Experimental Study On The Role Of Nrf2on Autophagy Induced By Temozolomide In U251Human Glioma Cell Line

Background and objective:Glioblastoma multiforme (GBM) is the most common and fatal intracranial tumor. The10-year median survival time for patients with GBM is approximately one year. Recent studies found that GBM is resistant to chemotherapy protocols that induce apoptosis but seems to be less resistant to therapies that induce autophagy. Thus, autophagy has potential utility as a target for GBM therapy. As the most effective cytotoxic drug, temozolomide (TMZ) induces significant autophagic cell death in GBM cells. However, the efficacy of TMZ is not satisfactory, which is closely related to the multidrug resistance of GBM. Therefore, how to solve the problem of drug resistance remains critical, in which how to enhance autophagy induced by TMZ and improve the effect of chemotherapy ultimately become one of the research priorities about combination chemotherapy. Multiple lines of evidence indicate that GBM and oxidative stress are closely linked. Oxidative stress affects many signaling pathways and may cause the induction of autophagy. The Nrf2/Keap1signaling pathway is the main pathway responsible for cell defense against oxidative stress and Nrf2is a critical protective transcription factor related with cancer multidrug resistance. However, the relation between Nrf2and autophagy is not well understood. Thus, the aim of the current study was to explore the role of Nrf2on autophagy induced by TMZ in U251human glioma cell line.Materials and methods:Under normal conditions, U251human glioma cells were cultured, Si-Nrf2、pEGFP-Nrf2plasmids were constructed and transfected by Lipofectamine2000according to the manufacturer’s protocol.5groups were separated as follows:group Lipo, two negative control groups (Si-control, pEGFP-control), group Si-Nrf2and pEGFP-Nrf2. Transient transfection effect was tested48hours after transfection using western blot. And meanwhile, Transmission Electron Microscopy (TEM), western blot and acridine orange staining using flow cytometry were performed to analyse the alteration of the base level of autophagy after transfection. The role of Nrf2on autophagy induced by TMZ (100μM) for3days in transfected U251cells was tested by western blot and acridine orange staining using flow cytometry.Results:1. In U251-Si-Nrf248hours after transfection the protein levels of Nrf2were significantly downregulated, while the protein levels of LC3B-Ⅱ increased. Knockdown of Nrf2also led to a significant increase of autophagic vacuoles and acidic vesicular organelles (AVOs).2. After the treatment with TMZ (100μM) for3days, the U251-Si-Nrf2transfected cells showed less viability rate and the protein levels of LC3B-Ⅱ and AVOs increased obviously.Conclusions:Our results suggest that knockdown of Nrf2may enhance autophagy induced by TMZ in the U251glioma cell line.