Knockdown of Nrf2 enhances autophagy induced by temozolomide in U251 human glioma cell line

Zhou,Yuan; Wang,Han-Dong; Zhu,Lin; Cong,Zi-Xiang; Li,Ning; Ji,Xiang-Jun; Pan,Hao; Wang,Jia-Wei; Li,Wan-Chun

doi:10.3892/or.2012.2115

Knockdown of Nrf2 enhances autophagy induced by temozolomide in U251 human glioma cell line

Authors:
- Yuan Zhou
- Han-Dong Wang
- Lin Zhu
- Zi-Xiang Cong
- Ning Li
- Xiang-Jun Ji
- Hao Pan
- Jia-Wei Wang
- Wan-Chun Li
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Affiliations: Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, P.R. China, Department of Neurosurgery, School of Medicine, Second Military Medical University (Shanghai), Jinling Hospital, Jiangsu, Nanjing 210002, P.R. China, Department of Pathology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, P.R. China
Published online on: October 31, 2012 https://doi.org/10.3892/or.2012.2115
Pages: 394-400

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Abstract

Glioblastoma multiforme (GBM) and oxidative stress are closely linked. Oxidative stress affects many signaling pathways and may cause the induction of autophagy. The NF-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) signaling pathway is the main pathway responsible for cell defense against oxidative stress and Nrf2 is a critical transcription factor related with cancer multidrug resistance. However, the relation between Nrf2 and regulation of autophagy is not well understood. In this study, we used temozolomide (TMZ), which inhibited the viability of GBM cells mainly by inducing autophagic cell death and explored the role of Nrf2 downregulation on autophagy induced by TMZ in GBM cells. In U251-Si-Nrf2 48 h after transfection the protein levels of Nrf2 were significantly downregulated, while the protein levels of LC3B-II increased by western blot analysis. Knockdown of Nrf2 also led to a significant increase of autophagic vacuoles and acidic vesicular organelles (AVOs), revealed by transmission electron microscopy (TEM) and acridine orange (AO) staining using flow cytometry. Collectively, these findings demonstrate that knockdown of Nrf2 can enhance the basal level of autophagy in the U251 glioma cell line. Furthermore, after the treatment with TMZ (100 µM) for 3 days, the U251-Si-Nrf2 transfected cells showed less viability rate by cell counting kit-8 (CCK-8) assay and the levels of autophagy increased obviously through analysis of western blot and AO staining using flow cytometry. Taken together, our results suggest that knockdown of Nrf2 may enhance autophagy induced by TMZ in the U251 glioma cell line, which should be further evaluated for novel anticancer activity.

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