Alkylating Agent–Induced NRF2 Blocks Endoplasmic Reticulum Stress–Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis

Abstract

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival; an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine (BSO) caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine (NAC) or glutathione-ethyl-ester (GSH-E), decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 targets overexpression and poor survival. In KEAP1 mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity.

Ophiobolin A kills human glioblastoma cells by inducing endoplasmic reticulum stress via disruption of thiol proteostasis

“…These results strongly suggest that NAC blocks OP-A cytotoxicity by eliminating its ability to form Michael adducts, particularly with the nucleophilic thiol groups of intracellular proteins. To further test whether OP-A directly reacts with the free thiol residues of the proteins, we performed dibromobimane (dBrB) assay, which is based on the ability of dBrB to react with free reduced thiols and generate a highly fluorescent protein-dBrB adduct [ 24 , 25 ]. When we used iodoacetamide (IAM), an alkylating agent that reacts with protein-SH groups to form stable S-carboxyaminodomethyl-cysteine adducts [ 25 , 26 ] as a positive control, IAM treatment effectively reduced free protein-SH levels in T98G cells (Figure 8E ).…”

“…The depletion of protein thiol groups (protein-SH) following OP-A exposure was measured using the previously described dibromobimane (dBrB) assays [ 24 , 25 ] with minor modifications. T98G cells plated in 12-well plates were treated with various concentrations of OP-A, harvested, resuspended in PBS, and sonicated.…”

Treatment of Human Placental Choriocarcinoma Cells with Formaldehyde and Benzene Induced Growth and Epithelial Mesenchymal Transition via Induction of an Antioxidant Effect

“…ROS are known to induce apoptosis through the C/EBP-homologous protein (CHOP) pathway of ER-stress [ 48 ], as well as to activate Nrf2 [ 49 , 50 ]. Nrf2 prevents apoptosis through inactivation of ER stress [ 51 , 52 ]. Although Nrf2 has been known as a transcription factor that regulates the expression of antioxidants and cytoprotective genes under oxidative stress, recent data has revealed that Nrf2 activity is also associated with oncogenic function and survival and metastasis of cancer cells [ 53 ], and blockage of Nrf2 inhibited the metastatic abilities such as migration and invasion of esophageal squamous cell carcinoma cells [ 54 ].…”

Modulation of proteostasis by transcription factor NRF2 and impact in neurodegenerative diseases

“…NRF2 modulates crucial enzymes of the GSH metabolism in the brain, such as cystine/glutamate transport, γ-glutamate cysteine synthetase (γ-GS), glutamate-cysteine ligase catalytic and modulator subunits (GCLC and GCLM), glutathione reductase (GR) and glutathione peroxidase (GPX) (reviewed in [55]). The relevance of NRF2 in the maintenance of GSH in the ER is supported by the finding that pharmacological or genetic activation of NRF2 results in increased GSH synthesis via GCLC/GCLM, while inhibiting the expression of these enzymes by NRF2-knockdown caused an accumulation of damaged proteins within the ER leading to the UPR activation [56].…”