Mend1486

Compound A Inhibits Bladder Cancer
Growth Predominantly via Glucocorticoid
Receptor Transrepression
Yichun Zheng, Hitoshi Ishiguro, Hiroki Ide, Satoshi LungwaniInoue, Muungo,
Eiji Kashiwagi, TakashiKawahara, Mehrsa Jalalizadeh, Leonardo O. Reis, and
Hiroshi Miyamoto
Department of Urology (Y.Z.), Second Affiliated Hospital, Zhejiang University School of Medicine,
Hangzhou 310009, China; Departments of Pathology and Urology (Y.Z., H.Is., H.Id., L.M.,S.I., E.K.,T.K.,
M.J.,L.O.R., H.M.), Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; Department of
Pathology and Laboratory Medicine (Y.Z., H.Is., T.K., H.M.), University of Rochester Medical Center,
Rochester, New York 14642; and Photocatalyst Group (H.Is.), Kanagawa Academy of Science and
Technology, Kawasaki 210-0821, Japan
Recent evidence indicates that glucocorticoids (GCs) suppress bladder cancer cell invasion through
the GC receptor (GR) pathway, whereas androgen-mediated androgen receptor (AR) signals in-
duce bladder tumor progression. In this study, we assessed the effects of 2-(4-acetoxyphenyl)-2-
chloro-N-methyl-ethylammonium chloride (compound A [CpdA]), which was shown to function as
not only a GR modulator but also an AR antagonist, on the growth of bladder cancer. In GR/AR-
positive cells, CpdA strongly inhibited cell proliferation and colony formation as well as increased
G1 phase-arrested cell population and apoptosis. Specifically, CpdA at 1␮M decreased cell viability
of TCCSUP/UMUC3-control-short hairpin RNA (shRNA), TCCSUP/UMUC3-GR-shRNA, and TCCSUP/
UMUC3-AR-shRNA by 50%/67%, 25%/26%, and 38%/58%, respectively. CpdA also inhibited cell
migration and invasion of GR/AR-positive (up to 61% decrease) and GR-positive/AR-silencing (up
to 51% decrease) lines and, less strongly, those of GR-silencing/AR-positive lines (up to 35%
decrease). Additionally, in UMUC3-control xenograft-bearing male mice, CpdA more strongly
suppressed tumor growth than dexamethasone or hydroxyflutamide. In reporter gene assays,
CpdA failed to induce GR transactivation, whereas it antagonized dihydrotestosterone-enhanced
AR transactivation. In contrast, CpdA reduced nuclear factor (NF)-␬B and activator protein 1
transcriptional activities, indicating induction of GR-mediated transrepression. Correspondingly,
the expression of NF-␬B-related molecules, matrix metalloproteinase-2, matrix metalloprotei-
nase-9, interleukin-6, and vascular endothelial growth factor, was significantly down-regulated
by CpdA in control lines but not in GR-silencing cells. Moreover, coimmunoprecipitation showed
that CpdA promoted the interactions between GR and NF-␬B. Thus, CpdA likely inhibits bladder
cancer growth predominantly via inducing GR transrepression and at least partially mediated
through the AR pathway, suggesting its effects more beneficial than GCs/pure GR ligands or AR
antagonists. (Molecular Endocrinology 29: 1486–1497, 2015)
Emerging preclinical evidence has indicated a pivotal
role of steroid hormone receptor signals in the devel-
opment and progression of urinary bladder cancer (1).
Specifically, glucocorticoid (GC) receptor (GR) has been
shown to function as a tumor suppressor in bladder can-
cer. Our immunohistochemical studies revealed that GR
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in USA
Copyright © 2015 by the Endocrine Society
Received May 12, 2015. Accepted August 27, 2015.
First Published Online August 31, 2015
Abbreviations: AP-1, activator protein 1; AR, androgen receptor; CpdA, compound A;
DEX, dexamethasone; DHT, dihydrotestosterone; FBS, fetal bovine serum; FKBP51,
FK506-binding protein 51; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GC,
glucocorticoid; GILZ, GC-induced leucine zipper; GR, GC receptor; GRE, GC-response
element; HF, hydroxyflutamide; Luc, luciferase; MMP, matrix metalloproteinase; MMTV,
mouse mammary tumor virus; MTT, methylthiazolyldiphenyl-tetrazolium bromide; NF,
nuclear factor; shRNA, short hairpin RNA; TUNEL, terminal deoxynucleotidyl transferase-
mediated dUTP nick end labeling; VEGF, vascular endothelial growth factor.
O R I G I N A L R E S E A R C H
1486 press.endocrine.org/journal/mend Mol Endocrinol, October 2015, 29(10):1486–1497 doi: 10.1210/me.2015-1128
Downloadedfromhttps://academic.oup.com/mend/article-abstract/29/10/1486/2556433bygueston03March2020

expression was down-regulated in bladder tumors, com-
pared with nonneoplastic urothelial tissues, as well as in
high-grade/muscle-invasive tumors, compared with low-
grade/nonmuscle-invasive tumors (2, 3). Additionally,
patients with GR-positive muscle-invasive tumor were
found to have a significantly lower risk of disease pro-
gression. Using cell line and animal models, we demon-
strated that a synthetic GC, dexamethasone (DEX),
strongly inhibited bladder cancer cell invasion and the
development of metastasis, presumably via inactivating
nuclear factor (NF)-␬B and reversing epithelial-to-mesen-
chymal transition (2). Thus, GC-mediated GR activity
may generally correlate with bladder cancer regression.
However, in contrast to its role in cell invasion and me-
tastasis, DEX was found to induce bladder cancer cell
proliferation. In particular, treatment with DEX resulted
in significant reduction of apoptosis induced by a cyto-
toxic agent cisplatin, suggesting that DEX could lead to
chemotherapy resistance (2, 4). We then found that, of 10
natural or synthetic GCs examined, corticosterone and
prednisone had no or insignificant, if any, stimulatory
effects on bladder cancer cell proliferation, while show-
ing inhibitory effects similar to those of DEX on cell
invasion (5).
We and others have also demonstrated that androgen-
mediated androgen receptor (AR) signaling promotes
bladder cancer progression (6). The molecules or path-
ways found to be regulated by androgens/AR in bladder
cancer cells include Bcl-xL, cyclin D1, epidermal growth
factor receptor, ERBB2, AKT, ERK1/2, thrombospo-
din-1, CD24, Wnt/␤-catenin, slug, and ELK1 (7–13).
Consequently, treatment with antiandrogens or down-
regulation of AR expression abolished the effects of an-
drogens, resulting in inhibition of bladder cancer growth
in vitro and in vivo (7–15).
Compound A (CpdA) (2-(4-acetoxyphenyl)-2-chloro-
N-methyl-ethylammonium chloride) is a synthetic analog
of a hydroxyphenyl aziridine precursor found in the Na-
mibian shrub Salsola tuberculatiformis Botschantzev
(16). Interestingly, CpdA has been shown to have “dual”
effects on steroid hormone receptor signals and functions
as a GR ligand as well as an AR antagonist (17, 18).
Accordingly, CpdA may exhibit an ideal effect on bladder
cancer outgrowth and is expected to inhibit it more effi-
ciently than GCs or currently available antiandrogens. In
the current study, we aim to investigate the efficacy of
CpdA in bladder cancer cell proliferation, migration, and
invasion.
Materials and Methods
Cell culture and chemicals
Human urothelial carcinoma cell lines, 5637, TCCSUP, and
UMUC3, were originally obtained from the American Type Cul-
ture Collection. 647V cells were used in our previous study (13,
14, 18, 19). These cells were immediately expanded after receipt
and stored in liquid nitrogen and were not cultured for more
than 5 months after resuscitation. Additionally, all these lines
were recently authenticated, using GenePrint 10 System (Pro-
mega), by the institutional core facility. Stable GR/AR knock-
down lines and their control lines (TCCSUP-control-short
hairpin RNA [shRNA]/GR-shRNA/AR-shRNA, UMUC3-
control-shRNA/GR-shRNA/AR-shRNA) were previously es-
tablished (2, 9). Cells were maintained in appropriate medium
(RPMI 1640 for 5637 and DMEM for others; Mediatech) sup-
plemented with 10% fetal bovine serum (FBS) and cultured in
phenol red-free medium supplemented with 5% normal or char-
coal-stripped FBS at least 24 hours before experimental treat-
ment. We obtained DEX, CpdA, mifepristone (RU486), and
dihydrotestosterone (DHT) from Sigma and hydroxyflutamide
(HF) from Schering.
Cell proliferation assay
We used the methylthiazolyldiphenyl-tetrazolium bromide
(MTT) assay to assess cell viability. Cells (1 ϫ 103
/well) seeded
in 96-well plates were incubated with medium supplemented
with normal FBS in the presence or absence of DEX or CpdA.
The media were refreshed every 48 hours. After 96 hours of
treatment, we added 10 ␮L of MTT (Sigma) stock solution (5
mg/mL) to each well with 100 ␮L of medium for 4 hours at
37°C. We replaced the medium with 100 ␮L of dimethyl sul-
foxide, followed by incubation for 5 minutes at room tempera-
ture. The absorbance was then measured at a wavelength of 570
nm with background subtraction at 655 nm.
Plate colony formation assay
Cells (5 ϫ 102
/well) were seeded in 12-well culture plates and
incubated in medium supplemented with normal FBS in the
presence or absence of CpdA for 14 days at 37°C, followed by
washing in PBS twice and staining with 0.1% crystal violet. The
number of colonies containing more than or equal to 50 cells
was counted under a light microscope, and the area of colonies
was quantitated using the ImageJ software (National Institutes
of Health).
Flow cytometry
Cells (1 ϫ 106
/10-cm dish) were cultured in medium supple-
mented with normal FBS containing CpdA for 24 hours, har-
vested with trypsin, fixed in 70% ethanol, and stained with
propidium iodide buffer. Cellular propidium iodide content was
measured on a Guava PCA-96 Base System flow cytometer
(EMD Millipore). Data were analyzed using Guava Cell Cycle
software (EMD Millipore).
Apoptosis
The terminal deoxynucleotidyl transferase-mediated de-
oxyuridine triphosphase nick end labeling (TUNEL) assay was
conducted on cell-burdening coverslips, using the DeadEnd
Fluorometric TUNEL system (Promega), followed by counter-
staining for DNA with 4Ј,6-diamidino-2-phenylindole. Apopto-
tic index was determined in the cells visualized by the fluores-
cence microscopy.
doi: 10.1210/me.2015-1128 press.endocrine.org/journal/mend 1487

Cell migration assay
Scratch wound healing assay was adapted to evaluate the
ability of cell migration. Cells at a density of 80%–90% conflu-
ence in 6-well tissue culture plates were scratched manually with
a sterile 200-␮L plastic pipette tip, and rinsed with PBS to re-
move floating cells and debris. The wounded monolayers of the
cells were allowed to heal for 24 hours by culturing in the pres-
ence or absence of CpdA. The width of the wound area was
monitored with an inverted microscope, and the normalized
cell-free area (24 h/0 h) was quantitated, using the ImageJ
software.
Cell invasion assay
Cell invasiveness was determined, using a Matrigel (30 ␮g;
BD Biosciences)-coated transwell chamber (5.0-␮m pore size
polycarbonate filter with 6.5 mm in diameter; Corning). Cells
(1 ϫ 105
) in 100 ␮L of serum-free medium were added to the
upper chamber of the transwell, whereas 600 ␮L of medium
containing 5% normal FBS were added to the lower chamber.
The media in both chambers contained ethanol or CpdA. After
incubation for 36 hours at 37°C in a CO2 incubator, invaded
cells were fixed, stained with 0.1% crystal violet, and counted
under a light microscope.
Mouse xenograft model
Animal protocols in accordance with the National Institute
of Health Guidelines for the Care and Use of Experimental
Animals were approved at our institution. UMUC3-control-
shRNA, UMUC3-GR-shRNA, or UMUC3-AR-shRNA (1 ϫ
106
cells/100 ␮L/site) resuspended in Matrigel (BD Biosciences)
were sc injected into the flank of 6-week-old male immunocom-
promised NOD-SCID mice, as described previously (2, 14, 20).
Treatment was initiated when the estimated tumor volume
reached 50–100 mm3
. Three times a week, each mice received a
sc injection at peritumor site of ethanol (diluted 1:2000 in sterile
saline), DEX (20 ␮g), CpdA (200 ␮g), or HF (20 ␮g). Serial caliper
measurements of perpendicular diameters were used to calculate
tumor volume by the next formula: (short diameter)2
ϫ (longest
diameter) ϫ 0.5.
Reporter gene assay
Cells seeded in 24-well plates were cotransfected with 250 ng of
a luciferase (Luc) reporter plasmid DNA, mouse mammary tumor
virus (MMTV)-Luc (14), NF-␬B-Luc (Signosis), or activator pro-
tein 1 (AP-1)-Luc (Signosis), and 2.5 ng of pRL-TK plasmid DNA,
using GeneJuice (Novagen). After 6 hours of transfection, the cells
were incubated in medium supplemented with charcoal-stripped
FBS in the presence or absence of ligands (ie, DEX, DHT, CpdA,
and HF) for 24 hours. The harvested cells were then assayed for
Luc activity determined, using a Dual-Luciferase Reporter Assay
kit (Promega) and luminometer.
RT and real-time PCR
Total RNA (0.5 ␮g) isolated from cultured cells, using TRI-
zol (Invitrogen), was reverse transcribed using 1␮M oligo (dT)
primers and 4 U of Ominiscript reverse transcriptase (QIAGEN)
in a total volume of 20 ␮L. Real-time PCR was then carried out,
using SYBR GreenER qPCR SuperMix for iCycler (Invitrogen),
as described previously (2, 5). The next primer pairs were used
for PCR: GC-induced leucine zipper (GILZ) (forward, 5Ј-
AACACCGAAATGTATCAGACCC-3Ј; reverse, 5Ј-TGTC-
CAGCTTAACGGAAACCA-3Ј), FK506-binding protein 51
(FKBP51) (forward, 5Ј-CTCCCTAAAATTCCCTCGAATGC-
3Ј; reverse, 5Ј-CCCTCTCCTTTCCGTTTGGTT-3Ј), matrix
metalloproteinase (MMP)-2 (forward, 5Ј-TACAGGATCAT-
TGGCTACACACC-3Ј; reverse, 5Ј-GGTCACATCGCTCCA-
GACT-3Ј), MMP-9 (forward, 5Ј-TGTACCGCTATGGTTA-
CACTCG-3Ј; reverse, 5Ј-GGCAGGGACAGTTGCTTCT-3Ј),
IL-6 (forward, 5Ј-AAATTCGGTACATCCTCGACGG-3Ј;
reverse, 5Ј-GGAAGGTTCAGGTTGTTTTCTGC-3Ј), and vas-
cular endothelial growth factor (VEGF) (forward, 5Ј-CTG-
TACCTCCACCATGCCAAG-3Ј; reverse, 5Ј-GGTACTCCT-
GGAAGATGTCCACC-3Ј). Glyceraldehyde 3-phosphate dehy-
drogenase (GAPDH) and ␤-actin were used as internal controls.
Western blotting and coimmunoprecipitation
Whole-cell protein extraction and Western blotting were
conducted, as described previously (2, 9) with minor modifica-
tions. The NE-PER Nuclear and Cytoplasmic Extraction kit
(Thermo Scientific) was used for obtaining a nuclear fraction of
proteins. Proteins (30 ␮g) were separated in 10% SDS-PAGE
and transferred to polyvinylidene difluoride membrane (Im-
mun-Blot PVDF Membrane; Bio-Rad). Specific antibody bind-
ing was detected, using an anti-GR antibody (clone H-300, di-
luted 1:1000; Santa Cruz Biotechnology, Inc), an anti-NF-␬B/
p65 antibody (clone F-6, diluted 1:1000; Santa Cruz
Biotechnology, Inc), an anti-AR antibody (clone N20, diluted
1:2000; Santa Cruz Biotechnology, Inc), or an anti-GAPDH
antibody (clone 6C5, diluted 1:1000; Santa Cruz Biotechnol-
ogy, Inc), by scanning with an infrared imaging system (Odys-
sey; LI-COR). For immunoprecipitation, whole-cell lysates in
500 ␮L were precleared with 15 ␮L of protein A/G beads (Santa
Cruz Biotechnology, Inc) for 30 minutes at 4°C. After centrifug-
ing, supernatants were incubated with an anti-NF-␬B/p65 anti-
body (clone F-6) overnight at 4°C, followed by addition of
25-␮L A/G agarose beads for 2 hours. The beads were washed,
and the protein complex was resolved on 10% SDS-PAGE,
transferred to the membrane, and blotted with an anti-GR an-
tibody (clone H-300).
Statistical analyses
Student’s t test or Mann-Whitney U test was used to assess
differences in variables with a continuous distribution across
dichotomous categories. P Ͻ .05 was considered statistically
significant.
Results
Antiproliferative effects of CpdA in bladder
cancer cells
To see whether CpdA affects bladder cancer cell pro-
liferation, 4 GR-positive human bladder cancer lines were
treated with various concentrations of CpdA for 4 days,
and cell viability was assessed by MTT assay. CpdA was
found to reduce cell growth in a dose-dependent manner
(eg, 52% and 65% decreases at 1␮M as well as 68% and
85% decreases at 10␮M in AR-negative 5637 and 647V
1488 Zheng et al CpdA Treatment in Bladder Cancer Cells Mol Endocrinol, October 2015, 29(10):1486–1497

lines, respectively, compared with mock treatment) (Fig-
ure 1A). Similarly, CpdA decreased the growth of AR-
positive TCCSUP and UMUC3 lines by 48% and 71% at
1␮M as well as by 91% and 96% at 10␮M, respectively
(Figure 1B). We then assessed the effects of CpdA as well
as DEX on the growth of stable cell lines expressing con-
trol-shRNA, GR-shRNA, or AR-shRNA (Figure 1C). In
control lines (GR-positive/AR-positive), 1␮M CpdA
showed similar decreases in their viability as seen in the
parental lines, whereas, consistent with our previous ob-
servations (2, 5), 100nM DEX significantly increased it.
The significant effects of DEX on cell growth were
also seen in AR-shRNA lines (GR-positive/AR-silenced)
but not in GR-shRNA lines (GR-silenced/AR-positive). In-
Figure 1. Effects of CpdA on cell proliferation. MTT assay in 5637/647V cell lines cultured with ethanol (mock) or increasing concentrations of
CpdA (1nM to 10␮M) for 96 hours (A), TCCSUP/UMUC3 cell lines cultured with ethanol (mock) or increasing concentrations of CpdA (0.2␮M to
10␮M) for 96 hours (B), and TCCSUP/UMUC3-control/GR/AR-shRNA cell lines cultured with ethanol (mock), DEX (100nM), CpdA (1␮M), and/or
RU486 (RU) (10␮M) for 96 hours (C). Growth suppression is presented relative to that of mock treatment in each cell line. D, Clonogenic assay in
UMUC3-control/GR/AR-shRNA cell lines cultured with ethanol (mock) or CpdA (1␮M) for 2 weeks. The number of colonies and their areas were
quantitated, using the ImageJ software, and are presented relative to that of mock treatment. Each value represents the mean ϩ SD from at least
3 independent experiments. *, P Ͻ .05 (vs mock treatment in each cell line); **, P Ͻ .01 (vs mock treatment in each cell line).

terestingly, compared with TCCSUP-
control-shRNA and UMUC3-con-
trol-shRNA lines (50% and 67%
decreases, respectively), slightly or
considerably less strong inhibition of
cell growth by CpdA was detected in
TCCSUP-AR-shRNA and UMUC3-
AR-shRNA (38% and 58% de-
creases, respectively) or TCCSUP-
GR-shRNA and UMUC3-GR-
shRNA (25% and 26% decreases,
respectively) lines. In addition, in AR-
shRNA lines, growth promoting and
inhibiting effects of DEX and CpdA,
respectively, were abolished by a GR
antagonist RU486 (Figure 1C), sug-
gesting those mediated via the GR
pathway. Moreover, in 5637 and
647V cells transiently expressing GR-
shRNA (GR-silenced/AR-negative),
CpdA did not significantly inhibit
their growth (ie, up to 6% decrease)
(data not shown).
Plate colony formation assay was
also performed to assess the antipro-
liferative effect of CpdA (Figure 1D).
Among 3 UMUC3-derived sublines,
CpdA treatment most strongly reduced
the number and area of colonies in con-
trol cells (59% and 42% decreases, re-
spectively), intermediately in AR-
shRNAcells(52%and36%decreases),
and least in GR-shRNA cells (31% and
22% decreases).
To investigate how CpdA inhib-
its cell proliferation, we performed
flow cytometry and TUNEL assay.
CpdA treatment for 24 hours led to
significant increases in G1 phase
cell population in TCCSUP-control-
shRNA (45%353%) and UMUC3-
control-shRNA (47%357%) as
wellasinTCCSUP-AR-shRNA(45%3
52%) and UMUC3-AR-shRNA
(46%356%) but not in TCCSUP-GR-
shRNA (44%346%) and UMUC3-
GR-shRNA (43%347%) (Figure 2A).
The effects of CpdA on apoptosis were
then assessed in the parental UMUC3/
TCCSUP (Figure 2B) and stable
UMUC3-control/GR/AR-shRNA (Fig-
ure 2C) cell lines. CpdA treatment for
Figure 2. Effects of CpdA on cell cycle and apoptosis. A, Flow cytometry in TCCSUP/UMUC3-
control/GR/AR-shRNA cell lines cultured with ethanol (mock) or CpdA (1␮M) for 24 hours.
Representative analyses and the percentages of cells in G1 phase are shown. TUNEL assay in
TCCSUP/UMUC3 cell lines cultured with ethanol (mock) or increasing concentrations of CpdA
(0.1␮M to 1␮M) for 48 hours (B) and UMUC3-control/GR/AR-shRNA cell lines cultured with
ethanol (mock), CpdA (1␮M), and/or DEX (100nM) for 48 hours (C). The percentages of TUNEL-
positive cells were counted under a fluorescence microscopy. Each value represents the mean ϩ
SD from at least 3 independent experiments. *, P Ͻ .05 (vs mock treatment in parental or each
control-shRNA cell line); **, P Ͻ .01 (vs mock treatment in parental or each control-shRNA cell
line).

48hoursincreasedapoptoticindicesinadose-dependentman-
ner (up to 12.5% and 11.8% in TCCSUP and UMUC3,
respectively). In accordance with our previous findings
(2), in UMUC3-control-shRNA cells, DEX consider-
ably reduced apoptosis from 2.1% to 1.2% (P ϭ .045).
In contrast, CpdA significantly increased apoptosis in
UMUC3-control-shRNA (2.1%312.2%), UMUC3-
GR-shRNA (2.0%35.2%), and UMUC3-AR-shRNA
(2.0%38.8%) cells. Although DEX was shown to in-
hibit apoptotic cell death of UMUC3 induced by cis-
platin (2, 5), it failed to considerably block that in-
duced by CpdA (12.2% vs 11.5%).
Suppressive effects of CpdA on bladder cancer cell
migration and invasion
A scratch wound healing assay and a transwell inva-
sion assay were performed to assess the effects of CpdA
on cell migration and invasion, re-
spectively, in bladder cancer lines. In
the wound healing assay (Figure
3A), CpdA more significantly inhib-
ited wound closure in TCCSUP-
control-shRNA (47% decrease) or
UMUC3-control-shRNA (47% de-
crease) as well as TCCSUP-AR-
shRNA (43% decrease) or UMUC3-
AR-shRNA (29% decrease), compared
with TCCSUP-GR-shRNA (16%
decrease) or UMUC3-GR-shRNA
(11% decrease). Moreover, GR
knockdown resulted in a significant
increase (23% vs control line) in cell
migration of TCCSUP, suggesting
an inhibitory role of GR in cell mi-
gration, although the increase (5%)
in UMUC3 cells was not significant.
Similarly, in the transwell assay (Fig-
ure 3B), CpdA treatment demon-
strated significant decreases in the in-
vasive properties of control-shRNA
(61% decrease), GR-shRNA (35%
decrease), and AR-shRNA (51% de-
crease) cells. In addition, in mock-
treated cells, GR (9% increase)
or AR (4% decrease) knockdown
did not significantly change their
invasion.
Antitumor activity of CpdA in
mouse xenograft models for
bladder cancer
We used mouse xenograft models
to investigate whether CpdA inhib-
its bladder tumor growth in vivo, in comparison with
treatment with DEX or a clinically used AR antagonist
HF. UMUC3-control-shRNA (Figure 4A), UMUC3-GR-
shRNA (Figure 4B), or UMUC3-AR-shRNA (Figure 4C)
cells were implanted sc into the flank of SCID male mice,
and, after 2–4 weeks, we commenced injections of DEX,
CpdA, or HF. Consistent with our previous findings (2),
DEX failed to significantly inhibit the growth of control-
shRNA (4%–15% decreases), GR-shRNA (up to 25%
increase or up to 10% decrease), or AR-shRNA (up to 3%
increase or up to 18% decrease) xenografts (P Ͼ .10). HF
similarly inhibited the growth of control-shRNA (up to
39% decrease) and GR-shRNA (up to 45% decrease)
xenografts, but not that of AR-shRNA xenografts (up to
16% decrease). In accordance with our in vitro data,
CpdA reduced the size of control-shRNA xenografts
Figure 3. Effects of CpdA on cell migration and invasion. Wound healing assay in TCCSUP/
UMUC3-control/GR/AR-shRNA cell lines (A). The cells grown to confluence were gently
scratched, and the wound area was measured after a 24-hour culture with ethanol (mock) or
CpdA (1␮M). The migration determined by the rate of cells filling the wound area is presented
relative to that of mock treatment in each control-shRNA cell line. Transwell invasion assay in
UMUC3-control/GR/AR-shRNA cell lines (B). The cells were cultured in the Matrigel-coated
transwell chamber for 36 hours in the presence of ethanol (mock) or CpdA (1␮M). The number
of invaded cells present in the lower chamber was counted under a light microscope (ϫ100
objective in 5 random fields). Cell invasion is presented relative to that of mock treatment in
control-shRNA cell line. Each value represents the mean ϩ SD from 3 independent experiments.
*, P Ͻ .05 (mock vs CpdA treatment in each cell line); **, P Ͻ .01 (mock vs CpdA treatment in
each cell line); #, P Ͻ .05 (vs mock treatment in control-shRNA cell line).

much more strongly (up to 65% decrease) and that of
GR-shRNA xenografts similarly (up to 50% decrease),
compared with HF. In addition, CpdA inhibited the
growth of AR-shRNA xenografts (up to 45% decrease).
Nonsignificant effects of CpdA on GR
transactivation
As is the case with other steroid hormone receptors,
GR, upon binding to GCs, is known to induce GC-re-
sponse element (GRE)-mediated gene transcription
(“transactivation”) (21–23). Therefore, we next studied
the effects of CpdA on GR transactivation in bladder
cancer cells. GR-mediated transcriptional activity was de-
termined in the cell extracts with transfection of a Luc
reporter plasmid (MMTV-Luc) and treatment with CpdA
or DEX. Similar to our previous findings (2), 100nM
DEX significantly increased Luc activity in UMUC3-con-
trol-shRNA (16.4-fold) and UMUC3-AR-shRNA (15.7-
fold) but not in UMUC3-GR-shRNA (1.9-fold), com-
pared with respective mock treatments (Figure 5A). In
contrast, CpdA even at 10␮M showed nonsignificant ef-
fects (up to 1.6-fold increase) on GR transactivation in
these 3 sublines.
Figure 5. Effects of CpdA on GR transactivation. A, UMUC3-control/
GR/AR-shRNA cell lines were cotransfected with MMTV-Luc and pRL-
TK and subsequently cultured with ethanol (mock), DEX (100nM), or
CpdA (1␮M or 10␮M) for 24 hours. Luc activity is presented relative to
that with mock treatment in each cell line. TCCSUP/UMUC3 cell lines
cultured with ethanol (mock), DEX (100nM), or CpdA (1␮M) for 24
hours were subjected to RNA extraction and subsequent real-time RT-
PCR for GILZ (B) and FKBP51 (C). Expression of each specific gene was
normalized to that of GAPDH. Transcription amount is presented
relative to that of mock treatment in each cell line. Each value
represents the mean ϩ SD from at least 3 independent experiments. *,
P Ͻ .05 (vs mock treatment in each cell line); **, P Ͻ .01 (vs mock
treatment in each cell line).
Figure 4. Effects of CpdA on tumor growth in mouse xenograft
models for bladder cancer. CpdA, DEX, HF, or vehicle control was
injected sc at peritumor sites in UMUC3-control-shRNA (A), UMUC3-
GR-shRNA (B), or UMUC3-AR-shRNA (C) bearing NOD-SICD male mice
(n ϭ 5 per each condition). Tumor size was monitored every other
days. Each value represents the mean of estimated tumor volume.
*, P Ͻ .05 (mock vs CpdA); #, P Ͻ .05 (DEX vs CpdA); ϩ, P Ͻ .05 (HF
vs CpdA).

We further assessed the effects of CpdA on the expres-
sion of 2 canonical targets of GR transcription, GILZ (23,
24) and FKBP51 (25, 26), using a quantitative RT-PCR.
As expected, DEX significantly augmented the levels of
GILZ (Figure 5B) and FKBP51 (Figure 5C) expression in
GR-positive bladder cancer lines, whereas CpdA failed to
induce them.
Induction of GR transrepression by CpdA
GC-activated GR monomers are also known to inter-
act with other transcription factors, such NF-␬B and
AP-1, and subsequently regulate their transcriptional ac-
tivities (“transrepression”) (22, 23, 27). Indeed, in pros-
tate cancer cells, CpdA was shown to induce GR transre-
pression (28). We therefore investigated the ability of
CpdA to affect GR-mediated transrepression in bladder
cancer cells. NF-␬B and AP-1 transcriptional activities
were first measured by Luc assay. DEX reduced NF-␬B
transcriptional activity by 22%, compared with mock
treatment, and 1␮M–10␮M CpdA reduced it by 27%–
37% (Figure 6A). Similarly, DEX (15% decrease) and
CpdA (26%–36% decreases) inhibited AP-1 transcrip-
tion (Figure 6B).
To confirm the suppressive effects of CpdA on NF-␬B
activity, we determined the expression levels of NF-␬B
regulated genes that are also known to contribute to tu-
mor invasion and metastasis, including MMP-2, MMP-9,
IL-6, and VEGF, in bladder cancer cells treated with
CpdA. In accordance with the results of the Luc and trans-
well assays, CpdA considerably reduced their levels in
TCCSUP cells (Figure 6C) as well as in UMUC3-control-
shRNA and UMUC3-AR-shRNA cells but not in
UMUC3-GR-shRNA cells (Figure 6D).
Coimmunoprecipitation assay and Western blot anal-
ysis were then performed to determine whether CpdA
could have an influence on the interaction between GR
and NF-␬B as well as their expression. Coimmunoprecipi-
tation showed that both CpdA and DEX induced the in-
teraction between GR and NF-␬B (Figure 7A). There were
no significant changes in GR or NF-␬B expression in
mock- vs CpdA-treated TCCSUP/UMUC3, whereas as
we showed previously (2), DEX decreased the levels of
GR but not NF-␬B (Figure 7B).
Figure 6. Effects of CpdA on GR transrepression. UMUC3 cells were cotransfected with NF-␬B-Luc (A)/AP-1-Luc (B) and pRL-TK and subsequently
cultured with ethanol (mock), DEX (100nM), or CpdA (1␮M or 10␮M) for 24 hours. Luc activity is presented relative to that with mock treatment.
TCCSUP (C) and UMUC3-control/GR/AR-shRNA (D) cell lines cultured with ethanol (mock) or CpdA (1␮M) for 24 hours were subjected to RNA
extraction and subsequent real-time RT-PCR for MMP-2, MMP-9, IL-6, and VEGF. Expression of each specific gene was normalized to that of
GAPDH and ␤-actin. Transcription amount is presented relative to that of mock treatment in TCCSUP or UMUC3-control-shRNA. Each value
represents the mean ϩ SD from at least 3 independent experiments. *, P Ͻ .05 (vs mock treatment in each cell line); **, P Ͻ .01 (vs mock
treatment in each cell line).
Figure 7. CpdA-induced interaction between NF-␬B and GR. A, Cell
lysates from UMUC3/TCCSUP cultured with ethanol (mock), 1␮M
CpdA, or 100nM DEX for 24 hours were immunoprecipitated with an
anti-NF-␬B antibody or IgG and then immunoblotted for GR. B,
TCCSUP/UMUC3 cell lines cultured with ethanol (mock), 1␮M CpdA,
or 10nM DEX for 24 hours were analyzed on Western blotting, using
an antibody to GR (95 kDa) or NF-␬B (65 kDa). GAPDH (37 kDa) served
as an internal control.

Antiandrogenic effects of CpdA
Using a reporter gene assay, antiandrogenic activity of
CpdA was assessed in bladder cancer cells. AR-mediated
transcriptional activity was determined in the cell extracts
with transfection of MMTV-Luc, as used for GR trans-
activation, and treatment with CpdA and/or a potent an-
drogen DHT. In UMUC3-GR-shRNA cells, DHT in-
creased Luc activity, and CpdA as well as an antiandrogen
HF, showing nonsignificant agonist activity, could block
DHT-induced AR transcription (Figure 8A). Subcellular
localization of AR was also examined in UMUC3 cells
treated with CpdA and/or DHT by Western blotting.
CpdA failed to prevent DHT-induced AR nuclear trans-
location (Figure 8B). However, CpdA by itself did not
induce nuclear translocation of endogenous AR in blad-
der cancer cells.
Discussion
Several GCs, such as DEX, prednisone, and hydrocorti-
sone, have clinically been used as cytotoxic agents, pre-
dominantly for lymphomas and castration-resistant pros-
tate cancer (29). GCs are also known to reduce acute
toxicity of other cytotoxic drugs, particularly hypereme-
sis during systemic chemotherapy, as well as to protect
normal tissue against their long-term effects (30). As a
result, GCs are often prescribed as comedication, without
expecting their antitumor activities, in patients with solid
tumor, including bladder cancer. We have recently dem-
onstrated that DEX strongly suppresses GR-positive
bladder cancer cell invasion and metastasis in vitro and in
vivo (2). Nonetheless, DEX was found to promote blad-
der cancer cell proliferation despite its induction of cell-
cycle arrest at G1 phase and inhibited antiproliferative
effects of cisplatin via prevention of apoptosis (2, 4). Sim-
ilar findings have been reported in other types of malig-
nancies, suggesting that DEX can
reduce the sensitivity of chemother-
apeutic agents (31–33). More re-
cently, we have found that cortico-
sterone and prednisone suppress
bladder cancer cell invasion without
promoting cell proliferation or re-
ducing cisplatin cytotoxicity (5).
Thus, in conjunction with the results
in our immunohistochemical studies
(2, 3), GC-mediated GR activation
is likely to associate with bladder
cancer regression. However, no GR
ligands have been shown to signifi-
cantly reduce the viability of bladder
cancer cells. In the current study, we
demonstrate that a dual GR/AR modulator CpdA
strongly inhibits bladder cancer cell proliferation, via in-
creasing both G1 phase population and apoptosis, as well
as its migration and invasion.
The action of GCs is often complex and is generally
dependent on a balance between transactivation and tran-
srepression of GR (23, 34). Therapeutic effects of GCs are
thought to be due to transrepression, whereas adverse
effects associated with GC therapy are often induced by
transactivation. However, none of natural or synthetic
GCs have been shown to produce only the beneficial
changes via transrepression without the negative effects
resulting from transactivation (34). Recent advances in
drug design and compound screening have enabled the
identification of “dissociated” GR ligands that selectively
modulate GR functions presumably via altering GR struc-
ture which is favorable for transrepression over transac-
tivation (23, 35). CpdA, with a GR binding affinity sim-
ilar to that of DEX (17), is such a dissociated compound
isolated from natural sources. Remarkably, the literature
data indicated that CpdA was unable to induce GR trans-
activation. For instance, no significant increase in the ex-
pression of a GR-dependent target FKBP51 was seen in
leukemia cells (26). We confirmed this in bladder cancer
cells by showing that CpdA did not enhance GRE reporter
activity as well as the expression of GILZ and FKBP51
genes. Previous investigation of molecular mechanisms
underlying the effect of CpdA further revealed that the
lack of transactivation by CpdA on GRE-driven promot-
ers correlated with its inability to provoke ligand-medi-
ated GR dimerization as well as GR phosphorylation at a
specific residue (eg, Ser211
) (18). Based on these findings,
it is anticipated that treatment with CpdA in vivo is asso-
ciated with fewer GC-induced side effects.
Again, CpdA is known to preferentially induce GR-
mediated transrepression the major mechanism of which
Figure 8. Effects of CpdA on AR. A, UMUC3-GR-shRNA cells were cotransfected with MMTV-
Luc and pRL-TK and subsequently cultured with ethanol (mock), DHT (1nM), CpdA (1␮M), and/or
HF (5␮M) for 24 hours. Luc activity is presented relative to that with mock treatment. Each value
represents the mean ϩ SD from at least 3 independent experiments. *, P Ͻ .05 (vs mock
treatment); #, P Ͻ .05 (vs DHT treatment). B, Nuclear protein fractions from UMUC3 cells
cultured with ethanol (mock), 1nM DHT, and/or 1␮M CpdA for 24 hours were analyzed on
Western blotting, using an antibody to AR (110 kDa). Histone 3 (15 kDa) served as an internal
control.

is believed to be the ability of the receptor to inhibit the
activity of other transcription factors, including NF-␬B
and AP-1. In prostate cancer cells, not only CpdA (28) but
also DEX (36) reduced their growth mainly via inhibiting
NF-␬B activation. We also previously demonstrated the
data suggesting that inactivation of NF-␬B and inhibition
of the production of NF-␬B-dependent cytokines, such as
IL-6, might be a central mechanism involved in DEX/GR-
mediated suppression of bladder cancer cell invasion (2).
Similarly, in the present study, we showed that CpdA
inhibited transcriptional activities of NF-␬B and AP-1 as
well as the expression of NF-␬B-regulated genes, includ-
ing MMP-2, MMP-9, IL-6, and VEGF, in GR-positive
cells. CpdA could also induce the interaction between GR
and NF-␬B. It is thus likely that CpdA treatment leads to
a shift of GR functions toward transrepression in bladder
cancer cells. However, the reduction of MMP-2/MMP-9/
IL-6/VEGF gene expression by CpdA might simply rep-
resent its inhibitory effect on cell invasion rather than GR
transrepression, although nonsignificant and significant
inhibitions of these genes’ expression (Figure 6D) and cell
invasion (Figure 3B), respectively, were seen in CpdA-
treated AR-positive/GR knockdown UMUC3. Androgen
deprivation has been shown to inhibit the expression of
cell invasion-related genes, such as MMP-9 and VEGF, in
bladder cancer cells (8, 14). Indeed, the expression levels
of these 4 genes were slightly lower in mock-treated
UMUC3-AR-shRNA than in mock-treated UMUC3-con-
trol-shRNA (12%–25% decreases) (Figure 6D). None-
theless, in the current transwell assay (Figure 3B), there
was no significant effect of AR knockdown on bladder
cancer cell invasion. Meanwhile, it was noteworthy that
the suppressive effects of CpdA on bladder cancer cell
invasion (eg, 61% decrease in UMUC3-control-shRNA)
were found to be even stronger than those of DEX, cor-
ticosterone, or prednisone in the transwell assays per-
formed under the same conditions (eg, 34%–50% de-
creases) (2, 5).
Cytotoxic and proapoptotic activities of CpdA have
been related to its effects on GR functions, such as its
expression and nuclear translocation, in addition to
transactivation and transrepression. Underlying mecha-
nisms for GC-induced apoptosis may also vary depending
on cell type. GC treatment has been shown to promote
apoptosis in several types of malignancies, including leu-
kemia, osteosarcoma, lung small cell carcinoma, and
prostate cancer (36, 37), whereas it inhibits apoptosis in
other types, such as breast cancer (38) and fibrosarcoma
(39). DEX treatment is also associated with inhibition of
apoptosis of bladder cancer cells in our previous (2, 5)
and current studies. It has been suggested that the levels of
GR expression and GR-mediated transactivation play an
important role in determining the promotion of apoptosis
vs survival in a cell-specific manner (37, 38). In our study,
DEX is found to induce both transactivation and transre-
pression of GR and reduces its expression in bladder can-
cer cells, whereas CpdA induces only GR transrepression
and does not modify GR expression. These differences
may have contributed to the distinct effects of DEX vs
CpdA as GR ligands on bladder cancer cell proliferation,
whereas both compounds similarly induce G1 arrest. Ad-
ditionally, down-regulation of GR expression by DEX
has been implicated in a limitation of GCs with long-term
use. In this respect, CpdA inducing no significant changes
in GR expression may also be superior to DEX.
CpdA has been found to antagonize androgen actions
via mechanisms similar to those for classical antiandro-
gens, such as flutamide (16, 28). Although CpdA inhibits
androgen-enhanced AR transactivation, it possesses par-
tial agonist activity and induces nuclear translocation of
AR in prostate cancer cells (28). Indeed, clinically used
antiandrogens, including flutamide and bicalutamide,
failed to prevent androgen-induced AR nuclear translo-
cation (40, 41). We demonstrated in bladder cancer cells
that CpdA restored DHT-induced AR transcription yet
did not affect DHT-mediated AR nuclear translocation.
However, in contrast to the observations in prostate can-
cer (28), CpdA appeared to have only nonsignificant ag-
onist activity in bladder cancer cells without inducing AR
nuclear translocation. Thus, CpdA was confirmed to act
as an AR antagonist in bladder cancer cells. More impor-
tantly, the inhibitory effects of CpdA on bladder cancer
growth were invariably stronger in control cells than in
AR-silencing cells stably expressing AR-shRNA and were
still seen in GR-silencing cells stably expressing GR-
shRNA. Because stable expression of GR-shRNA in blad-
der cancer cells does not completely knock down endog-
enous GR (2), there is a possibility that residual GR in the
cells may have contributed to the latter effect. Nonethe-
less, AR-mediated pathway is still thought to involve the
inhibition of bladder cancer cell proliferation, migration,
and invasion by CpdA, which further supports the role of
AR signals in bladder cancer progression.
Using mouse xenograft models for bladder cancer, we
showed in vivo evidence suggesting that CpdA inhibited
the progression of bladder cancer via both GR and AR
pathways. CpdA was also found to more significantly
suppress the growth of GR-positive and/or AR-positive
xenografts than DEX or HF. Our previous immunohisto-
chemical studies in bladder tissue microarrays revealed
that most (eg, 87%) of bladder cancers expressed the GR
(3), whereas AR positivity in tumors correlated with dis-
ease progression in patients with muscle-invasive bladder
cancer (13, 42). Therefore, CpdA has the potential to

efficiently regress tumor growth, especially in male pa-
tients with GR-positive/AR-positive bladder cancer
whose androgen levels are not low, and its effects may be
more beneficial than GCs/pure GR ligands or AR antag-
onists. However, the current study has compared the ef-
ficacy of CpdA with that of only one compound each (ie,
DEX and HF) in tumor growth. Moreover, we injected
each drug 3 times a week. Although no data appears to be
available about the half-life of each compound when in-
jected peritumorally in mice, potentially different half-
lives that are probably less than 48 hours may have af-
fected the results from our xenograft study.
In conclusion, CpdA was found to inhibit bladder can-
cer growth predominantly via inducing GR-mediated
transrepression as well as at least partially via inactivating
androgen-enhanced AR signals. The current data also
support previous observations indicating that bladder
cancer cells possess functional GR and AR. Further study
to extensively compare the antitumor activities of this
unique compound vs various GR ligands and AR antag-
onists in bladder cancer is required.
Acknowledgments
Address all correspondence and requests for reprints to: Hiroshi
Miyamoto, MD, PhD, The James Buchanan Brady Urological
Institute at the Johns Hopkins Hospital, 600 North Wolfe
Street, Marburg 148, Baltimore, MD 21287. E-mail:
hmiyamo1@jhmi.edu.
This work was supported part by the National Natural Sci-
ence Foundation of China Grant NFSC 81202022 and the Basic
Research Program of the Department of Education of Zhejiang
Province Grant Y201225369 (to Y.Z.).
Disclosure Summary: The authors have nothing to disclose.
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