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1
BI1071, a novel Nur77 modulator, induces apoptosis of cancer cells
by activating the Nur77-Bcl-2 apoptotic pathway
Xiaohui Chen1,#
, Xihua Cao2,#
, Xuhuang Tu1, Gulimiran Alitongbieke
1, Zebin Xia
2, Xiaotong Li
1,
Ziwen Chen1, Meimei Yin, Dan Xu
1, Shangjie Guo
1, Zongxi Li
1, Liqun Chen
1, Xindao Zhang
1,
Dingyu Xu1, Meichun Gao
1, Jie Liu
1, Zhiping Zeng
1, Hu Zhou
1, Ying Su
2,*, and Xiao-kun
Zhang1,2,*
1School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug
Target Research, Xiamen University, Xiamen 361102, China; 2Cancer center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA;
Running title: A new activator of the Nur77-Bcl-2 apoptotic pathway
Keywords: Nur77, Bcl-2, DIM, Mitochondria, Apoptosis
# These authors equally contributed to this work
*Corresponding Authors:
Dr. Xiao-kun Zhang
Dr. Ying Su
Cancer Center
Sanford Burnham Prebys Medical Discovery Institute
10901 N. Torrey Pines Road
La Jolla, CA 92037
USA
Phone: 858-646-3141
Fax: 858-646-3195
E-mail: [email protected] or [email protected]
This study was supported in part by grants from the Natural Science Foundation of China
(U1405229, 81672749, 31271453, 31471318) to X. Zhang, Regional Demonstration of Marine
Economy Innovative Development Project (16PYY007SF17) to X. Zhang, the Fujian Provincial
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2
Science & Technology Department (2017YZ0002-1) to X. Zhang and the National Institutes of
Health (R01 CA198982).
Disclose of Potential Conflicts of Interest: The authors declare no potential conflicts of interest.
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Abstract
Nur77 (also called TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily,
induces apoptosis by translocating to mitochondria where it interacts with Bcl-2 to convert Bcl-2
from an anti-apoptotic to a pro-apoptotic molecule. Nur77 posttranslational modification such as
phosphorylation has been shown to induce Nur77 translocation from the nucleus to mitochondria.
However, small molecules that can bind directly to Nur77 to trigger its mitochondrial
localization and Bcl-2 interaction remain to be explored. Here we report our identification and
characterization of DIM-C-pPhCF3+MeSO3
- (BI1071), an oxidized product derived from indole-
3-carbinol metabolite, as a modulator of the Nur77-Bcl-2 apoptotic pathway. BI1071 binds
Nur77 with high affinity, promotes Nur77 mitochondrial targeting and interaction with Bcl-2,
and effectively induces apoptosis of cancer cells in a Nur77- and Bcl-2-dependent manner.
Studies with animal model showed that BI1071 potently inhibited the growth of tumor cells in
animals through its induction of apoptosis. Our results identify BI1071 as a novel Nur77-binding
modulator of the Nur77-Bcl-2 apoptotic pathway, which may serve as a promising lead for
treating cancers with overexpression of Bcl-2.
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Introduction
Nur77 (NR4A1) (also known as NGFI-B and TR3) is perhaps the most potent apoptotic member
of the nuclear receptor superfamily (1-8). The death effect of Nur77 was initially recognized
during studying the apoptosis of immature thymocytes, T-cell hybridomas (9,10). Later we found
that Nur77 mediates the death effect of the retinoid-related molecule AHPN (also called CD437)
in cancer cells (11). Furthermore, we discovered a nongenomic action of Nur77, in which Nur77
migrates from the nucleus to the cytoplasm, where it targets mitochondria to trigger cytochrome
c release and apoptosis in cancer cells (12-14). Further studies demonstrated in various cancer
types that such a Nur77 mitochondrial apoptotic pathway is characterized by its interaction with
Bcl-2 and the conversion of Bcl-2 from an anti-apoptotic molecule to a pro-apoptotic molecule
(6,15). Given the pivotal role of Bcl-2 in regulating the apoptosis of cancer cells and in the
resistance of cancer cells to a variety of radio- and chemo-therapeutic agents, understanding how
the Nur77-Bcl-2 apoptotic pathway is regulated and discovering its small molecule modulators
may offer new strategies to develop effective cancer therapeutics. However, small molecules that
can activate the Nur77-Bcl-2 apoptotic pathway by binding to Nur77 to trigger Nur77
mitochondrial translocation and interaction with Bcl-2 have not been reported.
As an orphan nuclear receptor, Nur77 lacks a canonical ligand-binding pocket (LBP) (16,17),
which excludes small molecules from binding to Nur77 to regulate Nur77 functions via the
canonical LBP binding mechanism. Recent advance has revealed the existence of alternate small
molecule binding regions on the surface of nuclear receptors, and compounds that bind to
alternate sites other than LBP have been identified for some nuclear receptors (18,19), including
Nur77 (20-24). These developments inspire us to discover Nur77-binding compounds that can
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regulate the Nur77-Bcl-2 apoptotic pathway. Here we report that a salt form of a 3,3’-
diindolymethane (DIM) derivative (di(1H-indol-3-yl)(4-(trifluoromethyl)phenyl)methane)
(named BI1071 here) can bind to Nur77 to induce apoptosis of cancer cells through the Nur77-
Bcl-2 apoptotic pathway. BI1071 binds to Nur77 at submicromolar concentration and induces
apoptosis that is dependent on the expression of both Nur77 and Bcl-2. BI1071 also effectively
inhibits the growth of tumor cells in animals. Moreover, BI1071 binding to Nur77 induces not
only its mitochondrial targeting but also its interaction with Bcl-2. Our results therefore identify
BI1071 as the first Nur77-binding small molecule that promotes the Nur77-Bcl-2 apoptotic
pathway.
Material and Method
Cell culture
The following cell lines are used in our study. HCT116 colon cancer, MDA-MB-231, HS578T,
BT549, MCF-7, and T47D breast cancer, breast epithelial cell line MCF-10A, HeLa ovarian
cancer, mouse embryonic fibroblast (MEF) cells and HEK293T embryonic cells were cultured in
Dulbecco’s Eagle’s medium (DMEM), while ZR-75-1, HCC1937 breast cancer and SW480
colon cancer were cultured in RPIM 1640 medium containing 10% fetal bovine serum (FBS).
Human Colonic Epithelial cells (HCoEpiC) were cultured in Colonic Epithelial Cell Medium
(CoEpiCM, Cat. #2951). Cell lines HCT116 (ATCC, CCL-247), SW480 (ATCC, CCL-228), and
HEK293T (ATCC, CRL-11268) were obtained from American Type Culture Collection (ATCC).
Cell lines MCF-10A (SCSP-660), MDA-MB-231 (SCSP-5043), HeLa (TCHu187), HS578T
(TCHu127), BT549 (TCHu 93), MCF-7 (SCSP-531), ZR-75-1 (TCHu126), T47D (TCHu 87)
and HCC1937 (TCHu148) were obtained from Chinese Academy of Science Shanghai Cell Bank
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on Dec. 09, 2016. Cell line HCoEpiC was obtained from ScienceCell (Cat. #2950) on Oct, 05,
2018. MEF cells were isolated from embryonic day 13 wild-type (WT) and Nur77 knock out
(KO) mice. The cells were grown in the cell incubator with 5% CO2 at 37 °C. Sub-confluent
cells with exponential growth were employed throughout the experiments. Cells plated onto cell
culture dishes and kept in 10% FBS for 24 h were treated with compounds or tranfected with
plasmids. Cell transfection was carried out by using Lipofectamin 2000 according to the
manufacture's instruction. The cells were tested by using Mycoplasma Hoechst Stain Assay kit
(Beyotime, C0296) every six months. We added the Hoechest solution to stain the cells with 50%
density at room temperature for 30 minutes and then used the confocal microscope to oberserve
the cells. In the cells without mycoplasma infection, only the blue fluorescence of the nucleus
was obersed. Filamentous blue fluorescence can be obersved around the nucleus in mycoplasma
contaminated cell samples. The cells were prevented from mycoplasma infection by using
plasmocin (Invivogen, ant-mpt). No positive mycoplasma tests was observed during the time of
our experiments.
Plasmids
Plasmids pcmv-myc-Nur77, GFP-Nur77, GFP-Nur77/LBD, GST-Bcl-2, pcmv-myc-Bcl-2, Flag-
cmv-Bcl-2 were constructed as described (12-14,24). Plasmids pcmv-myc-Nur77/H372D, pcmv-
myc-Nur77/H372A, pcmv-myc-Nur77/Y453L, pcmv-myc-Nur77/C566K were constructed by
using PCR and QuickChang mutagenesis kit.
Antibodies and Reagents
Anti-Myc (9E10) (Cat. Sc-40), anti-Ki67 (Cat. ab15580) and anti-Hsp60 (Cat. ab46798)
antibodies were purchased from Abcam (UK); anti-β-actin (Cat. 4970S), anti-Cleaved caspase-3
(Cat. 9661S), and anti-Nur77 (Cat. 3960S) antibodies were purchased from Cell Signal
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Technology (Beverly, MA, USA); anti-Nur77 (M-210) (Cat. sc-5569), anti-PCNA (Santa Cruz
sc-7907), anti-a-tublin (Santa Cruz sc-8035), anti-Bcl-2 (Santa Cruz sc-783), anti-PARP (Santa
Cruz sc-7150), and anti-GST (sc-138) antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA); anti-Flag (Cat.F1804) antibody was purchased from
Sigma (St. Louis, MO, USA); Mito-tracker deep red (Cat. M22426), JC-1 Probe (Cat. T3168)
and mitoSOX Red Mitochondrial Superoxide Indicator (Cat. M36008) were purchased from
Thermo Fisher.
Generation of Nur77 and Bcl-2 Knock-out cells by CRISPR/Cas9 system
Knocking out Nur77 and Bcl-2 from HeLa cells employed the CRISPR/Cas9 system. gRNA
targeting sequence of Nur77 (5’-ACCTTCATGGACGGCTACAC-3’) and Bcl-2 (5’-
GAGAACAGGGTACGATAACC-3’) was cloned into gRNA cloning vector Px330 (Addgene,
71707) and confirmed by sequencing. The accession numbers of Nur77 and Bcl-2 are NM-
001202233 and NM-000633.2 respectively. To screen for cells lacking Nur77 or Bcl-2, HeLa
cells were transfected with control vector and gRNA expression vectors, followed by G418
selection (0.5mg/ml). Single colonies were subjected to Western blotting using anti-Nur77 and
anti-Bcl-2 antibody to select knockout cells.
Cell Viability determination and cell death assay.
Cell viability was analyzed by using colorimetric 3-(4,5-dimethylthiazol-dimethylthiazol-2-yl)-
2,5-diphenyletetrazolium Bromide (MTT) assay as described (12-14,24).
Mammalian one hybrid assay
HEK293T cells were co-transfected with pG5 Luciferase reporter together with the plasmid
encoding RXR-LBD fused with the Gal4 DNA-binding domain and other expression plasmids
as described (18,25). After transfection, cells were treated with DMSO or BI1071, and assayed
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by using the Dual-Luciferase Reporter Assay System (Promega). Transfection efficiency was
normalized to Renilla luciferase activity.
Cell fractionation
For cellular fractionation (12-14,24), cells were lysed in cold buffer A (10 mM HEPES-KOH
(pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) with a cocktail of proteinase
inhibitors on ice for 10 min as described. Cytoplasmic fraction was collected by centrifuging at
6000 rpm for 10 minutes. Pellets containing nuclei were resuspended in cold high-salt buffer C
(20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
0.5 mM dithiothreitol) with a cocktail of proteinase inhibitors on ice for 30 minutes.
GST-pull Down
GST or GST-Bcl-2 fusion protein (0.5 mg) was immobilized on glutathione-Sepharose beads and
incubated with purified His-Nur77-LBD (0.2 mg) in the presence of different concentration of
BI1071 as described (12-14,24). Bound Nur77-LBD was analyzed by Western blotting.
Western blotting and immunoprecipitation
Western blotting and co-immunoprecipitation (co-IP) were performed as described (12-14,24).
Generation of Nur77 and Bcl-2 Knock-out cells by CRISPR/Cas9 system
Knocking out Nur77 and Bcl-2 from HeLa cells employed the CRISPR/Cas9 system. gRNA
targeting sequence of Nur77 (5’-ACCTTCATGGACGGCTACAC-3’) and Bcl-2 (5’-
GAGAACAGGGTACGATAACC-3’) was cloned into gRNA cloning vector Px330 (Addgene,
71707) and confirmed by sequencing. To screen for cells lacking Nur77 or Bcl-2, HeLa cells
were transfected with control vector and gRNA expression vectors, followed by G418 selection
(0.5mg/ml). Single colonies were subjected to Western blotting using anti-Nur77 and anti-Bcl-2
antibody to select knockout cells.
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Apoptosis assay
Cells were plaked on six-well plates with a density of 1X106 per well. After 24 hours, the cells
treated with different concentration of BI1071 for 6 hours, and then the suspended and the
adherent cells were collected, stained with Annexin V-FITC for 10 minutes and with propidium
iodide for 5 minutes, and analyzed immediately by cytoFLEX Flow Cytometry System
(Beckman-Coulter, Miami, FL, USA) using FITC and PC5.5.
Determination of ∆Ψm and ROS
The determination of ∆Ψm and ROS was performed as previously described elsewhere (26). JC-
1 probe was used to measure mitochondrial depolarization in cells. Cells were first treated with
different concentration of BI1071 for 6 hours and then followed with the addition of JC-1
staining solution (5 μg/ml) for 20 minutes at 37°C. After washing with PBS twice, mitochondrial
membrane potentials were analyzed immediately by cytoFLEX Flow Cytometry System using
FITC and PE. Mitochondrial depolarization was measured by a change in the ratio of green/red
fluorescence intensity. ROS was monitored with the mitoSOX Red Mitochondrial Superoxide
Indicator and analyzed by cytoFLEX Flow Cytometry System using PE.
Immunostaining
Cells were fixed in 4% paraformaldehyde. For mitochondrial staining, cells were incubated with
anti-Hsp60 goat immunoglobulin G (IgG) (Santa Cruz Biotechnology, Santa Cruz, CA),
followed by anti-goat IgG conjugated with Cy3. The nuclei were visualized by DAPI staining.
Fluorescent images were collected and analyzed by using a fluorescence microscopy or MRC-
1024 MP laser-scanning confocal microscope (Bio-Rad, Hercules, CA).
Animal studies
The protocols for animal studies were approved by the Animal Care and Use Committee of
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Xiamen University, and all mice were handled in accordance with the “Guide for the Care and
Use of Laboratory Animals” and the “Principles for the Utilization and Care of Vertebrate
Animals”. For MMTV–PyMT mice breast cancer model, female MMTV-PyMT mice of 12
weeks old were randomly divided into two groups (n=7 each), treated with a daily oral dose of
BI1071 (5 mg/kg) for 18 days. Standard histopathological analysis of tumor tissue was
performed. BI1071 was dissolved in DMSO and diluted with normal saline containing 5.0%
(V/V) Tween-80 to a final concentration 0.5 mg/ml. Normal saline with DMSO and 5.0%
Tween-80 was employed as the vehicle control. For xenograft nude mouse study, male BALB/c
nude mice (6 weeks old) were subcutaneously injected with log growth-phase of SW620 cells (1
X 106 cells in 0.1 ml PBS). Mice were treated orally after 7 days of transplantation with BI1071
once a day. Body weight and tumor size were measured every 3 days. Tumors were measured
and weighted. Tissues isolated from the nude mice were fixed with 4% paraformaldehyde. TdT-
mediated dUTP nick end labeling assay was performed according to the manufacturer’s
instructions (In situ Cell Death Detection Kit; Roche).
Immunohistochemistry
4 µm thick sections were deparaffinized and rehydrated using xylene and a graded series of
ethanol (100, 95, 85, 75, 50%), followed by washing in PBS. Antigen retrieval was performed in
10 mM sodium citrate buffer (pH 6.0), which was microwaved at 100°C for 20 minutes. After
rinsed twice in PBS, sections were blocked at room temperature for 1 hour by using 10% normal
goat serum, followed by incubation with anti-ki67, anti-cleaved caspase 3 overnight at 4°C.
Colors were developed with a DAB horseradish peroxidase color development kit.
Docking experiments
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Schrodinger’s (www.schrodinger.com) Glide (27), a grid-based docking program was used for
the docking study of BI1071 to the protein. The crystal structure of Nur77-LBD in complex with
a cytosporone B analog (Protein Data Bank code 3V3Q) was used. Docking was performed with
the implemented standard routine in Glide. The Glide GScore was used as docking score to rank
the docking results. Poses were further visually investigated to check for their interactions with
the protein in the docking site. Schrödinger's Maestro was used as the primary graphical user
interfaces for the visualization of the crystal structure and docking results.
Surface plasmon resonance (SPR)
The binding kinetics between Nur77-LBD and compounds were performed on a BIAcore T200
instrument (GE Healthcare) at 25℃ (24). Nur77-LBD were diluted to 0.05 mg/mL in 50 mM
NaOAc (pH 5.0) and immobilized on a CM5 sensor chip (GE Healthcare) by amine coupling at
densities ~10000 RU according to the manufacturer’s instructions. Gradient concentrations of
compounds were injected into the flow cells in running buffer (PBS, 0.4% DMSO) at a flow rate
of 30 μL/min for 150 s of association phase followed by a 420 s dissociation phase and a 30 s
regeneration phase (10 mM Glycin-HCl, pH 2.5). The data were analyzed using BIAcore T200
Evaluation Software 2.0 and referenced for blank injections and reference Surface. The
dissociation constant (Kd) was fitted to the standard 1:1 interaction model and calculated using
the global fitting of the kinetic data from gradient concentrations.
Statistical analysis
Data were expressed as mean ± SD. Each assay was repeated in triplicate in three independent
experiments. The statistical significance of the differences among the means of several groups
was determined using Student’s t-test.
Results
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A salt form of DIM-C-pPhCF3 exhibits superior apoptotic effect
In our effort to identify small molecules that modulate the Nur77/Bcl-2 apoptotic pathway, we
evaluated an in-house compound library, which includes di(1H-indol-3-yl)(4-
(trifluoromethyl)phenyl)methane (DIM-C-pPhCF3, Fig. 1A) (28). We surprisingly observed that
the freshly prepared DIM-C-pPhCF3 solution was not as active as the aged solution in apoptosis
induction. In addition, the freshly made DIM-C-pPhCF3 solution is colorless, however it turns
reddish when it is aged or exposed to air at room temperature. Thus, we presumed that the
compound underwent oxidization and the oxidized DIM-C-pPhCF3 was more active than DIM-
C-pPhCF3. To test our hypothesis, DIM-C-pPhCF3 was oxidized in the presence of
methanesulfonic acid, and the products were subsequently purified to obtain the oxidized DIM-
C-pPhCF3: di((1H-indol-3-yl)(4-trifluoromethylphenyl)methylium methanesulfonate (DIM-C-
pPhCF3+MeSO3
-) (BI1071, Fig. 1A and Supplementary Methods for synthesis and purification).
BI1071 was then tested in comparison with DIM-C-pPhCF3 for growth inhibition and apoptosis
induction. Fig. 1B showed that BI1071 inhibited the growth of HCT116 colon cancer cells with
an IC50 of 0.06 M, which is about 25-fold more active than DIM-C-pPhCF3 (IC50=1.5 M).
Treatment of MDA-MB-231 cells with 0.5 M BI1071 for 6 hours effectively induced PARP
cleavage, an indication of apoptosis, while DIM-C-pPhCF3 had no effect under the same
condition (Supplementary Fig. S1A). Dose-dependent study demonstrated that BI1071 could
induce PARP cleavage at submicromolar concentrations in HCT116 cells (Fig. 1C) and other
cancer cell lines (Supplementary Fig. S1B).
Interestingly, BI1071 was effective in various breast cancer cell lines analyzed regardless of its
hormone dependency (Supplementary Fig. S2). Furthermore, BI1071 did not display apoptotic
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effect in the non-transformed mammary and normal colon cells (Supplementary Fig. S3A-B),
indicating that BI1071 selectively induces apoptosis in cancer cells. The apoptotic effect of
BI1071 was also confirmed by its induction of extensive nuclear condensation and fragmentation
revealed by DAPI staining in cells treated with 0.5 M BI1071 for 6 hours (Fig. 1D and
Supplementary Fig. S1C) and confirmed as well by PARP and caspase 3 cleavage assays (Fig.
1E). The effect of BI1071 on cell death was further assessed using flow cytometry-based
Annexin V/ Propidium iodide (PI) apoptosis assay. Dose-dependent study showed that about
31.63% of MDA-MB-231 cells were apoptotic when treated with 1 M of BI1071 for 6 hours,
while only 1.31% of cells were apoptotic in vehicle control cells (Fig. 1F).
Loss of mitochondrial membrane potential (Δψm) represents one of the hallmarks of apoptosis.
To assess whether the BI1071-induced apoptosis was related to the intrinsic mitochondrial
pathway, we used JC-1, the mitochondrial-specific dye, to monitor the changes of
mitochondrial membrane potential (29). MDA-MB-231 breast cancer cells treated with BI1071
were stained with JC-1. JC-1 dye accumulation in mitochondria is dependent of mitochondrial
membrane potential, accompanied by a shift of JC-1 fluorescence emission from green to red. In
comparison to healthy cells, apoptotic cells display an increase in the green/red fluorescence
intensity ratio. Analysis of both red and green fluorescence emissions by flow cytometry
revealed a dose-dependent BI1071 induction of mitochondrial membrane dysfunction. After
treatment with 1 M of BI1071 for 6 hours, the green to red ratio increased from 100% to 345%
(Fig. 1G). Mitochondrial dysfunction was also revealed by marked increase in intracellular
mitochondrial reactive oxygen species (mito-ROS) in MDA-MB-231 cells exposed to BI1071 in
a dose dependent manner (Fig. 1H). Collectively, these data suggested that BI1071 induced
mitochondrion-related apoptosis in cancer cells.
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BI1071 inhibits the growth of tumor cells in vivo
To assess the apoptotic effect of BI1071 in animals, SW620 colon cancer cells were inoculated
subcutaneously in the right and left hind-side flank of nude mice. Administration of tumor-
bearing nude mice with BI1071 inhibited the growth of SW620 xenograft tumor in a dose and
time-dependent manner (Figs. 2A-C). TUNEL assay revealed extensive apoptosis in BI1071-
treated tumor specimens as compared to control tumor (Fig. 2D). MMTV-PyMT-transgenic
mouse model of breast cancer was also used to evaluate the anti-cancer effect of BI1071.
Administration of the MMTV-PyMT mice with BI1071 (5 mg/kg) potently inhibited the growth
of PyMT mammary tumor (Fig. 2E and 2F). Western blotting of tumor tissues prepared from
treated and non-treated mice revealed that the expression levels of two proliferation markers,
PCNA and Ki67, were markedly reduced by BI1071 (Fig. 2G). Immunostaining also showed a
reduced expression of Ki67 and enhanced expression of cleaved caspase 3 in tumor tissue
specimens prepared from mice treated with BI1071 (Fig. 2H). There was not significant
difference in the body weight (without tumor weight) between the control mice and the BI1071-
treated mice in both animal models (Supplementary Fig. S4A-B). These data demonstrated that
BI1071 potently inhibited the growth of tumor cells in animals through its induction of apoptosis.
BI1071 induces Nur77 dependent apoptosis and Nur77 mitochondrial targeting
We next determined whether BI1071-induced apoptosis was Nur77-dependent by examining its
apoptotic effect in mouse embryonic fibroblast (MEF) and MEF lacking Nur77 (Nur77-/-
MEF).
BI1071 dose-dependently inhibited the growth of MEFs, but such an inhibitory effect was
significantly diminished in Nur77-/-
MEFs (Fig. 3A). Induction of PARP cleavage in MEFs by
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BI1071 was also attenuated in Nur77-/-
MEFs (Fig. 3B). The death effect of BI1071 was also
evaluated in Nur77 genome knockout HeLa cells generated by CRISPR/Cas9 technology.
Induction of PARP cleavage and caspase 3 activation by BI1071 were strongly suppressed in
Nur77-/-
HeLa cells (Fig. 3C). Annexin V/PI staining revealed a reduced apoptotic effect of
BI1071 in Nur77-/-
HeLa cells than in the parental HeLa cells (from 35.55% to 3.25%) (Fig. 3D).
Furthermore, unlike the parental HeLa cells, Nur77-/-
HeLa cells did not display BI1071-induced
mitochondrial membrane potential loss measured by JC-1 staining (Fig. 3E) or BI1071-induced
release of mitochondrial ROS (Fig. 3F). To further address the role of Nur77, we transfected the
ligand-binding domain (LBD) of Nur77, Nur77-LBD, into HEK293T cells and asked whether
the overexpression of Nur77-LBD could influence the effect of BI1071. Indeed, transfection of
Nur77-LBD enhanced the killing effect of BI1071, with 36% of the transfected HEK293T cells
undergoing apoptosis, while 4.5% of the non-transfected cells were apoptotic (Supplementary
Fig. S5). Together, these results demonstrated that BI1071 targets Nur77 to induce cancer cell
apoptosis.
Our observation that BI1071 induced mitochondria-dependent apoptosis prompted us to
determine whether BI1071 exerted its Nur77-dependent apoptosis by promoting Nur77
mitochondrial targeting. Immunostaining showed that Nur77 was mainly localized in the nucleus
of HCT116 cells. However, it was predominantly cytoplasmic when cells were treated with 0.5
M of BI1071 for 2 hours (Fig. 3G). To confirm the effect of BI1071 on Nur77 cytoplasmic
localization, HEK293T cells were transfected with GFP-Nur77 and subsequently treated with 0.5
M BI1071. Transfected GFP-Nur77 resided in the nucleus, however it was diffusely distributed
in both the cytoplasm and nucleus upon BI1071 treatment (Fig. 3H). Cells transfected with GFP-
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Nur77-LBD also responded well to BI1071. Only for cells treated with BI1071, GFP-Nur77-
LBD colocalized extensively with the mitochondria-specific Hsp60 protein revealed by confocal
microscopy (Fig. 3I) and co-accumulated with the Hsp60 protein in the heavy membrane fraction
shown by cellular fractionation (Fig. 3J). The effect of BI1071 on inducing Nur77 mitochondrial
targeting was further illustrated by cellular fractionation experiments showing that a significant
amount of transfected Myc-Nur77 accumulated in the mitochondria-enriched heavy membrane
(HM) fraction when cells were treated with BI1071 (Fig. 3K). Taken together, these data
demonstrated that BI1071 exerted its Nur77-dependent apoptotic effect by promoting Nur77
mitochondrial targeting.
BI1071 binds Nur77 to induce its mitochondrial targeting and apoptosis
Although Nur77 lacks a LBP and no endogenous ligands have yet been identified (16,17), recent
crystallographic studies have identified several regions on the surface of the Nur77 protein as
small molecule binding regions (20,22,30). We therefore determined whether BI1071 binds
directly to Nur77 to induce its mitochondrial targeting and apoptosis. Surface plasmon resonance
(SPR) analyses revealed that DIM-C-pPhCF3 bound to Nur77-LBD with a Kd of 3.0 M (Fig.
4A) and that BI1071 bound to Nur77-LBD protein with a Kd of 0.17 M (Fig. 4B), which
demonstrated that BI1071 binding to Nur77-LBD was 18-fold stronger than DIM-C-pPhCF3. We
also evaluated the effect of BI1071 on the transcriptional activity of Nur77/RXR-LBD
heterodimer. Co-transfection of pBind-RXR-LBD and Nur77 strongly activate the reporter
transcriptional activity when cells were treated with 9-cis-RA, a RXR ligand (Fig. 4C). BI1071
further dose-dependently induced the reporter activity (Fig. 4C), likely due to its binding to
Nur77. To exclude the possibility that BI1071 acted on RXR, Glu453 and Glu456 in the
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activation function 2 (AF2) region of RXR (31) were substituted with Ala and the resulting
mutant, RXR-LBD/E453,6A, was used to repeat the reporter assay. As expected, 9-cis-RA
failed to induce the reporter activity in cells transfected with Nur77 and pBind-RXR-LBD-
E453,6A. However, BI1071 could still activate the reporter gene transcription (Fig. 4C),
demonstrating that BI1071 induced reporter gene transcription through Nur77 binding but not
RXR. We also excluded the possibility of BI1071 binding to other nuclear receptors using
reporter assays (Supplementary Fig. S6A). We next employed molecular docking approach to
study how BI1071 bound to Nur77-LBD. Our docking studies showed that BI1071 docked better
to a binding region formed by helices H1, H5, H7 and H8, and loops H1-H2, H5-B1 and H7-H8.
The docked mode also suggested that the indole ring of BI1071 made key interaction with the
side chains of H372 and Y453 located in H1 and H5, respectively (Fig. 4D). For comparison, we
also docked DIM-C-pPhCF3 to the same region. As shown in Fig. 4D, BI1071 fit better to the
binding groove with the bis-indolyl rings embedded deeper in the groove. The bis-indolyl rings
of BI1071 also was positioned to form π-π interaction with Y453 and to make stronger
interaction with H372. To test this binding mode, H372 was mutated into either Ala or Asp.
When tested in the Gal4 reporter assay for its response to BI1071, Nur77/H372D (Fig. 4E) or
Nur77/H372A (Supplementary Fig. S6B) could not induce the reporter gene transcription in
response to BI1071 treatment, revealing a critical role of H372 in BI1071 binding. Nur77/H372A
also failed to respond to BI1071 to accumulate in the heavy membrane fraction in the cellular
fractionation assay (Fig. 4F). To further characterize the binding of BI1071 and its apoptotic
effect, we made 2 more mutants: mutant Nur77/Y453L, another key residue suggested by the
docking studies, and mutant Nur77/C566K, a residue located in another reported ligand-binding
region (20-24). BI1071 failed to induce PARP cleavage in cells transfected with Nur77/H372D
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or Nur77/Y453L, while it strongly induced PARP cleavage in cells transfected with Nur77 or
Nur77/C566K (Fig. 4G). Similarly, in cells transfected with Nur77/H372D or Nur77/Y453L, the
effect of BI1071 on inducing Mito-ROS generation (Fig. 4H), on apoptosis analyzed using dual
staining with fluorescent Annexin V and PI (Fig. 4I), or on loss of mitochondrial membrane
potential (Fig. 4J) was much attenuated when compared to cells transfected with Nur77 or
Nur77/C566K. Taken together, these data demonstrated that BI1071 exerted its Nur77-dependent
apoptotic effect by a direct Nur77 binding mechanism.
BI1071-induced Nur77 mitochondrial targeting and apoptosis is Bcl-2 dependent
We previously showed that the Nur77 mitochondria-dependent apoptotic pathway involved
Nur77 interaction with Bcl-2 (14). We therefore asked if Bcl-2 plays a role in BI1071-induced
apoptosis. Thus, the apoptotic effect of BI1071 was evaluated in MEFs and MEFs lacking Bcl-2
(Bcl-2-/-
MEF). BI1071 at 0.5 M effectively induced PARP cleavage in MEFs, while it had no
apparent effect on PARP cleavage in Bcl-2-/-
MEFs (Fig. 5A). This was confirmed by DAPI
staining showing that Bcl-2-/-
MEF cells were much more resistant than MEFs to the apoptotic
effect of BI1071 (Fig. 5B). In addition, the impaired effect of BI1071 in Bcl-2-/-
MEFs could be
rescued by re-expression of Bcl-2 (Supplementary Fig. S7). In response to 0.5 M of BI1071,
40% of MEFs displayed chromatin condensation and nuclear fragmentation, whereas only 14%
of Bcl-2-/-
MEF cells exhibited similar apoptotic features. We also used the CRISPR/Cas9
technology to generate Bcl-2 knockout HeLa cells and showed that the effect of BI1071 on
inducing PARP cleavage was almost completely suppressed in Bcl-2-/-
HeLa cells (Fig. 5C). The
role of Bcl-2 in mediating the death effect of BI1071 was also illustrated by Annexin V/PI
staining showing a reduced apoptotic effect of BI1071 in Bcl-2-/-
HeLa cells compared to its
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effect in the parental HeLa cells (from 24.59% to 7.95%), and in Bcl-2-/-
MEF cells compared to
the parental MEF cells (from 76.65% to 10.27%) (Fig. 5D). Induction of the mitochondrial
membrane potential loss by BI1071 was also suppressed in Bcl-2-/-
MEFs and Bcl-2-/-
HeLa cells
(Fig. 5E). Furthermore, BI1071-induced release of mitochondrial ROS was compromised by loss
of Bcl-2 (Fig. 5F). These results revealed a crucial role of Bcl-2 in mediating the Nur77-
dependent apoptotic effect of B1071, demonstrating that the compound acts through the Nur77-
Bcl-2 apoptotic pathway.
BI1071 promotes Nur77 interaction with Bcl-2
In this study we have showed that BI1071 can bind to Nur77 to induce its migration from the
nucleus to mitochondria, where it interacts with Bcl-2 and triggers Bcl-2-dependent apoptosis.
However, if the binding of BI1071 to Nur77 promotes the interaction between Nur77 and Bcl-2
is not clear. Therefore, we investigated whether BI1071 binding to Nur77 enhanced the Nur77
interaction with Bcl-2. In vitro GST-pull down assays showed that Nur77-LBD was pulled down
by GST-Bcl-2 in a BI1071 concentration-dependent manner (Fig. 6A). Cell-based Co-IP showed
that Nur77 (Fig. 6B) or Nur77-LBD (Fig. 6C) transfected in HEK293T cells interacted with Bcl-
2 when cells were treated with BI1071. Endogenous Nur77 could be specifically
immunoprecipitated together with endogenous Bcl-2 by anti-Bcl-2 antibody only when cells
were treated with BI1071 (Fig. 6D). Moreover, confocal microscopy analysis revealed that
BI1071 promoted extensive mitochondrial colocalization of transfected GFP-Nur77 (Fig. 6E and
Supplementary Fig. S8A) or GFP-Nur77-LBD (Fig. 6F and Supplementary Fig. S8B) with Bcl-2
in cells. To further address the role of BI1071 on inducing Nur77 interaction with Bcl-2, the
aforementioned Nur77 mutants were analyzed. Fig. 6G showed that Nur77/C566K like the wild-
type Nur77 interacted strongly with Bcl-2 in a BI1071 dependent manner. In contrast,
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Nur77/H372D and Nur77/Y453L failed to interact with Bcl-2 in the presence of BI1071. The
importance of the binding of BI1071 on inducing Nur77 interaction with Bcl-2 was also
illustrated by immunostaining showing extensive colocalization of transfected Flag Bcl-2 with
the wild-type Nur77 LBD, but not with Nur77 LBD H372A (Supplementary Fig. S8C). Thus,
binding of BI1071 to Nur77 promotes Nur77 interaction with Bcl-2 and mitochondrial
localization.
Discussion
BI1071 is a salt form of DIM-C-pPhCF3 (Fig. 1A) previously reported to induce Nur77-
dependent apoptosis (30,32). However, relative high concentrations (around 10 M) of DIM-C-
pPhCF3 are required for its induction of apoptosis and activation of Nur77. To our surprise, we
observed that aged DIM-C-pPhCF3. was generally more active than the freshly prepared one in
apoptosis induction. This led to our synthesis of the oxidized product of DIM-C-pPhCF3,
methanesulfonate salt of DIM-C-pPhCF3 (BI1071). Our evaluation of BI1071 revealed its
superior death effect in cancer cells. BI1071 was also very effective in other cancer cell lines and
in animal tumor models. The apoptotic effect of several DIM derivatives has been shown to be
Nur77-dependent and various pathways were proposed to account for their apoptotic effect (2).
However, the mechanism by which Nur77 mediates their death effect remains elusive, which is
conceivably due to the activation of multiple pathways by the high compound concentration used
in the studies. For instance, both transcriptional agonist such as DIM-C-pPhOCH3 and
transcriptional antagonist such as DIM-C-pPhOH were shown to induce Nur77-dependent
apoptosis (28). Our finding that oxidization of DIM-C-pPhCF3 could augment its death effect
offered an opportunity to delineate the mechanism by which Nur77 mediates the apoptotic effect
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of DIM-related small molecules. To this end, our studies showed that the potent apoptotic effect
of BI1071 was Nur-77 dependent (Fig. 3) and was a result of its induction of Nur77
mitochondrial targeting via a direct Nur77 binding mechanism. Furthermore, our results revealed
that the death effect of BI1071 was also dependent of Bcl-2 expression and that BI1071 could
induce Nur77 interaction with Bcl-2 leading to Nur77 colocalization with Bcl-2 at mitochondria
and apoptosis.
Binding studies showed that BI1071 could bind to Nur77 better than DIM-C-pPhCF3. This is
likely due to the difference in the structural conformations between the oxidized and the
unoxidized forms of DIM-C-pPhCF3, perhaps resulted from different atomic orbital
hybridization of the central C atom. In the oxidized form the central C is sp2-hybridized,
positively charged and bonded to 3 atoms with a co-planner arrangement, while in the
unoxidized form, C is sp3-hybridized and bonded to 4 atoms with a tetrahedral arrangement.
Differences in structural conformation and charge distributions can affect how molecules bind to
proteins. Our docking results suggested that BI1071 could interact more strongly with Nur77-
LBD than DIM-C-pPhCF3 due to the different conformations adopted by the compounds.
Mutagenesis studies confirmed that H372 and Y453 were 2 key residues involving in the binding
of BI1071 as suggested by the docking studies. Previously, we located the critical region in
Nur77 responsible for its interaction with Bcl-2 and identified a peptide NuBCP-9 as Bcl-2-
converting peptide, capable of inducing apoptosis of cancer cells in vitro and in animals (12).
NuBCP-9 is located at the C-terminal portion of H7. Interestingly our docking studies suggested
that H7 was part of the BI1071 binding region and BI1071 could potentially interact directly
with amino acids D499 and A450, structurally flanking the residues from which NuBCP-9 is
derived. Therefore, it is conceivable that the BI1071-bound Nur77 offers a more suitable Bcl-2
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interacting interface that promotes the formation of Nur77/Bcl-2 complex and thus augments the
biological effect of BI1071.
A critical step in the Nur77 mitochondrial apoptotic pathway is the interaction of Nur77 with
Bcl-2, which induces a conformation change in Bcl-2 and converts Bcl-2 from a pro-survival to a
killer (12,14). Members of the Bcl-2 family are critical regulators of apoptosis. As the funding
member of the Bcl-2 family, Bcl-2 acts as a survival molecule to protect cells from programmed
cell death. Bcl-2 overexpression is often observed in cancer cells and is associated with cancer
treatment resistance and poor prognosis (33-36). Thus, Bcl-2 has been an important drug target
(37,38). Two strategies are commonly used to develop therapeutic agents targeting Bcl-2: The
first relies on making use of antisense oligonucleotides to block Bcl-2 expression and the second
relies on designing and optimizing BH3 small-molecule or peptide mimetics that bind the Bcl-2
BH3-binding cleft, antagonizing its antiapoptotic activity (37,38). Like Bcl-2, Nur77 is
overexpressed in a variety of cancer cells and plays a dual role in mediating apoptosis and
survival of cancer cells (39,40). While the growth promoting effect of Nur77 appears to be
dependent on its nuclear action, the death effect of Nur77 involves its translocation from nucleus
to cytoplasm (41-43). Our results showed that cancer cells are sensitive to the treatment of
BI1071 as compared to normal cells, consisting with the fact that the level of Nur77 is elevated
in cancer cells. Thus, targeting Nur77 by BI1071 will have less effect on normal cells, and
therefore likely offer a high therapeutic index. The ability of Nur77 to interact with Bcl-2 to not
only suppress its anti-apoptotic function but also convert Bcl-2 into a pro-apoptotic molecule
(12,14) provides a promising strategy to target both Nur77 and Bcl-2 for cancer therapy. Agents
that can bind directly to Nur77 to promote Nur77 translocation and interaction with Bcl-2 are
unique in that they can simultaneously target both Nur77 and Bcl-2. The reported Nur77-derived
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peptide with 9 amino acids (NuBCP-9) and its enantiomer as Bcl-2-converting peptides has
demonstrated such a potential (12,44,45). In this regard, our identification of small molecules
that can directly bind Nur77 to activate the Nur77-Bcl-2 apoptotic pathway is significant and
BI1071 represents the first lead of this class of small molecules, which warrants further
evaluation.
Acknowledgements
The authors thank Dr. Marcia Dawson for her contributions in chemistry to this work and to her
memory this paper is dedicated. We also thank Dr. Lin Li for her critical reading of this
manuscript.
This study was supported in part by grants from the Natural Science Foundation of China
(U1405229, 81672749, 31271453, 31471318) to X. Zhang, Regional Demonstration of Marine
Economy Innovative Development Project (16PYY007SF17) to X. Zhang, the Fujian Provincial
Science & Technology Department (2017YZ0002-1) to X. Zhang and the National Institutes of
Health (R01 CA198982).
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Figure legends
Figure 1. A salt form of DIM-C-pPhCF3 exhibits superior apoptotic effect.
A. Structure of DIM-C-pPhCF3 and DIM-C-pPhCF3+MeSO3
- (BI1071). B. HCT116 cells treated
with the indicated concentration of BI1071 or DIM-C-pPhCF3 (labeled as CF3) for 3 days were
assessed by MTT assay. Data are shown as mean ± SD (n=6). C. HCT116 cells treated with the
indicated concentration of BI1071 for 6 hours were analyzed for PARP cleavage by Western
blotting. D. HCT116 cells treated with 0.5 M BI1071 for 6 hours were visualized by DAPI
staining. Apoptotic cells were counted in 200 cells. E. Level of PARP cleavage and cleaved
caspase 3 in MDA-MB-231 cells treated with the indicated concentration of BI1071 for 6 hours
was determined by Western blotting. F. Annexin V/PI staining of MDA-MB-231 cells treated
with the indicated concentration of BI1071 for 6 hours was analyzed by flow cytometry. G.
MDA-MB-231 cells treated with the indicated concentration of BI1071 for 6 hours were stained
with JC-1. Aggregated JC-1, red fluorescence (PE), and monomeric JC-1, green fluorescence
(FITC), were measured by flow cytometry. Statistical data were mean SEM of 5 independent
images. *P< 0.1, ***P< 0.001 (Student’s t test). H. Mitochondrial ROS production in MDA-
MB-231 treated with the indicated concentration of BI1071 for 6 hours was analyzed by flow
cytometry. For Western blots and flow cytometry experiments, one of three similar experiments
are shown.
Figure 2. Effect of BI1071 on tumor growth and apoptosis in animals.
A. Nude mice (n=6) injected with SW620 (2X106 cells) were administrated with the indicated
dose of BI1071 once a day and tumors were measured every three days. B-C. 12 days after
administration of BI1071, nude mice bearing SW620 tumor were sacrificed and tumors were
removed, weighted and showed (***P< 0.001, Student’s t test). D. Representative TUNEL
staining images illustrating the apoptotic effect of BI1071. The apoptotic cells were detected by
TUNEL assay in specimens of xenograft tumors. E. Representative images of MMTV–PyMT
mammary tumor model mice and tumors from mice administered with or without BI1071. For
MMTV–PyMT mammary tumor model, female wild-type MMTV-PyMT mice of 12 weeks old
were randomly divided into two groups (n=7), treated with daily oral doses of BI1071 (5 mg/kg)
for 18 days. F. Inhibition of PyMT tumor growth by BI1071. Mice treated with BI1071 as in E,
and tumors were weighted (n=7). G. Western blot analysis of the expression of PARP, Ki67 and
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PCNA in tumor tissues prepared from 3 MMTV-PyMT mice treated with or without BI1071 (5
mg/kg) for 18 days. H. Representative immunocytochemistry staining showing the expression of
Ki67 and cleaved caspase 3 in tumor tissues prepared from MMTV-PyMT mice treated with or
without BI1071 (5 mg/kg) for 18 days.
Figure 3. BI1071 induces Nur77 dependent apoptosis and Nur77 mitochondrial targeting.
A. MEFs and Nur77-/-
MEFs treated with the indicated concentration of BI1071 for 6 hours were
assessed by MTT assay. (**P< 0.01 and ***P< 0.001, Student’s t test). B. PARP cleavage in
MEFs or Nur77-/-
MEFs treated with 0.5 M BI1071 for 6 hours was determined by Western
blotting. C-F. HeLa or Nur77-/-
HeLa cells were treated with 0.5 M BI1071 for 6 hours. Cells
were then subjected to Western blotting for PARP cleavage and cleaved caspase 3 detecting (C),
Annexin V/PI staining for apoptosis measurement (D), JC-1 staining for measuring
mitochondrial membrane potential (E) or mito-SOX staining for determining the production of
mitochondrial ROS (F). One of three similar experiments is shown. NS, not significant; ***P<
0.001 (Student’s t test).G. Subcellular localization of endogenous Nur77 in HCT116 cells
treated with 0.5 M BI1071 for 2 hours was analyzed by confocal microscopy after
immunostained with anti-Nur77 antibody. Nucleus were visualized by DAPI staining. H. HeLa
cells transfected with GFP-Nur77 were treated with 0.5 M BI1071 for 2 hours and visualized
by confocal microscopy. I. HeLa cells transfected with GFP-Nur77-LBD were treated with
BI1071 (0.5 M) for 2 hours were immunostained with anti-Hsp60 antibody and visualized by
confocal microscopy. J. HEK293T cells were transfected with GFP-Nur77-LBD and treated with
0.5 M BI1071 for 2 hours. Cytosolic (Cyt) and HM fraction were then prepared and analyzed
by Western blotting. Expression of cytoplasmic IB and mitochondrial Hsp60 was shown to
indicate the purity of cytosolic and mitochondrial fraction, respectively. K. HEK293T cells were
transfected with Myc-Nur77. Whole cell lysate (WCL) and mitochondria-enriched heavy
membrane (HM) fractions were prepared from HEK293T cells treated with 0.5 M BI1071 for 2
hours, and analyzed by Western blotting. Expression of nuclear PARP and mitochondrial Hsp60
was shown to ensure the purity of HM fraction. For cellular fractionation experiments, one of
three similar experiments are shown.
Figure 4. BI1071 binds directly to Nur77.
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29
A, B. Binding of DIM-C-pPhCF3 (A) or BI1071(B) to purified Nur77-LBD by SPR. C.
HEK293T cells were transfected with Gal-4 reporter plasmid and Gal-4-RXR-LBD or Gal-4-
RXR-LBD/E453,6A together with Myc-Nur77, and treated with the indicated concentration of
BI1071 or 9-cis-RA for 12 hours. Reporter activities were measured. D. Molecular modeling of
the binding of BI1071 (in yellow sticks) and DIM-C-pPhCF3 (in green sticks) to Nur77-LBD. E.
HEK293T cells transfected with Gal-4 reporter plasmid and Gal-4-RXR-LBD together with
Myc-Nur77 or Myc-Nur77/H372D were treated with the indicated concentration of BI1071, and
reporter activities were measured. **P< 0.01, ***P< 0.001 (Student’s t test). F. HEK293T cells
were transfected with Myc-Nur77, Myc-Nur77/H372A and treated with BI1071 (0.5 M) for 2
hours. WCL and HM fractions were then prepared and analyzed by Western blotting. G−J.
Nur77-/-
HeLa cells were transfected with the indicated Nur77 and mutant plasmids and treated
with 0.5 M BI1071 for 6 hours. Cells were then subjected to Western blotting for PARP
cleavage (G), mito-SOX staining for determining the production of mitochondrial ROS (H),
Annexin V/PI staining for apoptosis (I), or JC-1 staining for measuring mitochondrial membrane
potential (J). One of three similar experiments are shown. NS, not significant; ***P< 0.001
(Student’s t test).
Figure 5. Bcl-2 dependent induction of apoptosis by BI1071.
A, B. MEFs or Bcl-2-/-
MEFs were treated with 0.5 M BI1071 for 6 hours. Cells were then
subjected to Western blot analysis for PARP cleavage (A) or DAPI staining for apoptosis (B).
Apoptotic cells were counted in 200 cells. ***P< 0.001 (Student’s t test). C. PARP cleavage in
HeLa or Bcl-2-/-
HeLa cells treated with 0.5 M BI1071 for 6 hours was analyzed by Western
blotting. D-F. MEFs or Bcl-2-/-
MEFs, HeLa cells or Bcl-2-/-
HeLa cells were treated with 0.5 M
BI1071 for 6 hours. Cells were subjected to Annexin V/PI staining for apoptosis measurement
(D), JC-1 staining for measuring mitochondrial membrane potential (E) or mito-SOX staining for
determining the production of mitochondrial ROS (F). One of three similar experiments are
shown. *P< 0.1, ***P< 0.001 (Student’s t test).
Figure 6. BI1071 promotes Nur77 interaction with Bcl-2.
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30
A. GST pull-down. Purified Nur77-LBD incubated with or without the indicated concentration
of BI1071 was pulled down by GST or GST-Bcl-2 protein and analyzed by Western blotting. B,
C. Co-immunoprecipitation assay. HEK293T transfected with Myc-Bcl-2 together with GFP-
Nur77 (B) or GFP-Nur77-LBD (C) were treated with or without 0.5 M BI1071 and analyzed by
co-immunoprecipitation assays using anti-Myc antibody. D. Interaction of endogenous Nur77
and Bcl-2 in MDA-MB-231 cells treated with or without 0.5 M BI1071 for 2 hours was
analyzed by co-immunoprecipitation assay using anti-Bcl-2 antibody. E, F. Colocalization of
Nur77 with Bcl-2. HEK293T cells were transfected with Myc-Bcl-2 together with GFP-Nur77
(E) or GFP-Nur77-LBD, treated with or without 0.5 M BI1071 for 2 hours, stained with anti-
Myc antibody, and visualized using confocal microscopy. G. HEK293T cells transfected with
the indicated expression plasmids were treated with 0.5 M BI1071 for 2 hours and analyzed by
co-immunoprecipitation assays using anti-Flag antibody. For Co-IP experiments, one of three
similar experiments are shown.
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Published OnlineFirst March 29, 2019.Mol Cancer Ther Xiaohui Chen, Xihua Cao, Xuhuang Tu, et al. cells by activating the Nur77-Bcl-2 apoptotic pathwayBI1071, a novel Nur77 modulator, induces apoptosis of cancer
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