Temozolomide (TMZ) is the main drug used to treat gliomas, which causes DNA damage leading to cell death mainly as a consequence of processing of the primary damage O6-methylguanine (O6meG). Several mechanisms of resistance were described, and the ability of MGMT (O-6-methylguanine-DNA methyltransferase) to directly reverse O6meG in the DNA molecule is the most significant. Moreover, the increase in the level of reactive oxygen species (ROS) has also been associated with resistance to TMZ and our group showed that glutathione (GSH), a crucial peptide in the maintenance of redox cell homeostasis, prevents cell death by TMZ. This mechanism involves a pathway modulated by the transcription factor NRF2. However, it is unclear whether NRF2 is involved directly in the reduction or tolerance of TMZ-induced lesions in the DNA or due to additional damage generated by redox stress. For that, we obtained glioma cells that express different levels of MGMT. Low expressing cells are still sensitive to TMZ and high expressing models are insensitive to TMZ as the level of MGMT is very high. Interestingly, NRF2 is only activated in the absence of MGMT. We hypothesize that a balance between the number of lesions that could be repaired by MGMT and the induction of NRF2/GSH pathway protects the cells from TMZ treatment.In this project, our main aim is to verify whether GSH is protecting the cells by (i) detoxifying TMZ, a product from TMZ decomposition such as MITC (3-methyl-(triazen-1-yl)imidazol-4-carboxamide) or even the diazonium ion or, alternatively, (ii) by reducing ROS induced by mitochondrial damage. By inactivating the GSH pathway with buthionine-sulfoximine (BSO) or stimulating the detoxification pathway with N-acetylcysteine (NAC), we observed that NAC reduces DNA damage (yH2AX) and cell cycle arrest induced by TMZ in MGMT expressing cells, suggesting that lesion levels are reduced and removed by MGMT. Also, activation of the GSH pathway could be leading to detoxification of TMZ. Therefore, the distinction between O6meG and ROS lesions in the nucleus and in the mitochondrial DNA is imperative to understand what is happening in damaged cells following anticancer drug treatment. These data may also help us to understand how the levels of DNA lesions are related to NRF2 activation in glioma cells. In this project, we also plan to investigate how autophagy and senescence are regulated in cells treated with TMZ in the presence and absence of MGMT and endogenous ROS scavengers. For example, the inhibition of autophagy flux by chloroquine could lead to a higher level of apoptosis induced by TMZ, and MGMT and NRF2 could be involved in the modulation of the autophagy initiated as a response to TMZ. Understanding the interplay among these pathways may help us to search for alternative therapy protocols for the most severe brain cancer glioblastoma multiforme.
News published in Agência FAPESP Newsletter about the scholarship: