title: GSH depletion and consequent AKT inhibition contribute to the Nrf2 knockdown-induced decrease in proliferation in glioblastoma U251 cells.
abstract: Nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal transcription regulator that controls the expression of numerous antioxidant and cytoprotective genes, was recently defined as a proto-oncogene. However, the role and mechanism of Nrf2 in glioma pathoetiology remain unclear. In the present study, we first evaluated the expression patterns of Nrf2 in normal human astrocytes and 3 glioblastoma (GBM) cell lines (U251, U87 and A172) and found that all 3 GBM cell lines overexpressed Nrf2, with the highest level observed in the U251 cells. We further assessed the biological effects of Nrf2 in U251 cells by specific knockdown of Nrf2 using lentivirus‑mediated RNA interference. We discovered that Nrf2 deficiency led to a decrease in U251 cell proliferation and caused intracellular redox imbalance [diminished glutathione (GSH) levels and increased reactive oxygen species (ROS) levels]. Both N-acetylcysteine and glutathione monoethyl ester (GMEE) supplementation completely eliminated the increased levels of ROS that were present in the Nrf2‑deficient U251 cells. However, only GMEE supplementation both reversed Nrf2 deficiency-induced cell growth arrest and restored intracellular GSH levels. Moreover, AKT and ERK1/2 signaling were both impaired in the Nrf2-knockdown U251 cells, but GMEE supplementation restored AKT signaling but not ERK1/2 signaling, and blocking AKT signaling with an AKT-specific inhibitor greatly diminished the GMEE-induced Nrf2-deficient cell proliferation. In conclusion, our findings revealed novel functions for Nrf2 in the regulation of redox status and cell proliferation, and that intracellular GSH levels and AKT signaling are required for this process, a new viewpoint by which to comprehend the role and underlying mechanism of Nrf2 in tumorigenesis.
authors.affiliation: Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Han-Dong
authors.initials: HD
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, Jinling Hospital, School of Medicine, Southern Medical University (Guangzhou), Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Neurosurgery, Nanjing Jingdu Hospital, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: He-Ming
authors.initials: HM
authors.lastname: Wu
authors.affiliation: Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Pan
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques / Q000379:methods*; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D000090063:Proto-Oncogene Mas; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation
chemical_list: C000715470:MAS1 protein, human; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D000090063:Proto-Oncogene Mas; D017382:Reactive Oxygen Species; C042431:S-ethyl glutathione; D051057:Proto-Oncogene Proteins c-akt; D005978:Glutathione