Department Of Biochemistry And Microbial 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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    Role of Helicobacter pylori Enriched Media in Inducing Oxidative Stress in Human Cell lines
    (Central University of Punjab, 2018) Samal, Pallavi; Dhiman, Monisha
    Helicobacter pylori is a gram-negative, helical, microaerophilic bacterium which colonizes the human gastrointestinal tract. Vacuolating cytotoxin A (VacA) is one of the major virulent factors. Reactive oxygen species (ROS) and Reactive nitrogen species (RNS) produced by the immune and epithelial cells damage the host cell thereby resulting in a persistent infection. The prolonged infection results in chronic inflammation, oxidative stress and DNA damage. The microbe affects the major macromolecules of the host tissues lipids, proteins and DNA which leads to lipid peroxidation, protein oxidation and DNA fragmentation hence making the oxidative stress a deleterious damage. Role of H. pylori enriched media (HPEM) in inducing oxidative stress in two human cell lines AGS (human gastric cell line) and THP-1(human monocytic cell line) was studied in present work. The AGS cells and THP-1 cells was treated with various concentrations of HPEM and oxidative stress was evaluated by examining the levels of protein carbonyls, TBARS (thiobarbituric acid reactive species) and nitric oxide by spectophotometric and Western blotting methods. The oxidative stress induced by HPEM showed damaging effects on the cell membrane, protein and produced significantly high nitric oxide (NO) when compared with the untreated controls. From the present work it can be concluded that HPEM exposure to THP-1 and AGS cells enhanced the oxidative stress which leads to cellular damage and is ultimately responsible for the severe H. pylori associated fatal complications during its pathogenesis.