School Of Basic And Applied Sciences
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Item A short review on cross-link between pyruvate kinase (PKM2) and Glioblastoma Multiforme(Springer, 2021-03-02T00:00:00) Verma, Harkomal; Cholia, Ravi P.; Kaur, Sharanjot; Dhiman, Monisha; Mantha, Anil K.Pyruvate kinase (PK) catalyzes the last irreversible reaction of glycolysis pathway, generating pyruvate and ATP, from Phosphoenol Pyruvate (PEP) and ADP precursors. In mammals, four different tissue-specific isoforms (M1, M2, L and R) of PK exist, which are translated from two genes (PKL and PKR). PKM2 is the highly expressed isoform of PK in cancers, which regulates the aerobic glycolysis via reprogramming cancer cell�s metabolic pathways�to provide an anabolic�advantage to the tumor cells. In addition to the established role of PKM2 in aerobic glycolysis of multiple cancer types, various recent findings have highlighted the non-metabolic functions of PKM2 in brain tumor development. Nuclear PKM2 acts as a co-activator and directly regulates gene transcription. PKM2 dependent transactivation of various oncogenic genes is instrumental in the progression and aggressiveness of Glioblastoma Multiforme (GBM). Also, PKM2 acts as a protein kinase in histone modification which regulates gene expression and tumorigenesis. Ongoing research has explored novel regulatory mechanisms of PKM2 and its association in GBM progression. This review enlists and summarizes the metabolic and non-metabolic roles of PKM2 at the cellular level, and its regulatory function highlights the importance of the nuclear functions of PKM2 in GBM progression, and an emerging role of PKM2 as novel cancer therapeutics. � 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.Item A short review on cross-link between pyruvate kinase (PKM2) and Glioblastoma Multiforme(Springer, 2021-03-02T00:00:00) Verma, Harkomal; Cholia, Ravi P.; Kaur, Sharanjot; Dhiman, Monisha; Mantha, Anil K.Pyruvate kinase (PK) catalyzes the last irreversible reaction of glycolysis pathway, generating pyruvate and ATP, from Phosphoenol Pyruvate (PEP) and ADP precursors. In mammals, four different tissue-specific isoforms (M1, M2, L and R) of PK exist, which are translated from two genes (PKL and PKR). PKM2 is the highly expressed isoform of PK in cancers, which regulates the aerobic glycolysis via reprogramming cancer cell�s metabolic pathways�to provide an anabolic�advantage to the tumor cells. In addition to the established role of PKM2 in aerobic glycolysis of multiple cancer types, various recent findings have highlighted the non-metabolic functions of PKM2 in brain tumor development. Nuclear PKM2 acts as a co-activator and directly regulates gene transcription. PKM2 dependent transactivation of various oncogenic genes is instrumental in the progression and aggressiveness of Glioblastoma Multiforme (GBM). Also, PKM2 acts as a protein kinase in histone modification which regulates gene expression and tumorigenesis. Ongoing research has explored novel regulatory mechanisms of PKM2 and its association in GBM progression. This review enlists and summarizes the metabolic and non-metabolic roles of PKM2 at the cellular level, and its regulatory function highlights the importance of the nuclear functions of PKM2 in GBM progression, and an emerging role of PKM2 as novel cancer therapeutics. � 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.Item OXIDATIVE STRESS INDUCED CELL PROLIFERATION AND DNA REPAIR MECHANISMS IN GLIOBLASTOMA CELLS: ROLE OF ENPP2 AND APE1(Central University of Punjab, 2018) Cholia, Ravi Parkash; Mantha, Anil K. and Kumar, RajGlioblastoma Multiforme (GBM) is a grade IV, most frequent, and invasive devastating brain tumor with poor prognosis, even with the advancement of multimodal therapies, patients have survival period of less than 15 months. GBM is a multifactorial disease with oxidative stress as a key accelerating player. In the present study, rat glioma C6 and human glioblastoma U-87 MG cell lines were exposed to non-cytotoxic concentrations i.e. 10 -35) peptide, 10 U/ml GO, and 50 M H2O2, respectively. Further, the ROS levels were measured via NBT and H2DCFDA assays. Our genome encounters exogenous and intracellular oxidants which result in the DNA damage; small DNA base lesions such as apurinic/apyrimidinic (AP) sites are generated following the oxidants exposure and repairing of these AP sites is the prerequisite to maintaining the genomic integrity. In the present study, it was observed that APE1 being a redox-sensitive protein, with the moderate level of oxidative stress [induced -35) peptide, GO, and H2O2] resulted in the elevation of APE1 expression as measured using Western blotting, RT-PCR, and (repair) activity was boosted after the treatment of oxidants. Oxidative stress also resulted in the secretion of APE1 extracellularly. Additionally, in this study dysregulated expression of BER-pathway enzymes were observed after the treatment of non-cytotoxic concentrations of the oxidants. Cancer shows higher metabolic properties as compared to the normal cells. Pyruvate Kinase M2 (PKM2) one of the isoform of pyruvate kinase (PK), is a key enzyme in the glycolytic pathway, which catalyses the terminal step of the glycolysis, converts phosphoenolpyruvate (PEP) into pyruvate. PKM2 also perform nonglycolytic functions via enhancing the expression of cyclin D, c-myc, and contributing towards the aggressiveness of GBM. In the present study, oxidative stress resulted in up-regulation of PKM2 level, as analyzed using Western blotting and majorly in the cytosolic regions as identified by immunocytochemistry. Ectonucleotide pyrophosphatase/phosphordiestrase2 (ENPP2) is the secretary protein, known to be involved in a variety of processes like embryonic development, blood vessel formation during development, inflammation, favoring PKM2 dimeric form, and progression of cancer through its enzymatic product LPA. ENPP2 is highly expressed in the GBM, and LPA receptors are also predominate in GBM and play a role in its growth and development. In the present study, elevated expression and activity of ENPP2 was observed after the treatment of non-cytotoxic doses of oxidants in C6 and U-87 MG cells as analyzed using Western blotting and immunocytochemistry. In addition, LPA treatment resulted in the induction of migratory potential of C6 and U-87 MG cells. LPA treatment also up-regulated the key transcription factors such as c-jun, p-c-jun, NF- B, and HIF-1- advocating for their involvement in the survival of GBM cells. LPA treatment resulted in the timedependent increase in the PKM2 and ENPP2 expression and subcellular translocation in the C6 and U-87 MG cells. However, LPA treatment resulted in the elevation of nuclear APE1 expression after 48 hr incubation period. Oxidants - 35) peptide, GO, and H2O2 treatment enhanced the secretory levels of ENPP2 in the extracellular media up to 48 hr, suggesting the protective role of ENPP2 against the oxidative stress. Co-localization of APE1, PKM2, and ENPP2 were observed in the C6 and U87 MG cells when treated with -35) peptide, GO, and H2O2 treatment suggesting the role of oxidative stress in the cross talk interaction of three proteins towards the aggressiveness of GBM. In addition, anti-APE1 inhibitors were synthesized activity, and one of the screened molecule GR5G-b showed ani-proliferative property along with dysregulated APE1 level and repair function; and also displayed potential in cell cycle arrest as analyzed by flow cytometry. Taken together, it can be concluded that oxidative stress enhances the aggressiveness of GBM cells via up-regulating the key proteins (APE1, PKM2, and ENPP2) and altering the functions associated as studied in C6 and U-87 MG cell lines. Further studies focusing towards blocking of their activities by designing, help in development of new therapeutic interventions for GBM.Item 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.