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

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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;

8

§

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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;

12

#

13

*Corresponding author at:

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

These authors have contributed equally to this work.

14

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

15

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

24

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

25

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.

27

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

28

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

29

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

33

flow

34

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

35

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

36

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.

12

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



85

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

95

(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

98

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

100

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

101

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

104

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

105

(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,

109

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

112

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

113

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

114

cells.

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

116

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,

140

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

146

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

161

(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.

183

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

198

cells

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

200

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

205

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

206

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

207

Animal

Model.

Four-week-old

nonobese

diabetic/severe

combined

208

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

220

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

223

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

224

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

229

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

230

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

231

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

232

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

234

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

238

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

241

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

242

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.

639

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

643

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

644

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

647

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

648

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)


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

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