudation pathways will also be evaluated in the current study

Seizure, spinal schwannoma, peripheral neuropathy and pulmonary stenosis – A rare combination in a patient of Neurofibromatosis 1

Neurofibromatosis 1 (NF1) is the most common neurocutaneous syndrome. It is estimated to occur in approximately 1 out of every 3300 infants. The manifestations of this condition are diverse and can arise from almost any system in the body. The neurofibroma is the hallmark lesion of NF1 that develops from peripheral nerves. Here, we are reporting an 18-year-old girl with NF1. Clinical diagnosis was made according to the diagnostic criteria established by the National Institutes of Health Consensus Development Conference in 1987. She presented with quadriparesis due to dumbbell-shaped spinal schwannoma in the cervical region. She had history of recurrent seizures in the past, with poor scholastic performance. There were clinical and electrophysiological features of peripheral neuropathy and clinical and echocardiographical features of pulmonary stenosis. These are uncommon features of NF 1. The presence of all these features in a single patient makes it a unique case

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H2O2, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl? cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl? CML cell lines and primary cells from CML patients significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NACmediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl? cells. Conclusion NAC enhances imatinib-induced apoptosis of Bcr-Abl? cells by endothelial nitric oxide synthasemediated production of nitric oxide

Involvement of ROS in Chlorogenic Acid-Induced Apoptosis of Bcr-Abl+ CML Cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl+ chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl+ cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chlinduced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells fromCML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl+ cells

Synergistic Apoptosis of CML Cells by Buthionine Sulfoximine and Hydroxychavicol Correlates with Activation of AIF and GSH-ROS-JNK-ERK-iNOS Pathway

<div><p>Background</p><p>Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress –induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism.</p><p>Materials and Methods</p><p>Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexinV-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action.</p><p>Results</p><p>Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells.</p><p>Conclusion/Significance</p><p>BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.</p></div

Apoptosis induced by combination of BSO and HCH is mediated by JNK dependent ERK1/2 activation.

<p>(<b>A</b>) K562 cells were treated as indicated for different time periods and cell lysates were subjected to western blot analysis with indicated antibodies. (<b>B</b>) K562 cells were pretreated with JNK inhibitor SP600125 (20 µM), p38 inhibitor SB203580 (20 µM), ERK inhibitor PD98059 (40 µM) for 1 h before treatment with BSO (100 µM) and HCH (10 µM) in combination. After 36 h of incubation, cells were subjected to Annexin V/PI binding assay by flow cytometry. Data represent mean ± SD of three experiments. *** p<0.001 compared to treatment in absence of MAPK inhibitors. (<b>C</b>) Knockdown of JNK by specific JNK1 siRNA attenuates apoptosis. JNK1 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry in K562 cells after transfection with indicated siRNAs (lower panel). Dot plots are representative of two similar experiments. (<b>D</b>) Knockdown of ERK by specific ERK2 siRNA attenuates apoptosis. ERK2 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry after knocking down ERK2 (lower panel). Dot plots are representative of two similar experiments (<b>E</b>) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 18 h. Protein expression and phosphorylation of JNK and ERK1/2 were analysed by western blot on whole cell lysates. (<b>F</b>) K562 cells were pre-incubated with or without 2 mM GME for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) in combination for 18 h. The level of each protein and phosphorylation status was analyzed as indicated by western blot. (<b>G</b>) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 24 h. Analysis of intracellular NO was done by flow cytometry after staining with DAF-FM. Histograms are representative of two similar experiments.</p

Nitric oxide is produced by inducible nitric oxide synthase (iNOS); iNOS expression is dependent on ERK 1/2 and JNK activation.

<p>(<b>A</b>) K562 cells were treated as indicated for varying time periods and the whole cell lysates were subjected to western blot analysis with indicated antibodies. (<b>B</b>) After transfection of K562 cells with indicated siRNAs followed by indicated treatments for 24 h, cells were stained with DAF-FM for measurement of NO. Representative of two similar histograms. (<b>C</b>) K562 cells were transfected with control siRNA or indicated NOS siRNAs for 48 h and then treated with combination of BSO (100 µM) and HCH (10 µM) for 24 h. Whole cell lysates were subjected to western blot for the expression of indicated proteins to confirm knockdown of representative NOS isoforms. (<b>D</b>) K562 cells were subjected to annexin V/PI binding assay by flow cytometry after 36 h. Representative of two similar histograms. (<b>E</b>) K562 cells were transfected with JNK1 or ERK2 siRNA. Cells were then treated with combination of BSO (100 µM) and HCH (10 µM) for 18 h. Cell lysates were then immunoblotted with anti-iNOS antibody. (<b>F</b>) iNOS siRNA transfected K562 cells were treated with combination of BSO (100 µM) and HCH (10µM) for 18 h and subjected to immunoblot analysis with indicated antibodies. (<b>G</b>) K562 cells were pre-incubated with GME for 1 h before treatment with combination of BSO and HCH for 18 h and cell lysates were then immunoblotted with anti-iNOS antibody. (<b>H</b>) GSH measurement was done in iNOS transfected K562 cells after combined treatment with BSO and HCH for 18 h. Data represent mean ± SD of three experiments. *** p<0.001 compared to vehicle control.</p