NRF2 Is Targeted By the Polyphenol Quercetin and Induces Apoptosis, in Part, through up Regulation of Pro Apoptotic Mirs

Nuclear factor-erythroid 2 related factor (NRF2) is involved in cell defense and survival against endogenous and exogenous stress. Constitutively active Nrf2 in malignant cells increases the expression of cytoprotective genes and consequently, enhances proliferation via metabolic reprograming and inhibition of apoptosis (Leinonem, Advances in cancer Research, 2014). NRF2 is persistently activated in many human tumors, including acute myelogenous leukemia thus, inhibition of NRF2 activity may be a promising strategy for leukemia therapy. Flavonoids, present in vegetables, fruit and propolis, might exert antitumoral effects through induction of apoptosis and chromatin remodeling (Link, Biochem Pharmacol., 2010). A previous study from our group showed that Quercetin (Qu), a natural polyphenolic flavonoid compound, induced apoptosis, partly due to its DNA demethylating activity, through HDAC inhibition and by the enrichment of H3ac and H4ac in the promoter regions of genes involved in the apoptosis pathway, leading to their transcription activation (Alvarez, Clinical epigenetics 2018). In the present study, we evaluated the effect of Qu as a modulator of NRF2. This study was performed in vivo in human xenograft acute myeloid leukemia (AML) models, and in vitro using leukemia cell lines. Qu treatment (50 µM Qu) for 48h downregulated HDAC4, NRF2 and p-NRF2 at protein levels (p<0.05; p<0.005; p<0.005 respectively). Imaging Flow Cytometry (AMNIS, ImageStream ISX mkII^TM) and Confocal Microscopy evidenced a decrease in NRF2 nuclear localization. Furthermore, combined treatment with the proteasome inhibitor MG132 prevented degradation of NRF2, indicating that treatment increased proteasomal degradation. Loss of NRF2 decreases HDAC4, a redox sensitive histone deacetylase, resulting in an increased expression of miR-1 and miR-206 (Singh, J Clin Invest. 2013). Herein, expression profile of 84 miRNAs (Apoptosis miRNA PCR array) were performed in samples from human xenograft model. Treatment up-regulated the expression profile of 5% (n=4) of the 84 miRNAs evaluated, corresponding exclusively to miRNAs that target anti-apoptotic genes and to miRNAs that have been demonstrated to have pro-apoptotic functions. Furthermore, expression levels of miR-1, miR-133a/b, which target anti-apoptotic genes and miR-206, a pro apoptotic miR, were validated in xenograft model samples, resulting in a significant up-regulation of the expression levels in treated animals compared to controls (p<0.05). In addition, lentiviral sh down regulation of NRF2, led to an increased apoptosis, decreased cell survival and an up regulation of miRNA 206 expression in Qu treated cells. In summary, Qu might induce programmed cell death in part, by decreasing NRF2 nuclear localization, by inducing NRF2 proteasomal degradation and down regulation of HDAC4 which led to up-regulation of pro apoptotic miRNAs.