Department of Animal Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, 151 001, India.
Department of Biochemistry and Microbial Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, India.
Metab Brain Dis. 2018 Aug;33(4):1307-1326. doi: 10.1007/s11011-018-0233-3. Epub 2018 May 2.
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 (HO) 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.
维持基因组完整性对于细胞存活和活力至关重要。活性氧（ROS）的过度产生会导致氧化应激，通过在 DNA 中产生小碱基损伤导致基因组不稳定，这些未修复的 DNA 损伤导致各种细胞后果，包括癌症。最近的数据支持这样的概念，即在各种癌细胞类型（包括神经胶质瘤细胞）中，氧化应激是促进增殖、存活和迁移的不可或缺的参与者。在这项研究中，我们证明了用非细胞毒性剂量的氧化剂（如淀粉样β[β（25-35）]肽、葡萄糖氧化酶（GO）和过氧化氢（HO））处理 24 小时和 48 小时时间点，可增加多功能酶脱嘌呤/脱嘧啶内切核酸酶（APE1）的表达水平和活性，APE1 是碱基切除修复（BER）途径的关键酶，可处理碱基损伤；并且还观察到氧化应激后 C6 和 U-87 MG 细胞中 BER 途径下游酶的表达水平发生变化，即 PARP-1、XRCC1、DNA polβ 和 ligase IIIα。氧化剂处理 C6 和 U-87 MG 细胞也导致细胞内糖酵解途径酶丙酮酸激酶 M2（PKM2）和转移诱导蛋白核苷酸外切酶/磷酸二酯酶 2（ENPP2）的表达升高，如 Western 印迹和免疫荧光显微镜研究所示。我们的研究还报告说，在 C6 和 U-87 MG 细胞中诱导氧化应激 24 小时和 48 小时，可分析细胞外分泌 APE1 和 ENPP2，如条件培养基中的 Western 印迹所示。然而，细胞外分泌的 APE1 的生物学意义仍不清楚。氧化应激还增加了 C6 和 U-87 MG 细胞条件培养基中 ENPP2 的 LysoPLD 活性。我们的结果还表明，氧化应激影响 C6 和 U-87 MG 细胞中 APE1、PKM2 和 ENPP2 的表达水平和定位。根据 24 小时和 48 小时时间点的共定位模式，可以认为氧化应激介导了 APE1、PKM2 和 ENPP2 之间的串扰。此外，当 C6 和 U-87 MG 细胞用 10μM 浓度的生物活性脂质溶血磷脂酸（LPA）处理时，C6 和 U-87 MG 细胞表现出强烈的迁移潜力，并且在氧化应激下也诱导迁移。总之，这些发现表明 C6 和 U-87 MG 细胞有潜力利用三种蛋白质，即 APE1、PKM2 和 ENPP2 来促进神经胶质瘤的迁移和存活。因此，关于氧化应激诱导的 APE1 与 PKM2 和 ENPP2 相互作用的知识为神经胶质瘤的治疗靶点开辟了新的途径。
