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Modulation of Orphan Nuclear Receptor Nur77-Mediated Apoptotic Pathway by Acetylshikonin and Analogues

¹ Institute for Biomedical Research, Xiamen University, Xiamen, China; ² Shanghai School of Pharmacy, Shanghai Jiaotong University, ³ Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; and ⁴ Cancer Center, The Burnham Institute for Medical Research, La Jolla, California

Requests for reprints: Jin-Zhang Zeng, Cancer Center Institute for Biomedical Research, Xiamen University, Xiamen 361005, China. Phone: 86-592-2187221; Fax: 86-592-2181879; E-mail: jzzeng{at}xmu.edu.cn and Xiao-kun Zhang, Cancer Center, The Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-3141; Fax: 858-646-3195; E-mail: xzhang{at}burnham.org.

	Abstract

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Shikonin derivatives, which are the active components of the medicinal plant Lithospermum erythrorhizon, exhibit many biological effects including apoptosis induction through undefined mechanisms. We recently discovered that orphan nuclear receptor Nur77 migrates from the nucleus to the mitochondria, where it binds to Bcl-2 to induce apoptosis. Here, we report that certain shikonin derivatives could modulate the Nur77/Bcl-2 apoptotic pathway by increasing levels of Nur77 protein and promoting its mitochondrial targeting in cancer cells. Structural modification of acetylshikonin resulted in the identification of a derivative 5,8-diacetoxyl-6-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones (SK07) that exhibited improved efficacy and specificity in activating the pathway. Unlike other Nur77 modulators, shikonins increased the levels of Nur77 protein through their posttranscriptional regulation. The apoptotic effect of SK07 was impaired in Nur77 knockout cells and suppressed by cotreatment with leptomycin B that inhibited Nur77 cytoplasmic localization. Furthermore, SK07 induced apoptosis in cells expressing the COOH-terminal half of Nur77 protein but not its NH₂-terminal region. Our data also showed that SK07-induced apoptosis was associated with a Bcl-2 conformational change and Bax activation. Together, our results show that certain shikonin derivatives act as modulators of the Nur77-mediated apoptotic pathway and identify a new shikonin-based lead that targets Nur77 for apoptosis induction. [Cancer Res 2008;68(21):8871–80]

	Introduction

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Nur77 (also known as TR3 or NGFI-B), an orphan member of the steroid/thyroid/retinoid receptor superfamily and an immediate-early response gene, plays a critical role in the regulation of differentiation, proliferation, apoptosis, and survival of many different cell types (1, 2). Although the growth-promoting effect of Nur77 has been described in various tumors (3, 4), Nur77 expression also mediates the killing effects of a variety of apoptotic stimuli such as synthetic retinoids, calcium ionophores, etoposide, phorbol ester, chenodeoxycholic acid derivatives, di-n-butyltin dichloride, histone deacetylase inhibitors, cadmium, and 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes in cancer cells (5–9). The apoptotic effect of Nur77 seems to be clinically relevant as the expression of the Nur77 subfamily member Nor-1 is positively correlated with survival in diffuse large B-cell lymphoma patients treated with chemotherapeutic drugs (10), and down-regulation of Nur77 is associated with the metastasis of primary solid tumors (11).

One of the mechanisms that regulate the opposing biological activities of Nur77, survival and death, is through its subcellular localization. Nur77 confers its growth-promoting activities through its action in the nucleus (12, 13), which requires its DNA-binding and transactivation. In contrast, the apoptotic effect of Nur77 is transcription-independent and occurs in the absence of its DNA-binding domain (5, 8, 14–16). In this case, Nur77 initiates apoptotic cascades by migrating to the mitochondria where it interacts with the Bcl-2 apoptotic machinery by converting Bcl-2 from a protector to a killer (16, 17). Such a Nur77/Bcl-2 apoptotic pathway has been used by a variety of apoptosis-inducing agents in prostate cancer, lung cancer, colon cancer, ovarian cancer, and gastric cancer cells (2, 8, 14, 15, 17, 18). In this regard, Nur77 and Bcl-2 are often overexpressed in cancer cells, providing an excellent opportunity to preferentially induce apoptosis of these cells by inducing Nur77 migration and Bcl-2 conversion (1, 17). Such an approach may be highly effective in inducing cancer apoptosis because it engages a simultaneous suppression of the survival function of Nur77 and Bcl-2, and activation of their proapoptotic potential (1, 17). Thus, agents that specifically activate the Nur77-mediated pathway may have therapeutic potential.

In an effort to identify new modulators of the Nur77/Bcl-2 apoptotic pathway, we have undertaken the screening of a natural product library prepared from Chinese herbal medicines. Here, we report that certain shikonin derivatives, active components of the Chinese medicinal plant, Lithospermum erythrorhizon (19, 20), could activate the Nur77-mediated apoptotic pathway in cancer cells. Further evaluation of acetylshikonin analogues showed that 5,8-diacetoxyl-6-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones (SK07) was more active than its parental compound in inducing Nur77 expression and apoptosis. Our mechanistic studies revealed that SK07 increased the levels of Nur77 protein in cancer cells through its posttranscriptional regulation and that it killed cancer cells by inducing Bcl-2 conformational change and Bax activation in a Nur77-dependent manner. Together, our studies identify new modulators of the Nur77/Bcl-2 apoptotic pathway and a potential target-based anticancer lead.

	Materials and Methods

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Reagents. Lipofectamine 2000, Vybrant apoptosis assay kit no. 2, and Trizol LS from Invitrogen, enhanced chemiluminescence reagents, goat anti-rabbit and anti-mouse secondary antibody conjugated to horseradish peroxidase from Thermo Fisher Scientific, Inc., anti-mouse IgG conjugated with Cy3 from Chemicon International, polyclonal anti-Nur77 (sc-5569), anti-Hsp60 (sc-7150), anti–Bcl-2 (sc-509), anti-Bax (sc-493), anti-Bax (6A7; sc-23959), FITC-labeled anti-rabbit IgG from Santa Cruz Biotechnology, anti–poly(ADP-ribose) polymerase (PARP; 556494), and anti-cytochrome c (556433) from BD Biosciences, monoclonal anti–β-actin antibody from Sigma, monoclonal anti-Nur77 antibody from R&D Systems, anti–Bcl-2 BH3 (AP1303a) from Abgent, anti–cleaved caspase-3 (Asp175) from Cell Signaling Technology, polyvinylidene difluoride membranes from Millipore, and a cocktail of proteinase inhibitors (80-6501-23) from Amersham were used in this study. All other chemicals used were commercial products of analytic grade obtained from Sigma.

Preparation of acetylshikonin and analogues. L. erythrorhizon grown in Liao-ning Province in Northeast China were extracted with hexane by Soxhlet apparatus and subjected to purification for shikonin derivative compounds (19, 20). Acetylshikonin (SK03) and two isomers of SK03 analogues, SK06 [5,8-diacetoxyl-2-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones] and SK07 [5,8-diacetoxyl-6-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones], were synthesized (Fig. 1A ). Molecular structures were identified using spectroscopic techniques including electron ionization mass spectrometry, Fourier transform IR spectroscopy, and nuclear magnetic resonance analysis.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Induction of Nur77 protein levels by acetylshikonin and analogues. A, structures of acetylshikonin and analogues. B, induction of Nur77 expression by shikonins in NIH-H460 lung cancer cells. Western blotting analysis was used to evaluate the effect of shikonins on Nur77 protein levels (top). NIH-H460 cells were treated with SK03, SK06, SK07 (5 or 10 µmol/L), or vehicle for 12 or 24 h. Nur77 protein expression was detected by using anti-Nur77 antibody. The blots were reprobed with anti–β-actin antibody for loading control. As for RT-PCR analysis (bottom) of Nur77 mRNA expression, the same cells were treated with SK03, SK06, or SK07 (5 µmol/L) for 3 h, whereas TPA (100 ng/mL) was used as a positive control. Total RNA were purified and subjected to RT-PCR analysis. Nur77 and β-actin products were simultaneously amplified by including primers for both Nur77 and β-actin in the same reaction system, in which β-actin expression level served as an internal control. C, induction of Nur77 expression by shikonins in HeLa cervical cancer cells. HeLa cells were treated with shikonins as indicated. Anti-Nur77 antibody was used to detect the expression of Nur77 protein by Western blotting analysis. D, dose-dependent and time-dependent induction of Nur77. Cells were treated with vehicle or with SK07 at 1, 5, or 10 µmol/L in serum-free medium for the indicated period of time. Nur77 expression was determined by Western blotting analysis using anti-Nur77 antibody.

Cell lysis and fraction. Cell lysates were prepared by lysing cells with modified radioimmunoprecipitation assay buffer containing 50 mmol/L of Tris-HCl (pH 7.4), 150 mmol/L of NaCl, 5 mmol/L of EDTA, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS with a cocktail of proteinase inhibitors on ice for 30 min. For cellular fractionation, cells were lysed in cold buffer A [10 mmol/L HEPES-KOH (pH 7.9) at 4°C, 1.5 mm MgCl₂, 10 mmol/L KCl, 0.5 mmol/L DTT] with a cocktail of proteinase inhibitors on ice for 10 min. The cytoplasmic fraction was collected by centrifuging at 6,000 rpm for 30 s. Pellets containing nuclei were resuspended in cold high-salt buffer C [20 mmol/L HEPES-KOH (pH 7.9), 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5 mmol/L DTT], with a cocktail of proteinase inhibitors on ice for 30 min. Cellular debris was removed by centrifugation at 12,000 rpm at 4°C for 15 min. Protein concentrations were determined using the Bradford method according to the instructions of the manufacturer (Bio-Rad).

Western blotting. Equal amounts of the lysates were electrophoresed on an 8% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes, which were then blocked with 5% nonfat milk in TBST [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.1% Tween 20] for 1 h, incubated with various primary antibodies for 2 h and detected with either anti-rabbit (1:5,000) or anti-mouse (1:5,000) secondary antibodies for 1 h using an enhanced chemiluminescence system. The dilutions of the primary antibodies were anti-Nur77 (sc-5569) in 1:1,000, anti-PARP in 1:1,000, anti–Bcl-2 in 1:1,000, and anti-Bax in 1:1,000. The blots were reprobed with anti–β-actin antibody for loading control.

Immunofluorescence microscopy. Cells mounted on glass slides were permeabilized with PBS containing 0.1% Triton X-100 and 0.1 mol/L of glycine for 15 min, and blocked with 1% bovine serum albumin in PBS for 30 min at room temperature, followed with incubation with various primary antibodies at 37°C for 1 h and detected by FITC-labeled anti-rabbit IgG (1:400) or anti-goat IgG conjugated with Cy3 (1:400) at room temperature for 30 min. Cells were costained with 4'6'-diamidino-2-phenylindole (DAPI) to visualize nuclei. The images were taken under a fluorescent microscope (Carl Zeiss) or an LSM-510 confocal laser scanning microscope system (Carl Zeiss). The primary antibodies included monoclonal anti-Nur77 antibody (R&D Systems, 1:200), anti–cytochrome c (1:500), anti–cleaved caspase-3 (1:500), anti-Hsp60 (1:500), anti–Bcl-2 (1:500), anti–Bcl-2 BH3 (1:500), and anti-Bax (6A7; 1:500).

Reverse transcription-PCR analysis. Total RNAs were isolated by Trizol LS. The first-strand synthesis was performed with RevertAid First-Strand cDNA synthesis kits (Fermentas) according to the instructions of the manufacturer. The primers includes those for Nur77 (forward primer, 5'-TCA TGG ACG GCT ACA CAG-3'; reverse primer, 5'-GTA GGC ATG GAA TAG CTC-3') and β-actin (forward primer, 5'-CTG GAG AAG AGC TAC GAG-3'; reverse primer, 5'-TGA TGG AGT TGA AGG TAG-3'). PCR reactions were performed in Eppendorf AG 22331 Hamburg (Eppendorf). Cycling conditions were 30 s denaturation at 94°C, 30 s annealing at 55°C and 30 s elongation at 72°C for 35 cycles. PCR products were electrophoresed on 2% agarose gels and gel images were captured with a Gel logic 200 system (Kodak).

Apoptosis assays. For DAPI staining, cells were incubated with vehicle or with different concentrations of acetylshikonin and analogues in serum-free medium for 24 h. Detached and attached cells were collected and incubated in PBS containing 50 µg/mL of DAPI and 100 µg/mL of DNase-free RNase A at 37°C for 20 min. Apoptotic cells were identified with typical morphology of shrinkage of the cytoplasm, membrane blebbing, and nuclear condensation and/or fragmentation. At least 300 cells from more than five random microscopic fields were counted by two independent investigators. For flow cytometry analysis, cells treated with vehicle, SK07, or SK03 in serum-free medium for 24 h were collected and stained with Annexin V/propidium iodide (PI) using Vybrant apoptosis assay kit no. 2 according to the instructions of the manufacturer, and analyzed by flow cytometry (Beckman Coulter EPICS ALTRA).

Nur77 mutants and transfection. Two different fragments of Nur77, N-Nur77 (A/B domain) and C-Nur77 (LBD domain), were generated by PCR and inserted into the EcoRI/SalI site of pEGFP vector. The Nur77 mutants were verified by sequencing. HEK293T cells transfected with Nur77, N-Nur77, or C-Nur77 were treated with 5 µmol/L of SK07 or vehicle in serum-free medium for 8 h, and stained with DAPI. Apoptosis of transfected cells were examined as described above.

Statistical analysis. Data were expressed as mean ± SD. Each assay was repeated in triplicate in three independent experiments. Statistical significance of differences between groups was analyzed by using Student's t test or ANOVA analysis. P < 0.05 was considered significant.

	Results

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Acetylshikonin and analogues increase levels of Nur77 protein through posttranscriptional regulation. To identify new modulators of the Nur77-mediated apoptotic pathway (17), we screened a natural product library consisting of 3,000 extracts and pure compounds prepared from known Chinese herbs with therapeutic indications (21). Our results show that several shikonin derivatives could increase the levels of Nur77 protein and apoptosis in various cancer cell lines, including NIH-H460 lung cancer, HeLa cervical cancer, HepG2 liver cancer, MCF7 breast cancer, and MGC-803 gastric cancer cells (data not shown). We report here our characterization of the acetyl derivative of shikonin, SK03 (Fig. 1A). Treatment of NIH-H460 and HeLa cells with SK03 resulted in the strong induction of Nur77 protein levels (Fig. 1B and C). In NIH-H460 cells that express high basal Nur77 protein levels, we consistently observed that SK03 could increase Nur77 levels by 3-fold to 5-fold in a concentration range between 5 and 10 µmol/L for 12 or 24 hours (Fig. 1B), whereas the effect of SK03 was even more significant in HeLa cells that express low basal Nur77 levels (Fig. 1C). Thus, acetylshikonin SK03 can enhance the levels of Nur77 protein in cancer cells.

In order to identify more effective and selective modulators of the Nur77-mediated apoptotic pathway, two acetylshikonin derivatives, SK06 and SK07, were synthesized (Fig. 1A). SK06 and SK07 were triacetate shikonins that were designed to explore whether modification of pharmacophore quinine, which is the common moiety of various shikonin derivatives, could improve their regulation of Nur77 activities. Analysis of their effect on levels of Nur77 protein in NIH-H460 (Fig. 1B) and HeLa (Fig. 1C) cells showed that the ability of SK03 in increasing Nur77 protein levels was retained in SK07 but impaired in SK06. Thus, the pharmacophore quinine in shikonins is an important determinant for their modulation of Nur77 protein levels in cancer cells.

The induction of Nur77 protein levels by SK07 was dose-dependent and time-dependent (Fig. 1D). Treatment of NIH-H460 cells with SK07 at 1, 5, and 10 µmol/L for 6 hours did not show any effect on Nur77 expression. However, the levels of Nur77 were increased when cells were treated for 12 hours. Although 10 µmol/L was required for optimal induction of Nur77 expression when cells were treated for 12 hours, 1 µmol/L of SK07 was sufficient for the maximal increase of Nur77 proteins when cells were treated with the compound for 24 hours. Such a time-dependent effect of SK07 on Nur77 expression was interesting because induction of Nur77 by growth factors and apoptotic stimuli, such as 3-Cl-AHPC, is often rapid and transient (22). We therefore determined whether shikonins could regulate Nur77 expression at the transcriptional level. NIH-H460 cells were treated with vehicle or with SK03, SK06, or SK07 at 5 µmol/L for 3 hours and their effect on Nur77 transcript was examined by reverse transcription-PCR (RT-PCR; ref. Fig. 1B). As a positive control, cells were treated with phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA; 100 ng/mL), which is known to induce Nur77 expression in NIH-H460 cells (4). Consistent with previous reports, TPA could significantly up-regulate Nur77 mRNA expression. However, we did not detect clear changes in Nur77 mRNA level when cells were treated with SK03, SK06, or SK07 (Fig. 1B), suggesting that the induction of Nur77 protein by SK03 and SK07 is not due to their transcriptional regulation of Nur77 expression.

Role of Nur77 in shikonin-induced apoptosis. We next determined whether Nur77 expression played a role in shikonin-induced apoptosis. NIH-H460 cells were treated with 5 µmol/L of SK03, SK06, SK07, or vehicle for 24 hours and apoptosis was analyzed by DAPI staining (Fig. 2A ). Although apoptosis was seldom seen in untreated cells (7%), a significant number of cells underwent apoptosis when treated with SK03 (22%) or SK07 (33%). In contrast, SK06 exerted much less effect on apoptosis (14%). Thus, the apoptotic effect of shikonins correlated with their induction of Nur77 protein in cancer cells, suggesting that Nur77 expression contributes to their death effect.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Induction of apoptosis by SK07 and the role of Nur77. A, DAPI staining. NIH-H460 cells cultured in serum-free medium were treated with SK03, SK06, SK07 (5 µmol/L), or vehicle for 24 h and subjected to DAPI staining. Apoptotic cells were compared between different treatments. *, P < 0.05 (vs. control); **, P < 0.01 (vs. control); ##, P < 0.01 (vs. SK03 or SK06). B, PARP cleavage and caspase-3 activation. NIH-H460 or HeLa cells were treated with 5 µmol/L of SK07 in serum-free medium for the indicated times. Total cell lysates were subjected to Western blotting assay for PARP cleavage using anti-PARP antibody (top). For the analysis of caspase-3 activation (bottom), NIH-H460 cells were treated with shikonins as described above and stained with an antibody recognizing the cleaved caspase-3. Nuclei were visualized by costaining with DAPI. C, transfection of Nur77 mediates the apoptotic effect of SK07. HEK293T cells were transfected with full-length Nur77, N-Nur77 (A/B domain), or C-Nur77 (E/F domain) and subjected to SK07 treatment (5 µmol/L) or vehicle in serum-free medium for 8 h. Apoptotic cells examined by DAPI staining were compared between transfected cells carrying different mutants. **, P < 0.01 (vs. control). D, the apoptotic effect of SK07 is impaired in Nur77 knockout MEFs. MEF cells or MEF Nur77–/– cells were treated with vehicle or the indicated concentrations of SK07 or SK03 for 24 h and stained with PI/Annexin V. Apoptosis was analyzed by fluorescence-activated cell sorting analysis.

The role of Nur77 in mediating the apoptotic effect of SK07 was also examined by studying the effect of Nur77 transfection in HEK293T cells that are resistant to SK07 (data not shown). Treatment with 5 µmol/L of SK07 did not induce a clear apoptosis of HEK293T cells. However, SK07 induced an extensive apoptosis in HEK293T cells transfected with GFP-Nur77 (32%; Fig. 2C). SK07 also induced the apoptosis of HEK293T cells transfected with the COOH-terminal region of Nur77 (C-Nur77) but not the NH₂-terminal Nur77 region (N-Nur77). The fact that the COOH-terminal E/F domain of Nur77 is sufficient for mediating the apoptotic effect of SK07 suggests that DNA binding and transactivation of Nur77 are not required for mediating the apoptotic effect of SK07.

To further determine the role of Nur77 expression, we evaluated the death effect of SK07 in MEF and Nur77 knockout MEF (MEF Nur77^–/–) cells by flow cytometry for Annexin V and PI staining. Annexin V staining serves as a measure of phosphatidylserine externalization and cells that are Annexin V⁺/PI^– represent early apoptotic cells (24). A representative experiment (Fig. 2D) showed that early apoptotic cells (Annexin V⁺/PI^–; 14.6% and 57%) were seen in MEF treated with 1 and 10 µmol/L of SK07, respectively. In contrast, Annexin V⁺/PI^– staining was significantly reduced in MEF Nur77^–/– cells (3.38% with 1 µmol/L and 23.7% with 10 µmol/L SK07 treatment). These results show that the apoptotic effect of SK07 largely depends on Nur77 expression, although other protein factors may mediate its effect when a high concentration of SK07 is used. Interestingly, SK03 treatment (10 µmol/L) resulted in a significant amount of late apoptotic cells (Annexin V⁺/PI⁺) in more than half of both MEF cells (54.6%) and MEF Nur77^–/– cells (66.8%). In addition, SK03 caused significant necrotic cell death in both MEF cells (31%) and MEF Nur77^–/– cells (11.9%), suggesting that SK03 may induce cell death mainly through Nur77-independent pathways.

SK07 induces Nur77 nuclear export and mitochondrial targeting. To determine whether acetylshikonin and its analogues could induce cytoplasmic localization of Nur77, the hallmark of the Nur77-mediated apoptotic pathway (17), we examined their effect on the subcellular localization of Nur77 in NIH-H460 cells. As shown in Fig. 3A , Nur77 immunostaining was significantly enhanced in cells treated with SK03 and SK07, but not with SK06, consistent with their induction of Nur77 protein level. For comparison, TPA also strongly induced Nur77 immunostaining. However, TPA-induced Nur77 immunostaining was confined to the nucleus, as reported previously (4). In contrast, most of the cells treated with SK03 and SK07 displayed a diffused distribution of Nur77. Predominant cytoplasmic localization of Nur77 could be seen in some treated cells, which was more apparent when treated with SK07 than with SK03. These results suggested that SK03 and SK07 induced the cytoplasmic localization of Nur77. Cytoplasmic localization of Nur77 observed in cells treated with SK07 might be due to its induction of Nur77 nuclear export, as leptomycin B (LMB), which is known to block CRM1-dependent nuclear export (25), significantly inhibited SK07-induced Nur77 cytoplasmic accumulation. To confirm the effect of shikonins on inducing cytoplasmic localization of Nur77, cytosolic and nuclear fractions of cells treated with SK03, SK06, or SK07 were prepared and analyzed for levels of Nur77 protein by immunoblotting. As shown in Fig. 3B, Nur77 was predominantly nuclear (92%) in control cells. However, in cells treated with SK03 and SK07, a significant amount (65% and 85%, respectively) of Nur77 protein was cytoplasmic. In contrast, only 30% of Nur77 protein resided in the cytoplasm when treated with SK06. Consistent with immunostaining results, pretreatment of cells with LMB reduced the amount of SK07-induced cytoplasmic Nur77 from 85% to 42%. Thus, SK07 is more effective than SK03 and SK06 in inducing cytoplasmic localization of Nur77.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Acetylshikonins induce cytoplasmic localization of Nur77. A, Nur77 translocation. NIH-H460 cells treated with SK03, SK06, or SK07 (5 µmol/L) for 24 h were subjected to immunostaining with anti-Nur77 antibody. Cells treated with 100 ng/mL of TPA (3 h) were used as positive control. Subcellular localization of Nur77 in cells treated with SK07 alone was compared with that in cells with cotreatment of SK07 and LMB (10 ng/mL). Cells were costained with DAPI to visualize the nuclei. B, cellular fractionation. Nuclear and cytoplasmic fractions were prepared from NIH-H460 cells with similar treatments as indicated in A, and subjected to Western blotting analysis. The purity of cellular fraction was confirmed by using anti-PARP antibody. Ratio of cytoplasmic Nur77 (C) to nuclear Nur77 (N) expression was quantified by BandScan 5.0. C and D, Nur77 mitochondrial targeting. NIH-H460 cells transfected with or without GFP-Nur77 were treated with 5 µmol/L of SK07 for 24 h. Endogenous Nur77 expression was immunostained with anti-Nur77 antibody. Cells were costained with anti-Hsp60 antibody to recognize the mitochondria and with DAPI to visualize the nuclei. Fluorescent microscopy was used to determine the overlap of Nur77 and Hsp60.

SK07-induced apoptosis is dependent on cytoplasmic localization of Nur77. To study whether the cytoplasmic localization of Nur77 was required for the killing activity of SK07, NIH-H460 cells were treated with 10 µmol/L of SK07 and subsequently stained with anti-Nur77 antibody and DAPI. Microscopic analysis showed that treatment of cells with SK07 resulted in a significant amount of cells displaying cytoplasmic localization of Nur77, many of them showing nuclear morphology typical of apoptosis (45%; Fig. 4A ). However, when cells were cotreated with LMB, SK07-induced Nur77 protein mainly resided in the nucleus, and cells displayed normal nuclear morphology. The inhibitory effect of LMB on SK07-induced apoptosis was confirmed by Annexin V staining (Fig. 4B). Flow cytometry analysis showed that NIH-H460 cells treated with 5 µmol/L of SK07 resulted in extensive early apoptosis (37.9% Annexin V⁺/PI^– cells). This level of apoptosis was dramatically reduced (4.9% Annexin V⁺/PI^– cells) when cells were cotreated with LMB. We also determined the effect of LMB on SK07-induced cytoplasmic localization of GFP-Nur77 and apoptosis. NIH-H460 cells transfected with GFP-Nur77 were exposed to 10 µmol/L of SK07 for 24 hours, and apoptosis was examined by DAPI staining. In the absence of treatment, GFP-Nur77 resided in the nucleus and no apparent apoptotic cells were detected (Fig. 4C). However, GFP-Nur77 could be found in the cytoplasm of cells treated with SK07, which was coincident with extensive nuclear condensation and fragmentation with 80% apoptosis. In the presence of LMB, SK07-induced apoptosis was decreased to 16%. Again, inhibition of apoptosis by LMB was accompanied by its inhibition of the cytoplasmic localization of GFP-Nur77. Together, these results show that the cytoplasmic localization of Nur77 plays a critical role in mediating the apoptotic effect of SK07.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 4. SK07-induced apoptosis is dependent on Nur77 expression and Nur77 nuclear export. A, Nur77 cytoplasmic localization and apoptosis. NIH-H460 cells were treated with 10 µmol/L of SK07 in serum-free medium in the presence or absence of LMB (10 ng/mL) for 24 h. Subcellular localization of endogenous Nur77 was examined by immunostaining using anti-Nur77 antibody (R&D) and apoptosis was determined by DAPI staining. B, analysis of the effect of LMB on SK07-induced apoptosis by PI/Annexin V staining. NIH-H460 cancer cells were treated with SK07 (5 µmol/L) in the presence or absence of LMB (10 ng/mL) for 24 h. Apoptosis was determined by PI/Annexin V staining. C, cytoplasmic localization of transfected Nur77 and apoptosis. NIH-H460 cells transfected with GFP-Nur77 expression vector were treated with 10 µmol/L of SK07 in serum-free medium in the presence or absence of LMB (10 ng/mL) for 24 h. Subcellular localization of GFP-Nur77 was examined by fluorescence microscopy and apoptosis was examined by DAPI staining. Quantification was conducted in nontransfected and transfected cells, respectively. **, P < 0.01 [vs. control or LMB (+)].

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 5. SK07 induces Bcl-2 conformational change and Bax activation. A, colocalization of Nur77 with Bcl-2. NIH-H460 cells treated with vehicle or 5 µmol/L of SK07 in the presence or absence of LMB (10 ng/mL) for 24 h were subjected to costaining with anti-Nur77 and anti–Bcl-2 antibodies. Fluorescent microscopy was used to analyze the subcellular localization of Nur77 and Bcl-2. B, Bcl-2 conformational change and Bax activation. NIH-H460 cells treated with 5 µmol/L of SK07 were examined for Bcl-2 conformational change by anti–Bcl-2(BH3) antibody. Its association with Bax activation was analyzed by costaining with anti-Bax(6A7) antibody. Cells were also stained with DAPI to visualize the nucleus. C, translocation of Bax to mitochondria. NIH-H460 cells treated with 5 µmol/L of SK07 were examined for Bax activation by antibody against Bax(6A7). Cells were costained with anti-Hsp60 antibody to reveal Bax colocalization with mitochondria. DAPI staining was used to visualize the nuclei. The colocalization of active Bax with mitochondria and their association with apoptotic nuclear morphology were compared. D, Bax activation and cytochrome c release. NIH-H460 cells treated with 5 µmol/L of SK07 were stained with anti-Bax(6A7) and anti–cytochrome c (Cyt c) antibodies. Bax activation was compared with its induction of cytochrome c release as indicated by diffusive cytochrome c staining. LMB was used to determine its effect on both Bax activation and cytochrome c release.

SK07 induces Bax activation. An unresolved issue in the Nur77/Bcl-2 apoptotic pathway is whether Bcl-2 conformational change is sufficient to induce apoptosis or whether the exposed BH3 domain of Bcl-2 acts as a BH3-only protein that induces apoptosis through activation of proapoptotic Bcl-2 family members and/or inhibition of antiapoptotic members (17, 26). Thus, we determined whether Bax activation was involved in SK07-induced apoptosis by immunostaining NIH-H460 cells with anti-Bax(6A7) antibody that recognizes activated Bax. Untreated NIH-H460 cells were not immunostained by the anti-Bax(6A7) antibody, suggesting that Bax was inactive in the cells (Fig. 5B). However, cells treated with SK07 displayed strong immunostaining with the anti-Bax(6A7) antibody, demonstrating that SK07 could activate Bax. Our observation that SK07-treated cells stained by both anti–Bcl-2(BH3) and anti-Bax(6A7) antibodies suggests that SK07-induced Bcl-2 conformational change may play a role in Bax activation.

Bax resides predominantly in the cytosol before its activation and apoptosis induction. During early apoptosis, Bax undergoes conformational change and oligomerization, resulting in its translocation from the cytosol to the mitochondria (27). We then examined whether Bax could associate with mitochondria in SK07-treated cells. Microscopic analysis showed an extensive colocalization of active Bax with mitochondrial-specific protein Hsp60 (Fig. 5C). Furthermore, cells with active Bax immunostaining often displayed nuclear condensation (Fig. 5C), suggesting that SK07-induced apoptosis might involve Bax activation. To further confirm this association, NIH-H460 cells were costained with anti-Bax(6A7) and anti–cytochrome c antibodies. In control cells, cytochrome c displayed a punctate immunostaining pattern, consistent with its mitochondrial localization (Fig. 5D). However, cytochrome c was diffusely distributed in SK07-treated cells with active Bax immunostaining. Thus, SK07-induced Bax activation was associated with cytochrome c release. LMB cotreatment, which prevented Nur77 nuclear export and Bcl-2 conformational change, almost completely inhibited SK07-induced Bax activation and cytochrome c release (Fig. 5D).

SK07 does not affect Bcl-2 and Bax expression. Because it has been widely reported that the ratio between Bcl-2 and Bax determines cell survival or death (26), we next examined whether the apoptotic effect of SK07 was due to its effect on Bcl-2 and Bax expression. The result showed that SK07 caused no apparent change in level of either Bcl-2 or Bax for treatment time ranging from 3 to 24 hours (Fig. 6 ). Thus, SK07 induces apoptosis through the induction of Bcl-2 conformational change and Bax activation rather than affecting Bcl-2/Bax ratio.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 6. SK07 does not affect Bax and Bcl-2 expression levels. Cells were treated with vehicle or 5 µmol/L of SK07 for the indicated periods of time. Total cell lysates were prepared and examined for Bcl-2 and Bax expression by Western blotting analysis. β-Actin expression was used as a loading control.

	Discussion

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Nur77 is a unique orphan member of the nuclear receptor superfamily (1). It is perhaps the most potent proapoptotic member in the nuclear receptor superfamily (16, 17). Recently, we showed a new paradigm in cancer cell apoptosis through Nur77 mitochondrial targeting (17, 30), which was later confirmed by other groups (15, 16, 18). We showed that apoptosis of cancer cells induced by certain apoptotic agents requires Nur77 expression and its translocation from the nucleus to mitochondria where Nur77 interacts with Bcl-2, resulting in a Bcl-2 conformational change that converts it into a cytodestructive molecule (17). These findings suggest that agents which induce cytoplasmic localization of Nur77 and its interaction with Bcl-2 may preferentially kill cancer cells with elevated Bcl-2 levels.

In the course of identifying new agents that modulate the Nur77-mediated apoptotic pathway, we report here our finding that certain shikonin derivatives could activate the Nur77 pathway through their induction of Nur77 expression, nuclear export, mitochondrial targeting, Bcl-2 conformational change, and Bax activation. These results are consistent with previous report that shikonin-like compounds can induce apoptosis through mitochondria and caspase pathway with unknown mechanisms (31). Our current finding that certain shikonins could regulate the Nur77-mediated apoptotic pathway sets the stage for rational design of shikonin analogues with improved anticancer efficacy and specificity. It is generally believed that the nonspecific toxicity of shikonins is caused by the electronic transfer of hydroxyl groups between 1- and C-11 positions and subsequently occurring alkylation (20). Our current study indicates that certain esteration of 1- carbonyl group of acetylshikonins such as SK07 can enhance apoptosis by promoting Nur77 expression and migration. We found that the introduction of acetyl groups into the pharmacophore quinoid moiety of SK03 exerted a great effect on its modulation of Nur77 activity. Induction of Nur77 protein levels by SK03 in cancer cells was retained in SK07 (Fig. 1B and C). Interestingly, the ability of inducing Nur77 cytoplasmic localization by SK03 was significantly enhanced in SK07 but was reduced in SK06 (Fig. 3A and B). The distinct abilities to induce Nur77 expression and Nur77 cytoplasmic translocation shown in different shikonin derivatives (SK07 and SK06) suggest that quinoid modification is an important determinant for their modulation of Nur77 activities, including its stability and nuclear export.

Our results showed that an increase of Nur77 protein levels and its cytoplasmic localization were associated with the apoptotic effect of shikonins. Treatment of NIH-H460 cells with SK07, which induced Nur77 expression and nuclear export, resulted in cytochrome c release from the mitochondria (Fig. 5D), caspase-3 activation, and PARP cleavage (Fig. 2B). SK07-induced apoptosis was dependent on Nur77 expression level as its killing effect was largely impaired in MEF Nur77^–/– cells when compared with MEF cells (Fig. 2D) and enhanced by Nur77 transfection (Figs. 2C and 4C). The apoptotic effect of SK07 could be observed in other cancer cells such as HeLa cells (Figs. 1C and 2B; data not shown), consistent with the role of Nur77 expression in different types of cancer cells (8, 14, 15, 18). Our results also showed that SK07-induced apoptosis was associated with cytoplasmic localization of Nur77 because cytoplasmic accumulation of both endogenous Nur77 and transfected Nur77 was accompanied with nuclear condensation and fragmentation (Fig. 4A and C). In addition, LMB blockage of Nur77 cytoplasmic localization significantly inhibited SK07-induced Bax activation, cytochrome c release (Fig. 5D), and apoptosis (Fig. 4). Consistently, deletion of the DNA-binding domain from Nur77 did not affect the apoptotic effect of SK07 in HEK293T cells (Fig. 2C).

In studying the mechanism by which cytoplasmic Nur77 induced apoptosis, we found that SK07-induced cytoplasmic Nur77 targeted mitochondria (Fig. 3C and D) probably through its interaction with Bcl-2 (Fig. 5A), leading to Bcl-2 conformational changes that exposes its BH3 domain (Fig. 5B). These results confirmed our previous finding that cytoplasmic Nur77 could target mitochondria and induce Bcl-2 conversion (17). Interestingly, our results showed that SK07-induced Bcl-2 conformational change was associated with Bax activation (Fig. 5B). This observation is significant, as it would suggest that the converted Bcl-2 requires Bax activation for apoptosis induction. Although it is unclear how converted Bcl-2 activates Bax, it can be envisioned that the converted Bcl-2, with its BH3 domain exposed, may act directly or indirectly on Bax, resulting in Bax activation and apoptosis.

Besides its improved efficacy in inducing apoptosis, SK07 may have improved selectivity towards the Nur77-mediated apoptotic pathway. Shikonins are known to modulate multiple signal transduction pathways, including inhibition of DNA topoisomerases (31), induction of reactive oxygen species release (32), and inhibition of survival pathways involving extracellular signal-regulated kinase, Akt, and nuclear factor B activities (33). Interestingly, the apoptotic effect of SK07 was largely impaired in Nur77 knockout cells (Fig. 2D). By contrast, SK03, the parent compound of SK07, induced a significant amount of late apoptotic and necrotic death cells in both MEF and MEF Nur77^–/– cells (Fig. 2D), suggesting that its biological activities largely depended on Nur77-independent pathways. Improved Nur77 selectivity, as that observed with SK07, may be therapeutically desirable as Nur77 is often overexpressed in cancer cells compared with normal cells. Such a tumor selectivity can be further ensured by the fact that the Nur77-mediated apoptotic pathway targets Bcl-2, the expression of which is also often elevated in cancer cells (17). Thus, SK07 may represent a new drug lead that induces cancer cell death by targeting the Nur77/Bcl-2 apoptotic pathway.

	Disclosure of Potential Conflicts of Interest

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No potential conflicts of interest were disclosed.

	Acknowledgments

 
Grant support: The 863 Program of China (no. 2007AA09Z404), National Natural Science Foundation of China (no. 30471939), Shanghai Key Project (no. 04DZ19115), Program of Shanghai Subject Chief Scientist (no. OZ041915), Key Science and Technology Planning Project of Fujian Province (no. 2007I0023 and no. 2008Y0062), NIH (CA87000, GM60544, and CA109345), the Susan G. Komen Breast Cancer Foundation, the U.S. Army Medical Research and Material Command, the California Tobacco-Related Diseases Research Program, and the California Breast Cancer Research Program (J-Z. Zeng and X-k. Zhang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Pinchas Cohen for providing Nur77 knockout MEFs and Dr. Ying Su for technical assistance.

	Footnotes

 
Note: J. Liu, W. Zhou, and S-S. Li contributed equally to this work.

Received 5/29/08. Revised 8/12/08. Accepted 8/16/08.

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