OXIDATIVE STRESS INDUCED CELL PROLIFERATION AND DNA REPAIR MECHANISMS IN GLIOBLASTOMA CELLS: ROLE OF ENPP2 AND APE1
Cholia, Ravi Parkash
MetadataShow full item record
Glioblastoma 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.