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,† Dajian Yang,*,‡ and Hongyi Qi*,† †

College of Pharmaceutical Sciences, Southwest University, 2 Tiansheng Road, Beibei District, Chongqing 400716, China Chongqing Academy of Chinese Materia Medica, 34 Nanshan Road, Nan’an District, Chongqing 400065, China § Radiotherapy Department, Chongqing Ninth People’s Hospital, Jialing Village 69, Beibei District, Chongqing 400700, China ‡

ABSTRACT: Ginsenoside 20(S)-Rh2 has been shown to induce apoptosis and differentiation of acute myeloid leukemia (AML) cells. However, the underlying molecular mechanisms are not fully understood. In our study, 20(S)-Rh2 induced the expression of orphan nuclear receptor Nur77 and death receptor proteins Fas, FasL, DR5, and TRAIL, as well as the cleavage of caspase 8 and caspase 3 in HL-60 cells. Importantly, shNur77 attenuated 20(S)-Rh2-induced apoptosis and Fas and DR5 expression. Meanwhile, 20(S)-Rh2 promoted Nur77 translocation from the nucleus to mitochondria and enhanced the interaction between Nur77 and Bcl-2, resulting in the exposure of the BH3 domain of Bcl-2 and activation of Bax. Furthermore, 20(S)-Rh2 promoted the differentiation of HL-60 cells as evidenced by Wright-Giemsa staining, NBT reduction assay, and detection of the myeloid differentiation marker CD11b by flow cytometry. Notably, shNur77 reversed 20(S)-Rh2-mediated HL-60 differentiation. Additionally, 20(S)-Rh2 also exhibited an antileukemic effect and induced Nur77 expression in NOD/ SCID mice with the injection of HL-60 cells into the tail vein. Together, our studies suggest that the Nur77-mediated signaling pathway is highly involved in 20(S)-Rh2-induced apoptosis and differentiation of AML cells. KEYWORDS: ginsenoside, immediate-early gene, apoptosis, myeloid differentiation, hematologic malignancy

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INTRODUCTION Leukemia as a kind of malignant clonal disease originates from hematopoietic stem cells and can be characterized by inhibiting apoptosis, losing control proliferation, and blocking the differentiation of abnormal hematopoietic cells.1 In 2016, an estimated 60 140 new cases were diagnosed, and over 24 400 patients died from leukemia in the United States.2 In China, there were 75 300 newly increased cases and 53 400 patient deaths in 2015.3 Acute myeloid leukemia (AML) is the most frequently diagnosed acute leukemia in adults, especially older patients (older than 65 years).4 Chemotherapy is one of the main treatments for AML. However, the efficacy of conventional chemotherapy is extremely limited due to the serious side effects, multidrug resistance, and expensive costs. Until recent years, still over 50% of young adult patients and almost 90% of older patients have been dying from AML.4,5 Thus, new alternative treatment approaches are urgently needed for AML therapy. Recently, Nur77 (also known as TR3 and NGFI-B) as a critical therapeutic target of AML has drawn more and more attention. It is encoded by immediate-early gene NR4A1, which can be transiently activated in response to diverse extracellular stimuli and belongs to highly conserved orphan nuclear receptors of the thyroid/steroid receptor superfamily.6,7 It is involved in numerous biological processes, including cell apoptosis, proliferation, and differentiation, and a variety of disease states, such as cardiovascular disorders, atherosclerosis, and cancer.6−9 The function of Nur77 in tumor development is complicated and controversial.10 On the one hand, Nur77 is © 2017 American Chemical Society

overexpressed in the tissue of prostatic cancer, lung cancer, and breast cancer and exhibits oncogenic activity.11,12 On the other hand, Nur77 shows remarkable antitumor activity in hematological tumors.13 A growing body of evidence has firmly established that deletion of orphan nuclear receptor Nur77 is closely related to the development of AML.12−17 Moreover, low expression of Nur77 is commonly detected in AML patients.14 Importantly, retroviral restoration of Nur77 in conditional knockout mice blocks the leukemogenicity of AML cells.12 Recently, the rescue of silenced Nur77 and NOR-1 by histone deacetylase (HDAC) inhibitor SNDX-275 has been shown to dramatically inhibit AML cells.16 Thus, Nur77 is a key suppressor of AML, and targeting Nur77 may provide potential approaches for AML therapeutic intervention. Ginseng is one of the most commonly used functional foods and herbal medicines in East Asia and North America and is derived from the roots and rhizomes of different slow-growing perennial plants that belong to the genus Panax of the family Araliaceae.18,19 Ginsenosides are a variety of triterpenoid saponins and considered to be the main active components of ginseng.20 20(S)-Rh2 is a protopanaxadiol (PPD) type of ginsenoside and one of the typical components in red ginseng. Accumulating evidence demonstrates that 20(S)-Rh2 may be a new promising anticancer agent.21−24 Notably, 20(S)-Rh2 has Received: Revised: Accepted: Published: 7687

May 16, 2017 August 8, 2017 August 9, 2017 August 9, 2017 DOI: 10.1021/acs.jafc.7b02299 J. Agric. Food Chem. 2017, 65, 7687−7697

Article

Journal of Agricultural and Food Chemistry

were fixed with 4% paraformaldehyde in PBS for 10 min and washed with 1 × PBS 3 times. Hoechst 33342 (10 mg/mL) dissolving in 1 × PBS was added into each well. The plates were protected from light exposure and kept at room temperature for 10 min. Finally, the plates were washed with 1 × PBS 3 times again. Cells with fluorescence were observed and photographed under a fluorescence microscope (Leica DM4000B, Wetzlar, Germany). Western Blotting Analysis. After treatment, total cellular protein was extracted with RIPA lysis buffer (Cell Signaling Technologies, USA) on ice. Mitochondrial protein and cytoplasmic protein were isolated with a commercial kit following the manufacturer’s instructions (Beyotime, Shanghai, China). Briefly, collected cells were suspended in mitochondrial isolation buffer with 1 mM phenylmethanesulfonyl fluoride and homogenized. Then, cell homogenates were centrifuged at 1000g for 10 min at 4 °C. The supernatant was transferred to a new Eppendorf tube and centrifuged at 3500g at 10 min at 4 °C, and the precipitation was a mitochondrial fraction. Then, the supernatant was transferred to another new Eppendorf tube and centrifuged at 14,000g for 10 min at 4 °C to obtain a cytosolic fraction. Both mitochondrial and cytosolic fractions were boiled in protein sample buffer and analyzed by Western blotting. Protein concentration was measured using a BCA protein assay kit. Protein samples were heated to 100 °C for 5 min and placed briefly on ice. Then, protein samples were separated on 8−12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE), followed by transfer to a PVDF membrane. The membrane was blocked for 2 h in 5% bovine serum albumin (Sangon Biotech, Shanghai, China) and incubated with primary antibody overnight at 4 °C. After incubation, the membrane was washed five times for 5 min in TBST and incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Finally, the membrane was detected by enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Sweden), following the manufacturer’s protocol. The protein levels were first normalized to β-actin and then normalized to control. Immunoprecipitation (IP) Assay. IP was performed to detect the interaction between Nur77 and Bcl-2 proteins in HL-60 cells at 6 h after 20(S)-Rh2 treatment according to the manufacturer’s instructions (Beyotime, Shanghai, China). Briefly, HL-60 cells (4 × 106) were washed twice with cold PBS and lysed with ice-cold RIPA lysis buffer for 10 min on ice. Then, the lysate was collected after centrifugation at 13,000 rpm for 10 min at 4 °C. Anti-Nur77 and antirabbit IgG antibodies were incubated with magnetic beads at 4 °C for 3 h. After three washes with 1× PBS, bead-bound proteins were subjected to SDS−PAGE and analyzed by Western blot analysis according to standard protocols. Immunofluorescence Staining. After treatment, HL-60 cells were centrifuged and collected in centrifuge tubes. Then, cells were fixed with 4% paraformaldehyde in PBS for 30 min and washed with 1 × PBS 3 times. Cell membrane permeabilization was performed with 0.1% Triton-X100 for 10 min. Following 2 h of incubation in a fresh goat serum (5%), cells were probed with specific primary antibodies against Bcl-2 and Bax at 4 °C overnight. After washing with 1× PBS 3 times again, cells were incubated with the corresponding fluorescentlabeled secondary antibodies. Finally, DAPI was used to costain the nuclei. Fluorescence images were captured under a fluorescence microscope (Leica DM4000B, Wetzlar, Germany). Construction and Production of Nur77-shRNA Lentivirus Vector. Nur77-specific small hairpin RNA (shNur77) and shRNA control with nontargeting sequence (shCTRL) were constructed based on the lentivirus-based RNAi vector pGPU6/GFP/Neo (GeneChem Co. Ltd.). The human shNur77 target is 5′-TACACAGGAGAGTTTGACA3′. Package of lentiviral vectors was performed by the cotransfection of 293T cells using the ViraPower Lentiviral Expression Systems (Invitrogen, USA). The lentiviral supernatant was obtained within 48−72 h after transfection. Wright-Giemsa Staining. Morphological assessment of HL-60 cells or peripheral blood obtained from NOD/SCID mice was performed using Wright-Giemsa (Leagene, Beijing, China) staining according to the manufacturer’s protocol on slides prepared by cytospin. The morphology of cells was examined under a light microscope.

received much attention due to the remarkable inhibitory effect on growth and differentiation of human leukemia cells.25−28 Recently, cellular stress response mechanisms have received much more attention as the molecular targets of ginsenosides.29 Thus, it is interesting to us to discover whether the Nur77mediated signaling pathway is involved in the inhibitory effect of 20(S)-Rh2 on AML. In the current study, we characterized the inducing effect of 20(S)-Rh2 on apoptosis and differentiation of AML cells. On the one hand, we determined whether 20(S)-Rh2-induced apoptosis was correlated with the Nur77-regulated death receptor pathway and Nur77 mitochondria localization and subsequent Bcl-2 transformation. On the other hand, we evaluated whether 20(S)-Rh2-induced differentiation was related to Nur77 and Nur77-mediated differentiation-associated transcription factors.

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MATERIALS AND METHODS

Chemicals and Antibodies. Ginsenoside 20(S)-Rh2 with a purity of 98% was purchased from Chroma-Biotechnology Co. (Chengdu, China), dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) at 100 mM and stored at −20 °C before use. Leptomycin B was purchased from Beyotime Biotechnology (Shanghai, China). The antibodies against Fas, FasL, DR5, TRAIL, caspase 3, cleaved caspase 3, and cleaved caspase 8 were obtained from Wanlei Biotechnology (Shenyang, China). The antibodies against Nur77, PARP, cleaved PARP, c-Jun, Jun-B, Bcl-2, and Bax were purchased from Santa Cruz Biotechnology (CA, USA). The antibodies against β-actin, mouse, or rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hoechst 33342 was obtained from Wanlei Biotechnology (Shenyang, China). Cell Culture. AML cell lines HL-60 and Kasumi-1 were purchased from Cell Bank of Chinese Academic of Science (Shanghai, China) and grown in RPMI-1640 medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% penicillin/streptomycin (Invitrogen, USA) in a 5% CO2 humidified incubator at 37 °C. Measurement of Cell Viability. The cell counting kit-8 (CCK-8) (Dojindo, Shanghai, China) assay was used to determine the cell viability. HL-60 and Kasumi-1 cells (8 × 104 cells/mL) were inoculated into 96-well plates at 100 μL. Then, cells were treated for 72 h and subsequently added with 10 μL of CCK-8 for each well. The OD value was detected at 450 nm with a microplate reader (Biotek, Winooski, VT, USA). Cell viability was presented as a percentage of that of untreated cells. Cell Proliferation Assay. The proliferation of HL-60 and Kasumi-1 cells were determined by the trypan blue dye exclusion test. Cells were seeded at a density of 5 × 104 cells/mL and treated with 20(S)-Rh2 at various concentrations for 24−120 h. Then, cells were stained with trypan blue (Sigma, USA), and the number of viable cells was counted using hemocytometer. Colony Formation Assay. Colony forming units were assayed in methylcellulose (Methocult H4100, StemCell Technologies Inc., Canada) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% penicillin/streptomycin (Invitrogen, USA). Vehicle or 20(S)-Rh2 was added to methylcellulose containing 2500 cells in a 24-well plate. Colonies were evaluated microscopically 14 days after plating. Cell Apoptosis Analyzed by Annexin V-FITC/PI DoubleStaining Assay. Cells were seeded at a concentration of 2 × 105 cells/mL and incubated for 48 h with 20(S)-Rh2 at various concentrations. After treatment, cells were harvested, washed, and resuspended in the binding buffer containing annexin V-FITC and propidiumiodide (PI). Flow cytometry analyses were performed on a BD LSRFortessa Cell Analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed using Flow Jo 7.6.1 software (Tree Star, Inc., Ashland, OR, USA). Hoechst 33342 Staining. After treatment, HL-60 and Kasumi-1 cells were centrifuged and collected in Eppendorf tubes. Then, cells 7688

DOI: 10.1021/acs.jafc.7b02299 J. Agric. Food Chem. 2017, 65, 7687−7697

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Figure 1. 20(S)-Rh2 inhibited the growth of HL-60 and Kasumi-1 cells: (A) cell viability. Cells were treated with 20(S)-Rh2 as indicated for 72 h. The cell viability was assessed by cell counting kit-8 assay. (B) Proliferation. Exponentially growing cells were treated with the indicated concentrations of 20(S)-Rh2 for 120 h. Cell proliferation was assessed using trypan blue exclusion assay. (C) Colony formation. Cells were cloned in methylcellulose and treated with 20(S)-Rh2 as indicated or vehicle. Two weeks later, colonies >50 μm in diameter were counted. The colony images were a representative of three independent experiments. Values are presented as the means ± SD (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 vs control. Nitro Blue Tetrazolium (NBT) Reduction Assay. After treatment, HL-60 cells were incubated with NBT (1.0 mg/mL) solution at 37 °C for 30 min. The cells capable of reducing NBT (J&K, Beijing, China) were measured by counting the number of cells containing the precipitated formazan particles. 12-O-Tetradecanoylphorbol-13-acetate was used to stimulate the formation of formazan. CD11b Detected by Flow Cytometry. Cells (2 × 105 cells/mL) exposed to the vehicle or 20(S)-Rh2 for 96 h were collected and washed twice with ice-cold PBS. The cells were then incubated with the direct fluorescein isothiocyanate-labeled anti-CD11b antibody (BD Biosciences, San Jose, CA, USA) on ice for 30 min, washed twice with PBS, and the level of antibody binding to the cells was quantified using flow cytometry (BD Biosciences, San Jose, CA, USA). Animal Model. Four-week-old nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were obtained from Charles River (Beijing, China). Mice were sublethally irradiated with 2.4 Gy, and 24 h later, HL-60 cells were injected into the tail vein (5 × 106 cells/mouse, n = 6 per group). Starting the next day, mice were gavaged of 20(S)-Rh2 dissolved in PBS at a dose of 20 mg/kg, once a day for 3 weeks. The control group (no treatment) received the

vehicle only. All experiments were performed under the supervision of the Institutional Animal Care and Use Committee of Southwest University according to an approved protocol. Mice were euthanized 3 weeks after the injection of HL-60 cells. Analysis of Blood Physiological Parameters. Blood was obtained from the posterior vein plexus in the orbit of mice. Then, blood was transferred into an anticoagulant tube, and we carried out blood physiological analysis on an Automatic Animal Blood Analyzer (Perlong, Chengdu, China). The main indicators include WBC (white blood cells), HGB (hemoglobin), and PLT (platelets). Statistical Analysis. All data were presented as the mean ± SD. The significant difference in the study was examined using Student’s t test or one-way ANOVA. A p-value of less than 0.05 was considered to be significant. All calculations were performed using Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA).

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RESULTS 20(S)-Rh2 Inhibited Cell Growth of HL-60 and Kasumi-1 Cells. To determine the inhibitory effect of 20(S)-Rh2 on cell growth, we first treated two AML cells HL-60 and Kasumi-1 7689

DOI: 10.1021/acs.jafc.7b02299 J. Agric. Food Chem. 2017, 65, 7687−7697

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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.

with 20(S)-Rh2 (5 to 100 μM) for 72 h and evaluated cell viability with CCK-8 assay. As shown in Figure 1A, obvious cytotoxicity was shown in both HL-60 and Kasumi-1 cells after 20(S)-Rh2 treatment. Moreover, 20(S)-Rh2 exhibited stronger inhibition on cell viability of HL-60 cells (IC50 = 25.59 μM) than that of Kasumi-1 cells (IC50 = 60.06 μM). Then, we determined the influence of 20(S)-Rh2 on the proliferation of HL-60 and Kasumi-1 cells. Figure 1B showed that a concentrationdependent inhibition on proliferation was observed in both cells treated with 20 to 80 μM 20(S)-Rh2 for 96 h. Notably, the proliferation was almost totally suppressed by 60 and 80 μM 20(S)-Rh2 in HL-60 cells and 80 μM 20(S)-Rh2 in Kasumi-1 cells. To determine the inhibitory effect on cell growth with a long-term treatment of 20(S)-Rh2, the methylcellulose-based colony formation assay was performed in both HL-60 and Kasumi-1 cells with 20(S)-Rh2 treatment for 2 weeks. As shown in Figure 1C, the number of colonies formed by both cells was remarkably suppressed by 20 and 40 μM 20(S)-Rh2 compared with that treated with vehicle alone (p < 0.001). Morphologically, the size of colonies also obviously reduced in both cells after 20 and 40 μM 20(S)-Rh2 treatment. 20(S)-Rh2 Induced Apoptosis in HL-60 and Kasumi-1 Cells. To characterize the cell death induced by 20(S)-Rh2, we first determined the apoptosis rate caused by 20(S)-Rh2 by flow cytometry in HL-60 and Kasumi-1 cells after probing by Annexin V-FITC/PI. As shown in Figure 2A, 20(S)-Rh2 (20 and 40 μM) significantly enhanced the apoptosis rate of both HL-60 and Kasumi-1 cells. An increase of approximately six percent of apoptosis rate was observed in both HL-60 and Kasumi-1 cells after treatment with 40 μM of 20(S)-Rh2. Then, we further

determined the apoptosis by Hoechst 33342 staining. Figure 2B showed that much more cells with condensed and fragmented nuclei were observed in both HL-60 and Kasumi-1 cells treated with 40 μM 20(S)-Rh2 than those in the control. 20(S)-Rh2-Induced Apoptosis Is Mediated by Nur77 and Death Receptor Pathway. Recent evidence shows that the loss of Nur77 played a critical role in the rapid development of lethal AML in mice.12−17 Moreover, the down-regulation of death receptor pathway proteins contributes to the Nur77 deficiency-related AML development.14 Thus, we then determined the effect of 20(S)-Rh2 on Nur77 expression. As shown in Figure 3A, Nur77 was induced by 20(S)-Rh2 as early as 1 h, and the maximum expression was observed at 6 h. Emerging evidence also demonstrates that Nur77 plays a critical role in the apoptosis of cancer cells, and the cell death receptor pathway is one of the molecular mechanisms modulated by Nur77.14 Thus, we further determined the role of Nur77 in 20(S)-Rh2-mediated apoptosis. In our study, Nur77 was inhibited by shNur77 (Figure 3B). Flow cytometry analysis demonstrated that the apoptosis rate induced by 20(S)-Rh2 (40 μM) obviously reduced after shNur77 transfection (Figure 3C). Meanwhile, Hoechst 33342 staining showed that silencing of Nur77 by shNur77 remarkably attenuated 20(S)-Rh2 (40 μM)induced apoptosis of HL-60 cells (p < 0.05) (Figure 3D). Then, activation of death receptor-related proteins Fas and FasL, as well as DR5 and TRAIL, was also monitored in HL-60 cells treated by 20(S)-Rh2 (40 μM). As shown in Figure 3E, 20(S)-Rh2 caused an increase of Fas protein as early as 1 h after treatment. Moreover, FasL, DR5, and TRAIL was observed to be induced at 3 h after 20(S)-Rh2 treatment. Moreover, we 7690

DOI: 10.1021/acs.jafc.7b02299 J. Agric. Food Chem. 2017, 65, 7687−7697

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Journal of Agricultural and Food Chemistry

Figure 3. 20(S)-Rh2-induced apoptosis is correlated with Nur77 and the death receptor pathway: (A) HL-60 cells were treated with 40 μM 20(S)-Rh2 for different times. 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 the 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 the means ± SD (*) p < 0.05. (E) HL-60 cells were treated with 40 μM 20(S)-Rh2 for different times. Then, cell lysates were subjected to Western blotting for analyzing the 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.

20(S)-Rh2 Promoted Nur77 Mitochondria Localization and Bcl-2 Transformation. Accumulating evidence demonstrates that relocalization of Nur77 from the nucleus to mitochondria is another primary mechanism for Nur77-mediated apoptosis.30,31 To determine the influence of Nur77 nuclear export on 20(S)-Rh2-induced apoptosis, HL-60 cells were pretreated by leptomycin B (LMB), a specific inhibitor of nuclear protein export. As shown in Figure 4A, 20(S)-Rh2 (40 μM) moderately increased c-PARP and c-caspase 3 levels, whereas LMB pretreatment obviously decreased the level of both

determined the expression of death receptor-induced apoptosis related caspase 8 protein. The cleaved-caspase 8 was increased from 3 h, and the maximum expression was shown at 6 h after 20(S)-Rh2 treatment. In addition, we also found that cleavedcaspase 3 was activated as early as 1 h after 20(S)-Rh2 treatment. Finally, we further determined the role of Nur77 in 20(S)-Rh2-mediated cell death receptor pathway. Our results of Western blotting demonstrated that the increase of Fas and DR5 induced by 20(S)-Rh2 was obviously mitigated in HL-60 cells transfected with shNur77 (Figure 3F). 7691

DOI: 10.1021/acs.jafc.7b02299 J. Agric. Food Chem. 2017, 65, 7687−7697

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Journal of Agricultural and Food Chemistry

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 6 h. 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 anti-immunoglobulin G (IgG) is set as a negative control. The blots are a representative of three independent experiments. (D) HL-60 cells were treated with or without 40 μM 20(S)-Rh2 for 6 h. Immunofluorescence staining of Bcl-2 and Bax were performed as described in Materials and Methods. The images are a representative of three independent experiments. The scale bar is 100 μm.

antibody in HL-60 cells treated by 20(S)-Rh2 (40 μM). These results suggest that 20(S)-Rh2 promotes the binding of Nur77 to Bcl-2, resulting in the exposure of the BH3 domain of Bcl-2 and subsequent activation of Bax. Nur77 Is Required for 20(S)-Rh2-Induced Differentiation of HL-60 Cells. To examine the effect of 20(S)-Rh2 on myeloid differentiation of AML cells, Wright-Giemsa staining was first performed to detect the morphologic changes of HL-60 cells treated with 20(S)-Rh2 (0, 5, 10, and 20 μM) for 96 h. As shown in Figure 5A, HL-60 cells treated with 20(S)-Rh2 exhibited typical morphological features of myeloid differentiation, such as a lower nucleocytoplasmic ratio and chromatin condensation. On the contrary, only slightly morphologic changes were observed in untreated HL-60 cells. Then, a NBT reduction assay was further performed to examine the generation of oxidative bursts during differentiation in HL-60 cells. Figure 5B showed that the NBT positive cells time-dependently increased from 24 to 96 h in HL-60 cells treated by 20 μM of 20(S)-Rh2 (p < 0.001). Moreover, over 40% of NBT positive cells was observed in HL-60 cells after 96 h of treatment by 20(S)-Rh2. A similar result was also shown in HL-60 cells treated by vitamin D3 (VD3, positive control) for 96 h (p < 0.001). These results indicate that 20(S)-Rh2 induced marked myeloid differentiation of HL-60 cells. Furthermore, we determined the influence of shNur77 on 20(S)-Rh2-induced differentiation of HL-60 cells. As shown in Figure 5C, transfection with shNur77 significantly reversed the increase of NBT positive cells in HL-60 cells treated by 20(S)-Rh2 (20 μM) for 48 h (p < 0.01) and 96 h (p < 0.001).

cleaved-PARP and cleaved-caspase 3, suggesting that 20(S)Rh2-induced apoptosis was highly dependent on the process of nuclear export. To examine whether 20(S)-Rh2 can affect the localization of Nur77 to mitochondria, we isolated the mitochondrial protein and cytosolic protein from HL-60 cells treated with or without 20(S)-Rh2 (40 μM). Our results of Western blotting showed that the level of Nur77 slightly increased in mitochondria, whereas it decreased in cytosol, indicating that 20(S)-Rh2 promoted the translocation of Nur77 to mitochondria (Figure 4B). It is well-established that Nur77 directly interacts with Bcl-2 in the mitochondria and subsequently causes a conformational change to expose the BH3 domain of Bcl-2, which alters the function of Bcl-2 from antiapoptotic to proapoptotic.32 To determine the influence of 20(S)-Rh2 on the interaction of Nur77 and Bcl-2, we performed coimmunoprecipitation, and the results showed that the interaction between Nur77 and Bcl-2 increased in HL-60 cells after 20(S)-Rh2 (40 μM) treatment (Figure 4C). To further determine influence of 20(S)-Rh2 on the conformational change of Bcl-2, we conducted immunofluorescent staining to detect the exposure of the BH3 domain of Bcl-2 with an antiBcl-2 (BH3) antibody. Meanwhile, a Bax (6A7) antibody was used to detect the activation of Bax, which also undergoes a conformational change during early apoptosis.33 Our results (Figure 4D) showed that HL-60 cells without 20(S)-Rh2 treatment displayed almost no or very weak immunofluorescent staining of the anti-Bcl-2 (BH3) antibody and anti-Bax (6A7) antibody. However, there was obvious immunofluorescent staining by the anti-Bcl-2 (BH3) antibody and anti-Bax (6A7) 7692

DOI: 10.1021/acs.jafc.7b02299 J. Agric. Food Chem. 2017, 65, 7687−7697

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with shNur77. Then, we further determined the expression of differentiation-related transcription factors c-Jun and JunB, which were reported to be the Nur77-mediated downstream mechanism.14 As shown in Figure 5E, JunB and c-Jun were significantly activated at 12 and 24 h after 20(S)-Rh2 (20 μM) treatment. Antileukemic Activity and Nur77-Inducing Effect of 20(S)-Rh2 in NOD/SCID Mice. The antileukemic activity of 20(S)-Rh2 was further investigated in vivo using NOD/SCID immunodeficient mice with the injection of HL-60 cells into the tail vein. In the third week after HL-60 cell injection, NOD/ SCID mice in the vehicle were dispirited with low appetite, showed down-bent gait, wrinkled fur, and slow movement, while those in 20(S)-Rh2 group behaved more normally (Figure 6A). Moreover, 20(S)-Rh2 significantly reduced splenomegaly (p < 0.05) compared with that of vehicle alone (Figure 6B). The determination of blood physiological parameters showed that WBC and HGB exhibited a decreasing trend in 20(S)-Rh2 group compared with that of those in vehicle group. However, there is no statistical difference (p > 0.05) for WBC, HGB, and PLT between these two groups (Figure 6C). The blood smear stained with Wright-Giemsa showed that HL-60-like immature cells are present in the vehicle group. Notably, those cells exhibited differentiation-related morphological features, such as reduced nuclei/cytoplasm ratio and horseshoe-shaped nuclei, in the 20(S)-Rh2 group (Figure 6D). To determine the potential influence of 20(S)-Rh2 on Nur77 expression in NOD/SCID mice with HL-60 cell injection, Western blotting analysis was performed with the spleen and liver tissues obtained from the vehicle group and 20(S)-Rh2 group, respectively. The results demonstrated that Nur77 expression of both the spleen and liver significantly increased in the 20(S)-Rh2 group compared with those in the vehicle group.

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DISCUSSION Ginsenosides are the major components of ginseng and exhibit a variety of pharmacological effects, including antifatigue, antiobesity, neuroprotection, antihyperglycemic, and antitumor.34 Among them, 20(S)-Rh2 exhibits remarkable inhibitory effects on various cancer cells, including AML cells.25−28 Accumulating evidence shows that the loss of Nur77 is highly correlated with the development of AML and that the restoration of Nur77 is recognized as a promising molecular target for AML intervention.12−17 In the current study, we aimed to clarify whether 20(S)-Rh2-induced apoptosis and differentiation of AML cells is dependent on Nur77-mediated mechanisms. We first confirmed the growth inhibition of 20(S)-Rh2 on AML cells in our experimental system. Our results demonstrated that 20(S)-Rh2 markedly inhibited the cell viability, proliferation, and colony formation ability of both HL-60 and Kasumi-1 cells. In particular, our results indicated that HL-60 exhibited higher sensitivity to 20(S)-Rh2 with an IC50 of 25.59 μM, which is consistent with the IC50 value (25 μM) obtained by Huang, J., et al.27 and much more potent than that (38.5 μM) reported by Chung, K. S., et al.28 Meanwhile, 20(S)-Rh2 was also shown to inhibit the proliferation of HL-60 cells, which is similar to the result of Chung, K. S., et al.28 Then, we examined the inducing effect of 20(S)-Rh2 on apoptosis in AML cells. Both flow cytometry and Hoechst 33342 staining results showed that moderate apoptosis was induced by 20(S)-Rh2 in both HL-60 and Kasumi-1 cells. Such results are consistent with observations that 20(S)-Rh2 induced apoptosis in HL-60 cells,28 K562, and KG1-α cells.25 Despite the critical

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 the 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 are a representative of three independent experiments.

To further characterize the differentiation mediated by 20(S)-Rh2, we determined the expression of CD11b, a marker of myeloid differentiation, by flow cytometry. Figure 5D showed that 20(S)-Rh2 (20 to 40 μM) concentration-dependently enhanced the CD11b expression of HL-60 cells, while the increase of CD11b expression remarkably reduced in HL-60 cells transfected 7693

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Figure 6. Antileukemic 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 with 20(S)-Rh2 or vehicle for 3 weeks (n = 6). (A) The physical status was compared. (B) The weight of the 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 the 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 the spleen and liver was analyzed by Western blotting. The blots are a representative of three independent experiments.

Our results demonstrated that 20(S)-Rh2 exhibited obvious induction on death receptor ligands, FasL and TRAIL, and their related receptors, Fas and DR5. Furthermore, caspase 8, a death receptor-induced apoptosis related protein was also induced by 20(S)-Rh2. These results indicate that the death receptor signaling pathway is involved in 20(S)-Rh2-mediated apoptosis of AML cells. To directly correlate Nur77 induction with 20(S)-Rh2-mediated apoptosis, lentiviral repression of Nur77 was applied in our study. Our results showed that shNur77 not only decreased 20(S)-Rh2-induced apoptosis but also reversed Nur77-induced expression of death receptors Fas and DR5, suggesting that the Nur77-mediated death receptor pathway is involved in 20(S)-Rh2-induced apoptosis. Another way of Nur77-mediated apoptosis is related to the mitochondria localization of Nur77 and subsequent Bcl-2 conformational change. Under certain conditions, Nur77 translocates from the nucleus to cytoplasm and then locates to the mitochondria, resulting in the increased mitochondrial membrane permeability and release of cytochrome C and caspase precursors.30 Further mechanistic studies revealed that Nur77 binds to the N-terminal region of Bcl-2 during this process, which is necessary for the mitochondrial localization of Nur77. Moreover, it has also been shown that this binding leads to the exposure of the BH3 domain of Bcl-2 due to the conformational change, resulting in the conversion of Bcl-2 from antiapoptotic to proapoptotic.32,39 In particular, Nur77 was found to selectively bind to Bcl-B, Bcl-2, and Bfl-1 in plasma cells and myeloma.40 Thus, we then investigated the potential effect of 20(S)-Rh2 on Nur77 translocation and Bcl-2 conformational change. We first found that 20(S)-Rh2-induced cleavage of RARP and caspase 3 was blocked by the nuclear export inhibitor LMB. Meanwhile, 20(S)-Rh2 obviously increased the enrichment of Nur77 in mitochondria. These results indicate that 20(S)-Rh2-induced apoptosis may be correlated with the Nur77 translocation from the nucleus to mitochondria.

role of apoptosis in 20(S)-Rh2-mediated growth inhibition of AML cells, it is largely unknown what molecular mechanisms are responsible for 20(S)-Rh2-induced apoptosis. Meanwhile, it is worthwhile to note that only a few percentage of cells underwent apoptosis after 20(S)-Rh2 treatment in our study, indicating the existence of other types of cell death. Recently, 20(S)-Rh2 was also found to inhibit hepatocellular carcinoma cells and cancer stem-like cells in skin squamous cell carcinoma by inducing autophagy and blocking β-catenin signaling.22,35 Accumulating evidence shows that the loss of Nur77 is highly correlated with the development of AML.12−17 We thereby examined the effect of 20(S)-Rh2 on Nur77 expression. Our result of Western blotting showed 20(S)-Rh2 rapidly and transiently induced the expression of Nur77, which is consistent with the variation characteristics of immediate-early genes.36 It is reported that the transcriptional activation of Nur77 in response to diverse extracellular stimuli involves two mechanisms, including immediate-early expression and delayed-early expression. Immediate-early expression of Nur77 occurs in the absence of de novo protein synthesis and primarily dependent on the regulation of CArG element-like sequences located between nucleotides −86 and −126 and the binding site of the Ets family. The delayed-early expression of Nur77 is dependent on de novo protein synthesis and occurs in the presence of the interaction of AP-1-like and GC-rich elements with other immediate-early genes, such as Fos/Jun and Zif268, that are transiently induced by extracellular stimuli.37,38 Emerging evidence indicated that the deletion of Nur77 led to the down-regulation of death receptor proteins, indicating that the abnormity in extrinsic apoptotic pathway plays a critical role in Nur77 deficiency-related AML development.14 Moreover, death receptor proteins were shown to be induced in HDAC inhibitor SNDX-275-mediated apoptosis of AML cells by the restoration of silenced Nur77 and NOR-1.16 We then determined the influence of 20(S)-Rh2 on death receptor proteins. 7694

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Figure 7. Proposed mechanism of the 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 the nucleus to mitochondria and subsequent interaction between Nur77 and Bcl-2, which leads to the exposure of the 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 Nur77-associated transcription factors, such as c-Jun and JunB.

which were reported to be the differentiation-related transcription factors regulated by Nur77.42−45 Furthermore, we found that shNur77 attenuated 20(S)-Rh2-induced differentiation of HL-60 cells, suggesting that Nur77-mediated downstream mechanism (i.g., activation of c-Jun and JunB) plays a key role in 20(S)-Rh2-induced differentiation. Taken together, the results obtained in this study indicates the critical role of Nur77 in 20(S)-Rh2-induced apoptosis and differentiation of AML cells. On the one hand, 20(S)-Rh2induced apoptosis was shown to be correlated with the Nur77mediated death receptor pathway and the mitochondria localization of Nur77 and subsequent Bcl-2 conformational change. On the other hand, 20(S)-Rh2-induced differentiation was demonstrated to be related to Nur77-mediated differentiationassociated transcription factors (Figure 7). Thus, our study demonstrates that 20(S)-Rh2 induced apoptosis and differentiation through Nur77-mediated signaling pathways and may serve as a potent chemotherapeutic agent for AML.

It is reported that the subcellular trafficking of Nur77 is regulated through the phosphorylation of Nur77 by multiple protein kinases, such as at Thr142 by ERK2 and at Ser351 by Akt,13,41 which may act upstream to 20(S)-Rh2-mediated Nur77 nuclear export. Furthermore, our results of immunoprecipitation and immunofluorescence staining revealed that 20(S)-Rh2 promoted the binding between Nur77 and Bcl-2 and rendered the exposure of the BH3 domain of Bcl-2, followed by the activation of Bax, suggesting that the Nur77mediated conformational change and function conversion of Bcl-2 may be involved in 20(S)-Rh2-induced apoptosis. It has also been shown that the loss of Nur77 led to the differentiation blockage of hematopoietic stem cells and myeloid progenitors, which played a critical role in the development of AML.14 Such impairment was found to be related to the down-regulation of the transcription factors JunB, which inhibited the leukemic self-renewal capacity, and c-Jun, which initiated the myelomonocytic differentiation.28 Meanwhile, Nur77 was also identified as a key modulator in the differentiation of Ly6C− monocytes.15 Thus, we then determined the ability of 20(S)-Rh2 to induce differentiation in HL-60 cells. Our results of the Wright-Giemsa staining and NBT reduction assay showed that 20(S)-Rh2 obviously induced the differentiation of HL-60 cells. Such an effect was further confirmed by the detection of myeloid differentiation marker CD11b. Similar observation was also obtained by Chung, K. S., et al.,28 who further found that 20(S)-Rh2-induced differentiation was related to the modulation of transforming growth factor-β (TGFβ) production. In our study, we also revealed that 20(S)-Rh2 markedly induced the expression of c-Jun and JunB,

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AUTHOR INFORMATION

Corresponding Authors

*(D.Y.) Tel/Fax: +86 23 89029011. E-mail: yangdajian@ foxmail.com. *(H.Q.) Tel/Fax: +86 23 68251225. E-mail: hongyiqi@swu. edu.cn. ORCID

Hongyi Qi: 0000-0003-0564-3892 Author Contributions ∥

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This work was supported by the NSFC Projects (No. 81373903; No. 81202946), Chongqing Research Program of Basic Research and Frontier Technology (No. cstc2017jcyjAX0299), TCM Project of Chongqing Health and Family Planning Commission (ZY201702120), and the Key Project of Fundamental Research Fund for the Central Universities (XDJK2016B040; XDJK2016B034). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AML, acute myeloid leukemia; CCK-8, cell counting kit-8; HDAC, histone deacetylase; IP, immunoprecipitation; LMB, leptomycin B; NBT, nitro blue tetrazolium; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; TGFβ, transforming growth factor-β; VD3, vitamin D3

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