Fenretinide-induced Apoptosis of Acute Myeloid Leukemia Cells via NR4A1 Translocation into Mitochondria and Bcl-2 Transformation

Abstract

OBJECTIVE: Fenretinide is reported to induce NR4A1-associated apoptosis in several types of cancer cells. However, it remains unclear about its specific role and the underlying mechanism in acute myeloid leukemia (AML). Therefore, this study aimed to explore the role and mechanism of fenretinide-induced apoptosis in AML.METHOD: Firstly, the NR4A1 mRNA level in the newly diagnosed AML patients was measured, then AML cells were treated with fenretinide at various time points and doses, and cell viability was investigated by using the cell-counting kit-8 (CCK-8) assay. Additionally, apoptosis and cell cycles were analyzed by using flow cytometry. Moreover, siNR4A1 was utilized to knockdown NR4A1 expression, and leptomycin B (LMB) was adopted to inhibit the nuclear export; afterwards, the apoptosis rate and expression of apoptotic proteins in AML cells were detected. In addition, the expression levels of NR4A1 in the nuclei and mitochondria of fenretinide-treated AML cells were also measured. Meanwhile, the interaction between NR4A1 and Bcl-2, as well as the Bcl-2 transformation, was also examined. The anti-leukemic effect of fenretinide on NOD/SCID mice was also determined through subcutaneous injection of HL-60 cells.RESULTS: NR4A1 expression in AML patients was markedly down-regulated compared with that in normal donors. Fenretinide induced the expression of NR4A1 and mitochondria-mediated apoptotic pathway-associated proteins in a time- and concentration-dependent manner. Importantly, both siNR4A1 alone or the combination of fenretinide with LMB could attenuate the fenretinide-induced apoptosis and expression of apoptotic proteins. Under the action of fenretinide, the NR4A1 protein expression was down-regulated in nuclear extracts whereas up-regulated in mitochondrial extracts. At the same time, fenretinide promoted NR4A1 translocation from nuclei into mitochondria, and enhanced the interaction between NR4A1 and Bcl-2, thereby exposing the BH3 domain of Bcl-2 to exert the anti-apoptotic effect. Moreover, fenretinide also exhibited an anti-leukemic effect and induced NR4A1 expression in the AML mouse model.CONCLUSIONS: Fenretinide exerts an obvious effect on AML cells both in vitro and in vivo. Besides, the NR4A1-mediated signaling pathway is highly involved in the fenretinide-induced apoptosis of AML cells.

hUCMSCs reduce theca interstitial cells apoptosis and restore ovarian function in premature ovarian insufficiency rats through regulating NR4A1-mediated mitochondrial mechanisms

“…To further investigate the mechanisms underlying hUCMSCs effects on the apoptosis of TICs, we next evaluated the function of the orphan nuclear receptor, NR4A1. Numerous studies have been studied on NR4A1, mostly present in the nucleus in normal condition, can transfer to the mitochondria when membrane permeabilization and cell apoptosis [39]. In our study, NR4A1 is expressed in TICs during proestrus of rats according to our immunohistochemical results demonstrating NR4A1 in the ovaries and immunofluorescence results of NR4A1 in the nucleus of TICs.…”

The Paradoxical Roles of Orphan Nuclear Receptor 4A (NR4A) in Cancer

“…Wu and coworkers first identified cytosporone b (Csn-B) as an NR4A1 ligand (Fig. 6); however, Csn-B also induced apoptosis in cancer cells via nuclear export of the receptor to the mitochondria, and this was also observed for THPN (55,56,132). Several Csn-B analogues also bound NR4A1 with K D values in the low micromolar range and not only induced mitochondrial localization of NR4A1 but also acted within the nucleus to repress expression of brain and reproductive organexpressed protein (BRE) in gastric cancer cells (56).…”

“…A recent study showed that DHE also targeted super enhancers of pro-oncogenic factors in leukemia, and this includes MYC and results in decreased H3K27 acetylation, thus providing a novel pathway for modulation of MYC expression in leukemia (54). Fenritinimide also induced expression of NR4A1 in AML cells, and this was accompanied by nuclear export of NR4A1, interaction with bcl-2 resulting in induction of apoptosis (55). This pathway is similar to that observed in solid tumor-derived cancer cells and will be further discussed in a subsequent section of the review.…”

Targeting Sphingolipids for Cancer Therapy

“…Fenretinide treatment induces cell death through the following mechanisms: apoptosis (increased cleavage of caspases and PARP proteins; induction of NR4A1 expression, which interacts with Bcl-2, exposing aBH3 domain and a pro-apoptotic function; and induction of ATF3 expression, ATF4 expression, and NOXA transcription) ( 9 , 11 – 14 , 16 , 18 , 43 ); autophagy (increased LC3-II levels) ( 9 ); endoplasmic reticulum stress and accumulation of reactive oxygen species (ROS) ( 11 , 14 , 16 – 18 , 43 ); repression of mammalian target of rapamycin (mTOR) signaling and a subsequent reduction in Erk1/2 activity ( 9 ); ceramide production ( 9 , 17 ); antitumor activity against CSCs (reduced CD44, ALDH, Nanog, Sox2, and POU5F1 expression) ( 10 , 16 , 18 ); induction of cell cycle arrest (decreased p-AURA, CDC25, cyclin E2, and cyclin A2 levels and increased p16 levels) ( 9 , 11 , 12 , 18 ); and p38-MAPK signaling ( 19 ) ( Figure 3 ). Several articles have shown that fenretinide preferentially targets CSCs when sphere formation and stemness markers are analyzed ( 9 – 11 , 14 – 16 ).…”