School Of Basic And Applied Sciences

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    Oxidative stress stimulates invasive potential in rat C6 and human U-87 MG glioblastoma cells via activation and cross-talk between PKM2, ENPP2 and APE1 enzymes.
    (Springer, 2018) Cholia, Ravi P.; Dhiman, Monisha; Kumar, Raj; Mantha, Anil K.
    Maintaining genomic integrity is essential for cell survival and viability. Reactive oxygen species (ROS) overproduction results in oxidative stress leading to the genomic instability via generation of small base lesions in DNA and these unrepaired DNA damages lead to various cellular consequences including cancer. Recent data support the concept "oxidative stress is an indispensable participant in fostering proliferation, survival, and migration" in various cancer cell types including glioblastoma cells. In this study we demonstrate that treatment of non-cytotoxic doses of oxidants such as amyloid beta [Aβ(25-35)] peptide, glucose oxidase (GO), and hydrogen peroxide (H2O2) for 24 h and 48 h time points found to increase the expression level and activity of a multifunctional enzyme Apurinic/apyrimidinic endonuclease (APE1), a key enzyme of base excision repair (BER) pathway which takes care of base damages; and also resulted in modulation in the expression levels of downstream BER-pathway enzymes viz. PARP-1, XRCC1, DNA polβ, and ligase IIIα was observed upon oxidative stress in C6 and U-87 MG cells. Oxidants treatment to the C6 and U-87 MG cells also resulted in an elevation in the intracellular expression of glycolytic pathway enzyme Pyruvate kinase M2 (PKM2) and the metastasis inducer protein Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) as analyzed using Western blotting and Immunofluorescence microscopic studies. Our study also reports that oxidative stress induced for 24 h and 48 h in C6 and U-87 MG cells resulted in extracellular secretion of APE1 and ENPP2 as analyzed using Western blotting in conditioned media. However, the biological significance of extracellular secreted APE1 remains elusive. Oxidative stress also elevated the ENPP2's LysoPLD activity in conditioned media of C6 and U-87 MG cells. Our results also demonstrate that oxidative stress affects the expression level and localization of APE1, PKM2, and ENPP2 in C6 and U-87 MG cells. As evidenced by the colocalization pattern at 24 h and 48 h time points, it can be attributed that oxidative stress mediates crosstalk between APE1, PKM2, and ENPP2. In addition, when C6 and U-87 MG cells were treated with lysophosphatidic acid (LPA), a bioactive lipid that negatively regulates ENPP2's LysoPLD activity at 10 μM concentration, demonstrated strong migratory potential in C6 and U-87 MG cells, and also induced migration upon oxidative stress. Altogether, the findings demonstrate the potential of C6 and U-87 MG cells to utilize three proteins viz. APE1, PKM2, and ENPP2 towards migration and survival of gliomas. Thus the knowledge on oxidative stress induced APE1's interaction with PKM2 and ENPP2 opens a new channel for the therapeutic target(s) for gliomas.
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    Assessment of antioxidant potential of phytochemicals in human glioblastoma (U-87 MG) cells
    (Central University of Punjab, 2014) Kaur, Manpreet; Mantha, Anil K.
    Imbalance between production of reactive oxygen/nitrogen species (ROS/RNS) leads to oxidative stress and has been well documented for mitochondrial dysfunction, a prime cause towards pathogenesis of neurological diseases and cancer. Glioblastoma Multiforme (GBM) is a highly aggressive, invasive and primary brain tumor which shows resistance to chemotherapy and radiotherapy. Superoxide dismutase (SOD) is an antioxidant enzyme that scavenges the production of superoxide radicals and dismutases into H?O? which is further converted into H?O and O? by catalase (CAT) enzyme. Apurinic/Apyrimidinic endonuclease (APE1) is a central enzyme of base excision repair (BER) pathway with two important functions; DNA repair and redox regulation of transcription factors (TFs) responsible for cell survival. In this study, it was seen that oxidative stress induced by endogenously found oxidants H?O? and glucose oxidase (GO) enhanced the activities of both CuZn-SOD and MnSOD in U-87 MG cells. In addition, CuZn-SOD levels were found to be increased in H?O?-induced oxidative stress and MnSOD levels were found to be increased in both H?O? and GO- induced oxidative stress. Further, pretreatment with phytochemicals Curcumin and Quercetin modulated the activities and expression of both forms of SOD studied. The BER-pathway enzyme, APE1 level was found to be decreased in mitochondria of oxidative stress induced U-87 MG cells by H?O? and GO, and in contrast APE1 level was found to be increased in cytosol, which indicates that oxidative stress affects the expression level and sub-cellular localization of APE1. Taken together, these results indicate that in GBM it is more likely that activated SOD a key player of antioxidant system and APE1 a key player in BER-pathway might be facilitating cancer cells to survive in oxidative stress environment.
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    Design and synethesis of APE1 inhibitors as putative anticancer agents
    (Central University of Punjab, 2014) Kaur, Gagandeep; Kumar, Raj
    Success in chemotherapy has not been attained completely yet and has remained a worried issue from years. Various reasons drive this failure, but the much talked about is failure due to emergence of resistance to chemotherapeutic drugs due to various factors. One of the major reasons here we have targeted is the resistance developed against DNA damaging chemotherapy due to over activation of APE1 enzyme evolved in BER pathway, which is the major repair pathway responsible for 95% of the DNA repair. Design and synthesis of APE1 inhibitors using rational approach fulfilling the pharmacophoric requirements has been carried out in this research work. Molecular modelling studies were performed to confirm that designed compounds fit well into the repair active cavity. 14 compounds have been designed and synthesized having pyrazolo-quinazolines core structure. The anticancer potential of the 8 representative compounds was evaluated against rat C-6 glial cell line at different concentrations. All synthesized compounds showed good anticancer activity against rat C-6 glial cell lines. The inhibitory potential of the compounds obtained from the MTT assay results helped us to formulate the SAR studies. Further ROS measurement was also carried out using DCFDA assay. Compounds showing good MTT results were also found to be potential antioxidants which conclude their mechanism of anticancer activity through APE1 inhibition. The active compounds may be taken further for lead optimisation and mechanistic interventions for their in vitro binding studies on APE1 in future.
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    APE1: A Molecule of Focus with Neuroprotective and Anti-Cancer Properties
    (OMICS Publishing Group, 2013) Mantha, Anil K.
    Apurinic/Apyrimidinic endonuclease (APE1) is a multi-functional, central enzyme of base excision repair (BER) pathway that takes care of oxidized base damage (AP sites and strand breaks) caused by both endogenous and exogenous oxidative DNA damaging agents. In repair function, APE1 exhibits majorly abasic (AP) endonuclease activity and stable interaction(s) with BER-pathway participant proteins. Second function of APE1 is redox activation of various transcription factors (TFs e.g., c-jun, NF-kB, p53 and HIF1α) and also named as redox effector factor 1(Ref-1). In redox function, APE1 reductively activates TFs involved in regulation of gene expression for cell survival mechanisms through stable pair-wise interaction(s). Recent studies have indicated that APE1 also possesses other distinct functions such as RNA metabolism, riboendonuclease activity and protein-protein interaction for maintaining cellular homeostasis. Altered APE1 expression has been reported in various cancers and neurodegenerative diseases. Taken together such findings advocates the necessity to delineate the underlying molecular mechanism(s) for understanding its role in various biological functions, that could be translated to its application in therapeutics against human diseases like cancer, neurodegenerative diseases and other pathologies such as cardiovascular diseases.
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    Ginkgolide B Revamps Neuroprotective Role of Apurinic/Apyrimidinic Endonuclease 1 and Mitochondrial Oxidative Phosphorylation Against Ab 25–35 -Induced Neurotoxicity in Human Neuroblastoma Cells
    (Wiley, 2015) Kaur, Navrattan; Dhiman, Monisha; Perez-Polo, J. Regino; Mantha, Anil K.
    Accumulating evidence points to roles for oxidative stress, amyloid beta (Aβ), and mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD). In neurons, the base excision repair pathway is the predominant DNA repair (BER) pathway for repairing oxidized base lesions. Apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional enzyme with DNA repair and reduction–oxidation activities, has been shown to enhance neuronal survival after oxidative stress. This study seeks to determine 1) the effect of Aβ25–35 on reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels, 2) the activities of respiratory complexes (I, III, and IV), 3) the role of APE1 by ectopic expression, and 4) the neuromodulatory role of ginkgolide B (GB; from the leaves of Ginkgo biloba). The pro-oxidant Aβ25–35 peptide treatment increased the levels of ROS/RNS in human neuroblastoma IMR-32 and SH-SY5Y cells, which were decreased after pretreatment with GB. Furthermore, the mitochondrial APE1 level was found to be decreased after treatment with Aβ25–35 up to 48 hr, and the level was increased significantly in cells pretreated with GB. The oxidative phosphorylation (OXPHOS; activities of complexes I, III, and IV) indicated that Aβ25–35 treatment decreased activities of complexes I and IV, and pretreatment with GB and ectopic APE1 expression enhanced these activities significantly compared with Aβ25–35 treatment. Our results indicate that ectopic expression of APE1 potentiates neuronal cells to overcome the oxidative damage caused by Aβ25–35. In addition, GB has been shown to modulate the mitochondrial OXPHOS against Aβ25–35-induced oxidative stress and also to regulate the levels of ROS/RNS in the presence of ectopic APE1. This study presents findings from a new point of view to improve therapeutic potential for AD via the synergistic neuroprotective role played by APE1 in combination with the phytochemical GB. © 2015 Wiley Periodicals, Inc.