Abstract 282A: The Nrf2 transcription factor is a positive regulator of differentiation of acute myeloid leukemia cells induced by vitamin D derivatives and plant polyphenols
Authors: Novik Victoria , Bobilev Irene , Kutner Andrzej , Levi Itai , Shpilberg Ofer , Sharoni Yoav , Studzinski George P. , Danilenko Michael 
Published: June 2012

Abstract
Differentiation therapy is an alternative approach to cytotoxic chemotherapy of acute myeloid leukemia (AML), based on the induction of leukemic blasts to mature and, thus, to restore the normal cellular phenotype and cell cycle arrest. Vitamin D derivatives (VDDs) – 1α,25-dihydroxyvitamin D3 (1,25D) and its low-calcemic analogs – are powerful differentiation agents, which have potential for treatment of AML; however, currently available VDDs have not been found to be safe for human use at concentrations needed to induce cell differentiation. We have previously shown that plant polyphenolic antioxidants (PAOx), such as carnosic acid (CA) from rosemary, curcumin from turmeric and silibinin from milk thistle markedly potentiate differentiation induced by low, near physiologic concentrations of 1,25D in HL60 human myeloblastic cells. Here, we demonstrate similar enhanced differentiation responses to the combinations of PAOx with the low-calcemic analog (24R)-1,24-dihydroxyvitamin D3 (PRI-2191) in both AML cell lines and leukemic blasts derived from patients with AML. The major aim was to determine the role of the Nrf2/antioxidant response element (Nrf2/ARE) pathway in the differentiation-enhancing effect of PAOx. We found that CA and CUR transactivated the ARE-luciferase reporter gene, induced the ARE-responsive genes, NADP(H)-quinone oxidoreductase and the γ-glutamylcysteine synthetase heavy subunit, and elevated cellular glutathione levels in HL60 and U937 cells. Interestingly, both 1,25D and PRI-2191 potentiated the effects of PAOx on ARE transactivation, induction of Nrf2/ARE-responsive genes and glutathione production. Stable transfection of wild type (wt) Nrf2 resulted in the enhancement, while transfection of dominant-negative (dn) Nrf2 produced suppression of differentiation induced by the 1,25D/CA combination and, surprisingly, by 1,25D alone. These effects were associated with a corresponding increase or decrease in vitamin D receptor and retinoid × receptor-α protein levels, and in vitamin D responsive element transactivation. Cells transfected with wtNrf2 and dnNrf2 also displayed analogous up or down changes in the levels of the AP-1 family proteins (c-Jun and ATF2) and AP-1 transcriptional activity. Pretreatment with AP-1 decoy oligodeoxynucleotide markedly attenuated the enhanced differentiation observed in wtNrf2-transfected cells, suggesting that the pro-differentiation action of Nrf2 is mediated by functional AP-1. Our findings suggest that the Nrf2/ARE pathway plays an important role in the cooperative induction of AML cell differentiation by 1,25D and PAOx, and is involved, at least in part, in the basic mechanism of the induction of differentiation by VDDs.
Supported by RO1-CA117942-04 grant from the NIH-NCI (to both GPS and MD) and by Israel Science Foundation grant 778/07 (to both MD and YS).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 282A. doi:10.1158/1538-7445.AM2011-282A