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

Abstract

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 trans mission 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.

NRF2 and glutathione are key resistance mediators to temozolomide in glioma and melanoma cells

“…Recently, it was demonstrated that NRF2 silencing in glioma cell lines inhibited cell proliferation [33], decreased cellular migration [34], and induced autophagic process [35]. Here we went further and showed that NRF2 silencing results in greater sensitivity and higher apoptosis induction upon TMZ treatment.…”

The effect of transforming growth factor β1 on the crosstalk between autophagy and apoptosis in the annulus fibrosus cells under serum deprivation

“…After reaching 90% confluence, the medium was changed to DMEM containing 1% FBS for 12 h to synchronize the cells and the cells were subjected to serum starvation. Autophagy incidence was measured by quantifying the development of acidic vesicular organelles (AVOs) after AO staining (0.5 lg/mL) for 15 min at 37°C [39], and immediately determined as percentage of AO positive cells analyzed with a FACS Canto II software (BD, USA).…”

Cytoprotection “gone astray’’: Nrf2 and its role in cancer

“…Accordingly, in other tumor entities, an increase of selective autophagy has been demonstrated that may be linked to the enhanced Nrf2 activity resulting from other amplification mechanisms 218,219. By contrast, Nrf2 has been shown to block autophagy induction by temozolomide in glioma or by mitoquinone in breast cancer cells,220,221 and a recent study demonstrated that tBHQ-mediated induction of autophagy is required for full activation of Nrf2 in hepatocytes 222. These findings point to a complex interrelation between autophagy and cellular redox control.…”