Role of Orphan Nuclear Receptor Nur

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Ginsenoside 20(S)-Rh2 Induces Apoptosis and Differentiation of Acute Myeloid Leukemia Cells: Role of Orphan Nuclear Receptor Nur77 Chengqiang Wang, Hui He, Guojun Dou, Juan Li, Xiaomei Zhang, Mingdong Jiang, Pan Li, Xiaobo Huang, Hongxi Chen, Li Li, da-jian yang, and Hongyi Qi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02299 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 14, 2017

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Journal of Agricultural and Food Chemistry

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Ginsenoside 20(S)-Rh2 Induces Apoptosis and Differentiation of Acute Myeloid

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Leukemia Cells: Role of Orphan Nuclear Receptor Nur77

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Chengqiang Wang†, #, Hui He†, #, Guojun Dou†, Juan Li†, Xiaomei Zhang§,

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Mingdong Jiang‡, Pan Li‡, Xiaobo Huang‡, Hongxi Chen‡, Li Li†, Dajian Yang§, *,

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Hongyi Qi†, *

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†

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District, Chongqing 400716, China;

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§

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Chongqing 400065, China

College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road, Beibei

Chongqing Academy of Chinese Materia Medica, 34 Nanshan Road, Nan'an District,

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‡

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Beibei District, Chongqing 400700, China;

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#

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*Corresponding author at:

Radiotherapy Department, Chongqing Ninth People's Hospital, Jialing village 69,

These authors have contributed equally to this work.

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Dajian Yang: Chongqing Academy of Chinese Materia Medica, 34 Nanshan

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Road, Nan'an District, Chongqing 400065, China. Tel./Fax: +86 23 89029011; E-mail:

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[email protected]

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Hongyi Qi: College of Pharmaceutical Sciences, Southwest University, 2

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Tiansheng Road, Beibei District, Chongqing 400716, China. Tel./Fax: +86 23

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68251225; E-mail: [email protected]

1

ACS Paragon Plus Environment


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Running title: 20(S)-Rh2 Induces Apoptosis and Differentiation via Nur77

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ABSTRACT

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Ginsenoside 20(S)-Rh2 has been shown to induce apoptosis and differentiation of

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acute myeloid leukemia (AML) cells. However, the underlying molecular

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mechanisms are not fully understood. In our study, 20(S)-Rh2 induced the expression

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of orphan nuclear receptor Nur77 and death receptor proteins Fas, FasL, DR5 and

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TRAIL, as well as the cleavage of caspase 8 and caspase 3 in HL-60 cells.

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Importantly, shNur77 attenuated 20(S)-Rh2-induced apoptosis and Fas and DR5

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expression. Meanwhile, 20(S)-Rh2 promoted Nur77 translocation from nucleus to

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mitochondria and enhanced the interaction between Nur77 and Bcl-2, resulting in the

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exposure of BH3 domain of Bcl-2 and activation of Bax. Furthermore, 20(S)-Rh2

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promoted the differentiation of HL-60 cells as evidenced by Wright-Giemsa staining,

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NBT reduction assay and detection of the myeloid differentiation marker CD11b by

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flow

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differentiation. Additionally, 20(S)-Rh2 also exhibited anti-leukemic effect and

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induced Nur77 expression in NOD/SCID mice with the injection of HL-60 cells into

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the tail vein. Together, our studies suggest that Nur77-mediated signaling pathway is

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highly involved in 20(S)-Rh2-induced apoptosis and differentiation of AML cells.

cytometry.

Notably,

shNur77

reversed

20(S)-Rh2-mediated

HL-60

38 39

KEYWORDS:

40

differentiation, hematologic malignancy

Ginsenoside,

immediate-early

gene,

2


Apoptosis,

myeloid

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3



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INTRODUCTION

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Leukemia as a kind of malignant clonal disease, originates from hematopoietic stem

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cells and can be characterized by inhibiting apoptosis, losing control proliferation, and

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blocking the differentiation of abnormal hematopoietic cells.1 In 2016, an estimated

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60 140 new cases were diagnosed, and over 24 400 patients died from leukemia in

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United States.2 In China, there were 75 300 newly increased cases and 53 400 death

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patients in 2015.3 Acute myeloid leukemia (AML) is the most frequently diagnosed

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acute leukemia in adults, especially older patients (older than 65 years).4

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Chemotherapy is one of the main treatments for AML. However, the efficacy of

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conventional chemotherapy is extremely limited due to the serious side effects,

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multidrug resistance and expensive costs. Until recent years, there are still over 50 %

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of young adult patients and almost 90 % of older patients that die from AML.4, 5 Thus,

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new alternative treatment approaches are urgently needed for AML therapy.

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Recently, Nur77 (also known as TR3 and NGFI-B) as a critical therapeutic target of

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AML has drawn more and more attention. It is encoded by immediate-early gene

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NR4A1, which can be transiently activated in response to diverse extracellular stimuli,

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and belongs to highly conserved orphan nuclear receptors of the thyroid/steroid

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receptor superfamily.6, 7 It is involved in numerous biological processes, including cell

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apoptosis, proliferation and differentiation, and a variety of disease states, such as:

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cardiovascular disorders, atherosclerosis and cancer.6-9 The function of Nur77 in

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tumor development is complicated and controversial.10 On the one hand, Nur77 is 4


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overexpressed in the tissue of prostatic cancer, lung cancer and breast cancer and

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exhibits oncogenic activity.11,

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antitumor activity in hematological tumor.13 A growing body of evidence has firmly

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established that deletion of orphan nuclear receptor Nur77 is closely related to the

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development of AML.12-17 Moreover, low expression of Nur77 is commonly detected

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in AML patients.14 Importantly, retroviral restoration of Nur77 in conditional

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knockout mouse blocks the leukemogenicity of AML cells.12 Recently, rescue of

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silenced Nur77 and NOR-1 by histone deacetylase (HDAC) inhibitor SNDX-275 has

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been shown to dramatically inhibit AML cells.16 Thus, Nur77 is a key suppressor of

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AML and targeting Nur77 may provide potential approaches for AML therapeutic

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intervention.

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On the other hand, Nur77 shows remarkable

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Ginseng is one of the most commonly used functional food and herbal medicine in

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East Asia and North America and is derived from the roots and rhizomes of different

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slow-growing perennial plants that belong to the genus Panax of the family

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Araliaceae.18, 19 Ginsenosides are a variety of triterpenoid saponins and considered to

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be the main active components of ginseng.20 20(S)-Rh2 is a protopanaxadiol (PPD)

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type of ginsenoside and one of the typical components in red ginseng. Accumulating

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evidence demonstrates that 20(S)-Rh2 may be a new promising anticancer agent.21-24

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Notably, 20(S)-Rh2 has received much attention due to the remarkable inhibitory

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effect on growth and differentiation of human leukemia cells.25-28 Recently, cellular

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stress response mechanisms are received much more attention as the molecular targets

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of ginsenosides.29 Thus, it is interesting to us that whether Nur77-mediated signaling 5



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pathway is involved in the inhibitory effect of 20(S)-Rh2 on AML.

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In the current study, we characterized the inducing effect of 20(S)-Rh2 on apoptosis

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and differentiation of AML cells. On the one hand, we determined whether

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20(S)-Rh2-induced apoptosis was correlated with Nur77-regulated death receptor

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pathway and Nur77 mitochondria localization and subsequent Bcl-2 transformation.

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One the other hand, we evaluated whether 20(S)-Rh2-induced differentiation was

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related to Nur77 and Nur77-mediated differentiation-associated transcription factors.

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MATERIALS AND METHODS

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Chemicals and Antibodies. Ginsenoside 20(S)-Rh2 with purity of 98% was

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purchased from Chroma-Biotechnology Co. (Chengdu, China), dissolved in DMSO

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(Sigma-Aldrich, St. Louis, MO, USA) at 100 mM and stored at −20°C before use.

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Leptomycin B was purchased from Beyotime Biotechnology (Shanghai, China). The

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antibodies against Fas, FasL, DR5, TRAIL, caspase 3, cleaved caspase 3 and cleaved

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caspase 8 were obtained from Wanlei Biotechnology (Shenyang, China). The

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antibodies against Nur77, PARP, cleaved PARP, c-Jun, Jun-B, Bcl-2 and Bax were

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purchased from Santa Cruz Biotechnology (CA, USA). The antibodies against β-actin,

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mouse or rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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Hoechst 33342 was obtained from Wanlei Biotechnology (Shenyang, China).

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Cell Culture. AML cell lines HL-60 and Kasumi-1 were purchased from Cell Bank

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of Chinese Academic of Science (Shanghai, China) and grown in RPMI-1640 medium

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(Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine 6


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serum (FBS) (Invitrogen, USA) and 1% penicillin/streptomycin (Invitrogen, USA) in

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a 5% CO2 humidified incubator at 37°C.

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Measurement of Cell Viability. The cell counting kit-8 (CCK-8) (Dojindo,

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Shanghai, China) assay was used to determine the cell viability. HL-60 and Kasumi-1

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cells (8 × 104 cells/mL) were inoculated into 96-well plate by 100 µL, respectively.

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Then, cells were treated for 72 h and subsequently added with 10 µL CCK-8 for each

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well. The OD value was detected at 450 nm with a microplate reader (Biotek,

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Winooski, VT, USA). Cell viability was presented as a percentage of that of untreated

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cells.

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Cell Proliferation Assay. The proliferation of HL-60 and Kasumi-1 cells were

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determined by trypan blue dye exclusion test. Cells were seeded at a density of 5 ×

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104 cells/mL and treated with 20(S)-Rh2 at various concentrations for 24-120 h. Then,

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cells were stained with trypan blue (Sigma, USA) and the number of viable cells was

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counted using haemocytometer.

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Colony Formation Assay. Colony forming units were assayed in methylcellulose

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(Methocult H4100, StemCell Technologies Inc, Canada) supplemented with 10 %

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fetal bovine serum (FBS) (Invitrogen, USA) and 1 % penicillin/streptomycin

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(Invitrogen, USA). Vehicle or 20(S)-Rh2 was added to methylcellulose containing

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2500 cells in 24-well plate. Colonies were evaluated microscopically 14 days after

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plating.

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Cell Apoptosis Analyzed by Annexin V-FITC/PI double-staining assay. Cells 7



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were seeded at a concentration of 2×105 cells/ml and incubated for 48 h with

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20(S)-Rh2 at various concentrations. After treatment, cells were harvested, washed

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and re-suspended in the binding buffer containing annexin V-FITC and

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propidiumiodide (PI). Flow cytometry analyses were performed on a BD LSRFortessa

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Cell Analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed using Flow

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Jo 7.6.1 software (Tree Star, Inc., Ashland, OR, USA).

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Hoechst 33342 Staining. After treatment, HL-60 and Kasumi-1 cells were

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centrifuged and collected in Eppendorf tubes. Then, cells were fixed with 4%

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paraformaldehyde in PBS for 10 min and washed with 1 × PBS for 3 times. Hoechst

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33342 (10 mg/mL) dissolving in 1 × PBS was added into each well. The plates were

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protected from light exposure and kept at room temperature for 10 min. Finally, the

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plates were washed with 1 × PBS for 3 times again. Cells with fluorescence were

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observed and photographed under fluorescence microscope (Leica DM4000B, Wetzlar,

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Germany).

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Western Blotting Analysis. After treatment, total cellular protein was extracted

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with RIPA lysis buffer (Cell Signaling Technologies, USA) on ice. Mitochondrial

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protein and cytoplasmic protein were isolated with commercial kit following the

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manufacturer's instruction (Beyotime, Shanghai, China). Briefly, collected cells were

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suspended in mitochondrial isolation buffer with 1-mM phenylmethanesulfonyl

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fluoride and homogenized. Then, cell homogenates were centrifuged at 1000× g, 10

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minutes at 4 °C. Supernatant was transferred to a new Eppendorf tube and centrifuged

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at 3500× g, 10 minutes at 4 °C, and the precipitation was mitochondrial fraction. Then, 8


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the supernatant was transferred to another new Eppendorf tube and centrifuged at

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14,000× g, 10 minutes at 4 °C to obtain cytosolic fraction. Both mitochondrial and

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cytosolic fractions were boiled in protein sample buffer and analyzed by western

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blotting. Protein concentration was measured using BCA protein assay kit. Protein

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samples were heated to 100°C for 5 min and placed briefly on ice. Then, protein

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samples were separated on 8-12 % sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis (SDS-PAGE), followed by transferred to a PVDF membrane. The

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membrane was blocked for 2 h in 5% bovine serum albumin (Sangon Biotech,

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Shanghai, China), and incubated with primary antibody overnight at 4°C. After

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incubation, the membrane was washed five times for 5 min in TBST, and incubated

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with HRP-conjugated secondary antibody for 2 h at room temperature. Finally, the

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membrane was detected by enhanced chemiluminescence (ECL) detection reagent

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(GE Healthcare, Sweden), following the manufacturer’s protocol. The protein levels

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were first normalized to β-actin, and then normalized to control.

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Immunoprecipitation (IP) assay. IP was performed to detect the interaction

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between Nur77 and Bcl-2 proteins in HL-60 cells at 6 h after 20(S)-Rh2 treatment

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according to the manufacturer's instruction (Beyotime, Shanghai, China). Briefly,

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HL-60 cells (4 × 106) were washed twice with cold PBS and lysed with ice-cold RIPA

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lysis buffer for 10 min on ice. Then, the lysate was collected after centrifugation at

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13,000 rpm for 10 min at 4°C. Anti-Nur77 and anti-rabbit IgG antibodies were

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incubated with magnetic beads at 4 °C for 3 h. After three washes with 1 × PBS,

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bead-bound proteins were subjected to SDS-PAGE and analyzed by Western blot 9



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analysis according to standard protocols.

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Immunofluorescence Staining. After treatment, HL-60 cells were centrifuged and

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collected in centrifuge tubes. Then, cells were fixed with 4% paraformaldehyde in

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PBS for 30 min and washed with 1 × PBS for 3 times. Cell membrane

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permeabilization was performed with 0.1% Triton-X100 for 10 min. Following 2 h

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incubation in a fresh goat serum (5%), Cells were probed with specific primary

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antibodies against Bcl-2 and Bax at 4 °C overnight. After washed with 1 × PBS for 3

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times again, cells were incubated with the corresponding fluorescent-labeled

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secondary antibodies. Finally, DAPI was used to co-stain the nuclei. Fluorescence

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images were captured under fluorescence microscope (Leica DM4000B, Wetzlar,

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Germany).

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Construction

and

Production

of

Nur77-shRNA

Lentivirus

Vector.

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Nur77-specific small hairpin RNA (shNur77) and shRNA control with nontargeting

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sequence (shCTRL) were constructed based on the lentivirus-based RNAi vector

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pGPU6/GFP/Neo

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5′-TACACAGGAGAGTTTGACA-3′. Package of lentiviral vectors was performed by

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the co-transfection of 293T cells using the ViraPower Lentiviral Expression Systems

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(Invitrogen, USA). Lentiviral supernatant was obtained within 48-72 h after

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transfection.

190 191

(GeneChem

Co.

Ltd.).

The

human

shNur77

target

is

Wright-Giemsa Staining. Morphological assessment of HL-60 cells or the peripheral

blood

obtained

from

NOD/SCID

mice

10


was

performed

using

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Wright-Giemsa (Leagene, Beijing, China) staining according to the manufacturer’s

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protocol on slides prepared by Cytospin. The morphology of cells was examined

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under a light microscope.

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Nitro blue Tetrazolium (NBT) Reduction Assay. After treatment, HL-60 cells

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were incubated with NBT (1.0 mg/mL) solution at 37°C for 30 min. The cells capable

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of reducing NBT (J＆K, Beijing, China) were measured by counting the number of

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cells

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12-O-Tetradecanoylphorbol-13-acetate was used to stimulate the formation of

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formazan.

containing

the

precipitated

formazan

particles.

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CD11b Detected by Flow Cytometry. Cells (2×105 cells/ml) exposed to vehicle or

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20(S)-Rh2 for 96 h were collected and washed twice with ice-cold PBS. The cells

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were then incubated with the direct fluorescein isothiocyanate-labeled anti-CD11b

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antibody (BD Biosciences, San Jose, CA, USA) on ice for 30 min, washed twice with

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PBS and the level of antibody binding to the cells was quantified using flow

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cytometry (BD Biosciences, San Jose, CA, USA).

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Animal

Model.

Four-week-old

nonobese

diabetic/severe

combined

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immunodeficiency (NOD/SCID) mice were obtained from Charles River (Beijing,

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China). Mice were sublethally irradiated with 2.4 Gy and 24 h later HL-60 cells were

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injected into the tail vein (5×106 cells/mouse, n=6 per group). Starting the next day,

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mice were gavaged of 20(S)-Rh2 dissolved in PBS at a dose of 20 mg/kg, once a day

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for 3 weeks. Control group (no treatment) received vehicle only. All experiments were

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performed under the supervision of the Institutional Animal Care and Use Committee 11



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of Southwest University according to an approved protocol. Mice were euthanized

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three weeks after the injection of HL-60 cells.

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Analysis of Blood Physiological Parameters. Blood was obtained from the

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posterior vein plexus in the orbit of mice. Then, blood was transferred into

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anticoagulant tube and carried out blood physiological analysis on Automatic Animal

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Blood Analyzer (Perlong, Chengdu, China). The main indicators include WBC (white

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blood cells), HGB (hemoglobin) and PLT (platelets).

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Statistical analysis. All data were presented as mean ± SD. The significant

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difference in the study was examined using Student’s t-test or one-way ANOVA. A

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p-value of less than 0.05 was considered to be significant. All calculations were

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performed using Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA).

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RESULTS

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20(S)-Rh2 Inhibited Cell Growth of HL-60 and Kasumi-1 Cells. To determine

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the inhibitory effect of 20(S)-Rh2 on cell growth, we first treated two AML cells

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HL-60 and Kasumi-1 with 20(S)-Rh2 (5 to 100 µM) for 72 h and evaluated cell

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viability with CCK-8 assay. As shown in Figure 1A, obvious cytotoxicity was shown

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in both HL-60 and Kasumi-1 cells after 20(S)-Rh2 treatment. Moreover, 20(S)-Rh2

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exhibited stronger inhibition on cell viability of HL-60 cells (IC50 = 25.59 µM) than

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that of Kasumi-1 cells (IC50 = 60.06 µM). Then, we determined the influence of

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20(S)-Rh2 on the proliferation of HL-60 and Kasumi-1 cells. Figure 1B showed that a

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concentration-dependent inhibition on the proliferation was observed in both cells 12


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treated with 20 to 80 µM of 20(S)-Rh2 for 96 h. Notably, the proliferation was almost

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totally suppressed by 60 and 80 µM of 20(S)-Rh2 in HL-60 cells and 80 µM of

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20(S)-Rh2 in kasumi-1 cells. To determine the inhibitory effect on cell growth with a

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long-term treatment of 20(S)-Rh2, the methylcellulose-based colony formation assay

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was performed in both HL-60 and kasumi-1 cells with 20(S)-Rh2 treatment for two

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weeks. As shown in Figure 1C, the number of colonies formed by both cells was

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remarkably suppressed by 20 and 40 µM of 20(S)-Rh2 compared with that treated

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with vehicle alone (p50 µm in diameter were counted. The colony

633

images were a representative of three independent experiments. Values are presented

634

as means ± SD. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 vs. control.

635

Figure 2. 20(S)-Rh2 induced apoptosis of both HL-60 and Kasumi-1 cells: Cells were

636

treated with 20(S)-Rh2 as indicated for 48 h. Apoptotic cells were determined by flow

637

cytometry (A) and Hoechst 33342 staining (B). The scale bar is 100 µm. (*) p < 0.05,

638

(**) p < 0.01 and (***) p < 0.001 vs. control.

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Figure 3. 20(S)-Rh2-induced apoptosis is correlated with Nur77 and death receptor

640

pathway: (A) HL-60 cells were treated with 40 µM 20(S)-Rh2 for different time. Then,

641

cell lysates were subjected to western blotting for the detection of Nur77 protein level.

642

The blots were a representative of three independent experiments. (B) The level of

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Nur77 was measured by Western blotting after HL-60 cells were transfected with

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shCTRL or shNur77 for 24 h. The blots were a representative of three independent

645

experiments. (C) HL-60 cells were transfected with shNur77 or shCTRL for 24 h and 32


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then treated with or without 40 µM 20(S)-Rh2 for 48 h. shCTRL was used as negative

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control. Apoptotic cells were determined by flow cytometry. (D) HL-60 cells were

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treated as described in “(C)”. The morphologic change was evaluated by Hoechst

649

33342 staining. The scale bar is 100 µm. Data are presented as means ± SD. (*) p
50 µm in diameter were counted. The colony images were a representative of three independent experiments. Values are presented as means ± SD. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 vs. control. 159x201mm (300 x 300 DPI)


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Figure 2. 20(S)-Rh2 induced apoptosis of both HL-60 and Kasumi-1 cells: Cells were treated with 20(S)-Rh2 as indicated for 48 h. Apoptotic cells were determined by flow cytometry (A) and Hoechst 33342 staining (B). The scale bar is 100 µm. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 vs. control. 174x159mm (300 x 300 DPI)



Figure 3. 20(S)-Rh2-induced apoptosis is correlated with Nur77 and death receptor pathway: (A) HL-60 cells were treated with 40 µM 20(S)-Rh2 for different time. Then, cell lysates were subjected to western blotting for the detection of Nur77 protein level. The blots were a representative of three independent experiments. (B) The level of Nur77 was measured by Western blotting after HL-60 cells were transfected with shCTRL or shNur77 for 24 h. The blots were a representative of three independent experiments. (C) HL-60 cells were transfected with shNur77 or shCTRL for 24 h and then treated with or without 40 µM 20(S)-Rh2 for 48 h. shCTRL was used as negative control. Apoptotic cells were determined by flow cytometry. (D) HL-60 cells were treated as described in “(C)”. The morphologic change was evaluated by Hoechst 33342 staining. The scale bar is 100 µm. Data are presented as means ± SD. (*) p< 0.05. (E) HL-60 cells were treated with 40 µM 20(S)-Rh2 for different time. Then, cell lysates were subjected to western blotting for analyzing death receptor pathway related proteins. (F) HL-60 cells were transfected with shNur77 or shCTRL for 24 h and then treated with or without 40 µM 20(S)-Rh2 for 3 h. Then, cell lysates were subjected to Western blotting for analyzing Fas and DR5. The blots were a representative of three independent experiments.


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178x244mm (300 x 300 DPI)



Figure 4. 20(S)-Rh2 promoted Nur77 mitochondria localization and Bcl-2 transformation: (A) Cleavage of both PARP and caspase-3 was examined after treatment of HL-60 cells with 40 µM 20(S)-Rh2 in the presence or absence of nuclear protein export inhibitor LMB for 48 h using western blot analysis. β-actin served as a loading control. (B) After treatment of HL-60 cells with or without 40 µM 20(S)-Rh2 for 24 h, cell lysates were subjected to western blotting for analyzing Nur77 and HSP60 in mitochondria (M) and Nur77 and α-tubulin in cytoplasm (C). (C) HL-60 cells were treated with or without 40 µM 20(S)-Rh2 for 6h. Cell lysates were immunoprecipitated with anti-Nur77 antibody (IP-Nur77) and then Bcl-2 was detected by western blotting. Input, cell lysates without IP process is set as a positive control. IgG, IP with antiimmunoglobulin G (IgG) is set as a negative control. The blots were a representative of three independent experiments. (D) HL-60 cells were treated with or without 40 µM 20(S)-Rh2 for 6h. Immunofluorescence staining of Bcl-2 and Bax were performed as described in Materials and Methods. The images were a representative of three independent experiments. The scale bar is 100 µm. 181x129mm (300 x 300 DPI)


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Figure 5. Nur77 is required for 20(S)-Rh2-induced differentiation of HL-60 cells. 20(S)-Rh2 induced differentiation of HL-60 cells and expression of differentiation-related transcription factors: (A) HL-60 cells (20 × 104 cells/mL) were treated with various concentrations of 20(S)-Rh2 as indicated for 96 h. Cells in slides prepared by Cytospin were stained with Wright-Giemsa staining. (B) HL-60 cells were incubated with 20 µM 20(S)-Rh2 for the indicated times. The NBT positive cells were detected with the NBT reduction assay as described in Materials and Methods. Data are presented as means ± SD. (***) p < 0.001. (C) HL-60 cells were transfected with shNur77 or shCTRL for 24 h and then treated with 20 µM 20(S)-Rh2 for the indicated times. The NBT positive cells were detected with the NBT reduction assay as described in Materials and Methods. (**) p < 0.01. (***) p < 0.001. (D) HL-60 cells were transfected with shNur77 or shCTRL for 24 h and then treated with 20(S)-Rh2 as indicated for 96 h. The CD11b level was detected by flow cytometry as described in Materials and Methods. (E) Cells were treated with 40 µM ginsenoside 20(S)-Rh2 for the indicated times and then harvested. The expression of transcription factors c-Jun and JunB was determined by Western blotting. The blots were a representative of three independent experiments.



150x244mm (300 x 300 DPI)


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Figure 6. Anti-leukemic activity and Nur77-inducing effect of 20(S)-Rh2 in NOD/SCID mice. NOD/SCID mice with the injection of HL-60 cells into the tail vein were gavaged of 20(S)-Rh2 or vehicle for 4 weeks (n=6). (A) The physical status was compared. (B) The weight of spleen and liver was compared between 20(S)-Rh2 and vehicle groups. (*) p < 0.05. (C) Blood physiological parameters (WBC, HGB and PLT) were analyzed as described in Materials and Methods. Values are presented as means ± SD. (D) The blood smear was stained with Wright-Giemsa staining. The images were a representative of three independent experiments. (E) Nur77 expression in spleen and liver was analyzed by Western blotting. The blots were a representative of three independent experiments. 161x120mm (300 x 300 DPI)



Figure 7. Proposed mechanism of induction of apoptosis and differentiation by 20(S)-Rh2 in AML cells: 20(S)-Rh2 enhances Nur77 expression in AML cells. Then, 20(S)-Rh2 induces apoptosis of AML cells through two Nur77-associated pathways: (1) activation of Nur77-mediated death receptor pathways (Fas and DR5); (2) promotion of Nur77 translocation from nucleus to mitochondria and subsequent interaction between Nur77 and Bcl-2, which leads to the exposure of BH3 domain of Bcl-2, resulting in the conversion of Bcl-2 from antiapoptotic to proapoptotic. 20(S)-Rh2-induced differentiation of AML cells is correlated with Nur77associated transcription factors, such as c-Jun and JunB. 154x142mm (300 x 300 DPI)


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Graphic Abstract 119x43mm (300 x 300 DPI)


Role of Orphan Nuclear Receptor Nur

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