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Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing
Published on: June 9, 2017
GSH depletion and consequent AKT inhibition contribute to the Nrf2 knockdown-induced decrease in proliferation in
Yue Jia1, Han-Dong Wang1, Qiang Wang1
1Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
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.
Insights
Nuclear factor erythroid 2-related factor 2 (Nrf2) drives glioma cell proliferation and redox balance. Nrf2 deficiency impairs growth and redox homeostasis, highlighting its proto-oncogene role in glioblastoma.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating antioxidant and cytoprotective genes.
- Nrf2 has been identified as a proto-oncogene, but its specific role in glioma pathogenesis is not fully understood.
Purpose of the Study:
- To investigate the expression and function of Nrf2 in glioblastoma (GBM) cells.
- To elucidate the underlying mechanisms by which Nrf2 influences GBM cell proliferation and redox status.
Main Methods:
- Nrf2 expression was analyzed in normal astrocytes and human GBM cell lines (U251, U87, A172).
- Nrf2 was specifically knocked down in U251 cells using lentivirus-mediated RNA interference.
- The effects of Nrf2 knockdown on cell proliferation, intracellular redox balance (GSH and ROS levels), and signaling pathways (AKT and ERK1/2) were assessed.
- Supplementation with N-acetylcysteine and glutathione monoethyl ester (GMEE) was used to evaluate their impact on ROS levels, cell growth, and GSH restoration.
- The role of AKT signaling was further investigated using an AKT-specific inhibitor.
Main Results:
- All tested GBM cell lines overexpressed Nrf2, with the highest levels in U251 cells.
- Nrf2 knockdown in U251 cells reduced cell proliferation and disrupted intracellular redox balance, decreasing glutathione (GSH) and increasing reactive oxygen species (ROS).
- GMEE supplementation restored GSH levels and reversed Nrf2 deficiency-induced cell growth arrest, while also normalizing ROS levels.
- Nrf2 knockdown impaired both AKT and ERK1/2 signaling; GMEE restored AKT signaling, but not ERK1/2.
- Inhibition of AKT signaling diminished the proliferative effects of GMEE in Nrf2-deficient cells.
Conclusions:
- Nrf2 plays a crucial role in regulating redox status and promoting cell proliferation in glioblastoma.
- Intracellular GSH levels and AKT signaling are essential components of Nrf2's function in glioma tumorigenesis.
- These findings offer a new perspective on Nrf2's mechanism in cancer development and suggest potential therapeutic targets.