The R-enantiomer of the nonsteroidal antiinflammatory drug etodolac binds retinoid X receptor and induces tumor-selective apoptosis

The Burnham Institute, La Jolla, CA 92037, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2005; 102(7):2525-30. DOI: 10.1073/pnas.0409721102
Source: PubMed
Prostate cancer is often slowly progressive, and it can be difficult to treat with conventional cytotoxic drugs. Nonsteroidal antiinflammatory drugs inhibit the development of prostate cancer, but the mechanism of chemoprevention is unknown. Here, we show that the R-enantiomer of the nonsteroidal antiinflammatory drug etodolac inhibited tumor development and metastasis in the transgenic mouse adenocarcinoma of the prostate (TRAMP) model, by selective induction of apoptosis in the tumor cells. This proapoptotic effect was associated with loss of the retinoid X receptor (RXRalpha) protein in the adenocarcinoma cells, but not in normal prostatic epithelium. R-etodolac specifically bound recombinant RXRalpha, inhibited RXRalpha transcriptional activity, and induced its degradation by a ubiquitin and proteasome-dependent pathway. The apoptotic effect of R-etodolac could be controlled by manipulating cellular RXRalpha levels. These results document that pharmacologic antagonism of RXRalpha transactivation is achievable and can have profound inhibitory effects in cancer development.


Available from: Michele Bernasconi
-enantiomer of the nonsteroidal
antiinflammatory drug etodolac binds retinoid X
receptor and induces tumor-selective apoptosis
Siva Kumar Kolluri*
, Maripat Corr
, Sharon Y. James*
, Michele Bernasconi*
, Desheng Lu
**, Wen Liu*,
Howard B. Cottam
**, Lorenzo M. Leoni
, Dennis A. Carson
, and Xiao-kun Zhang*
*The Burnham Institute, La Jolla, CA 92037; and
Department of Medicine and **The Rebecca and John Moores Cancer Center, University of California at
San Diego, La Jolla, CA 92093
Contributed by Dennis A. Carson, December 30, 2004
Prostate cancer is often slowly progressive, and it can be difficult
to treat with conventional cytotoxic drugs. Nonsteroidal antiin-
flammatory drugs inhibit the development of prostate cancer, but
the mechanism of chemoprevention is unknown. Here, we show
that the R-enantiomer of the nonsteroidal antiinflammatory drug
etodolac inhibited tumor development and metastasis in the trans-
genic mouse adenocarcinoma of the prostate (TRAMP) model, by
selective induction of apoptosis in the tumor cells. This proapo-
ptotic effect was associated with loss of the retinoid X receptor
) protein in the adenocarcinoma cells, but not in normal
prostatic epithelium. R-etodolac specifically bound recombinant
, inhibited RXR
transcriptional activity, and induced its
degradation by a ubiquitin and proteasome-dependent pathway.
The apoptotic effect of R-etodolac could be controlled by manip-
ulating cellular RXR
levels. These results document that pharma-
cologic antagonism of RXR
transactivation is achievable and can
have profound inhibitory effects in cancer development.
cancer prostate R-etodolac ubiquitin chemoprevention
everal epidemiological studies have shown that the use of
nonsteroidal antiinflammatory drugs (NSAIDs) is associated
with a reduced incidence of clinically detectable prostate cancer
(1–3). The side effects of cyclooxygenase (COX) inhibitors pre-
clude the use of these agents in many elderly men (4). Thus, there
is a major need to determine whether there are chemopreventative,
and antimetastatic effects of NSAIDs that can be separated from
COX inhibition. Controversy has permeated this field, because
many of the COX-independent actions of NSAIDs are measurable
only at concentrations that are not safely achievable in vivo, and
because the members of this structurally diverse group of drugs are
metabolized extensively and can exert different mechanisms of
action (5, 6).
Various NSAIDs have been demonstrated to induce apoptosis in
malignant cells (7–9). Etodolac is a commercially available NSAID
containing a racemic mixture, in which the S-enantiomer has COX
inhibitory activity, whereas the R-enantiomer does not (10). Unlike
all other chiral NSAIDs, the two enantiomers of etodolac are not
metabolically interconvertible. Moreover, the R-enantiomer is me-
tabolized much more slowly than the S-enantiomer, and it accu-
mulates to 10-fold higher concentrations than the S-enantiomer in
plasma (11). In a recent study, sufficient plasma levels of R-etodolac
were achieved after oral gavage in a xenograft prostate cancer
model to diminish the growth of the transplanted tumor (12).
The in vivo effect of R-etodolac was associated with enhancement
of peroxisome proliferator-activated receptor
) transac-
tivation (12). PPAR
, as well as other nuclear hormone receptors,
forms heterodimers with the retinoid X receptor
) (13,
14), which has been implicated in the pathogenesis of prostate
cancer (15). The effect of RXR
may be due to its induction of
apoptosis through its interaction with other proteins (16–19). An
activating ligand of RXR
,9-cis-retinoic acid (RA), is a lipophilic
acid similar to R-etodolac. Thus, we hypothesized that the COX-
independent effects of R-etodolac in malignant prostate cells might
be attributed to its binding and modulation of RXR
Here, we show that R-etodolac induced apoptosis of prostate
cancer cells, but not normal prostatic epithelial cells. The R-
etodolac-induced apoptosis was associated with reduction of RXR
levels selectively in the tumor cells. Direct interaction of R-etodolac
and RXR
was demonstrated by in vitro binding of radiolabeled
R-etodolac with the purified recombinant ligand-binding domain
(LBD) of RXR
and the ability of the drug to protect RXR
protein from trypsin digestion. In intact cells, R-etodolac antago-
nized RXR
transcriptional activity and induced its ubiquitination
and degradation. Furthermore, suppre ssion of RXR
expre ssion
reduced the apoptotic effect of R-etodolac. These results demon-
strate that RXR
acts as a receptor that mediates the COX-
independent anticancer effects of R-etodolac.
Materials and Methods
Drug Preparation. R-etodolac was prepared from pharmaceutical-
grade tablets of racemic etodolac to a purity of 97% as described
(see Supporting Materials and Methods, which is published as
supporting information on the PNAS web site) (20). The drug was
tritiated by Sibtech (Newington, CT) and purified with HPLC. The
resulting material had a specific activity of 20–25 Ci (1 Ci 37
GBq)mmol, and it was stored in acetonitrile at a concentration of
0.45 mCiml at 20°C. The RXR-selective retinoids SR11237,
SR11345, and SR11246 were described in ref. 21 and provided by
M. Dawson (The Burnham Institute). Staurosporine, MG-132
(Calbiochem) and 9-cis-RA (Sigma) were purchased commercially.
Cell Lines. LNCaP, PrEC, CV-1, ZR-75–1, and HEK 293T cells were
maintained in standard media. The F9 murine embryonal carci-
noma cell line with both alleles of RXR
disrupted was provided
by P. Chambon (Institut de Genetique et de Biologie Moleculaire
Freely available online through the PNAS open access option.
Abbreviations: HA, hemagglutinin; NSAID, nonsteroidal antiinflammatory drug; COX,
cyclooxygenase; RXR, retinoid X receptor; TRAMP, transgenic mouse adenocarcinoma of
the prostate; LBD, ligand-binding domain; PPAR
, peroxisome proliferator-activated re-
; RA, retinoic acid; CAT, chloramphenicol transferase; siRNA, small interfering RNA;
RAR, RA receptor; RARE, RA-response element.
S.K.K. and M.C. contributed equally to this work.
H.B.C., M.C., L.M.L., and D.A.C. were consultants for Salmedix, Inc., which is a biotechnol-
ogy company that is developing R-etodolac for the treatment of cancer. L.M.L. is currently
employed by Salmedix.
Present address: Cancer Research UK Medical Oncology Unit, Queen Mary’s University of
London, London EC1M 6BQ, England.
Present address: Division of Oncology and Infectiology, University Children’s Hospital
Zurich, August-Forel Strasse 1, 8008 Zurich, Switzerland.
Present address: Salmedix, Inc., 9380 Judicial Drive, San Diego, CA 92122.
To whom correspondence may be addressed. E-mail: or xzhang@
© 2005 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0409721102 PNAS
February 15, 2005
vol. 102
no. 7
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et Cellulaire, College de France, Illkirch, France) (22). See Sup-
porting Materials and Methods for details.
Murine Studies. Transgenic mouse adenocarcinoma of the prostate
(TRAMP) and C57BL6 mice were purchased from The Jackson
Laboratory and bred at UCSD. All animal protocols received prior
approval by the institutional review board. Plasma R-etodolac levels
were measured on a group of seven 15-week-old C57BL6 male
mice who were fed R-etodolac (1.25 mgkg) chow for 2 weeks by a
bioanalytical LCMS-based method developed by Maxxam Ana-
lytics (Mississauga, ON, Canada). Chiral HPLC (23) was used to
confirm the lack of R-toS-etodolac in vivo interconversion.
We started 46 male TRAMP mice at 9–12 weeks of age on chow
with R-etodolac 1.25 mgkg or control food (prepared by Dyets,
Bethlehem, PA) randomized by cage. At 30 weeks, or after ap-
pearance of a gross palpable tumor mass, the animals were sacri-
ficed and necropsies were performed. The urogential system, the
periaortic lymph nodes, and the major organs were removed and
weighed. The prostatic tissues were dissected and separated into
individual lobes and weighed. Tissues were fixed in 10% formalin
embedded in paraffin, sectioned in step sections at 50-
m intervals,
and stained with hematoxylin and eosin. The prostate sections were
scored for carcinoma grade on a 1–6 scale (see Supporting Materials
and Methods). The liver, lung, and lymph node sections were scored
for the pre sence or absence of tumor. The weights of the different
tissues, and the frequencies of metastases, in the drug treated and
control animals were compared by the Mann–Whitney test or
Fisher’s exact test, with P 0.05 considered significant.
Ligand Binding. The human RXR
LBD (223–462), prepared as a
polyhistidine-tagged fusion protein in pET15b (Novagen) (1
was incubated with radiolabeled ligand in the presence of different
concentrations of unlabeled 9-cis-RA or R-etodolac at 4°C for 14 h.
LBD was captured by nickel-coated beads. Bound
radiolabeled ligand was determined in a scintillation counter.
Immunohistochemistry and Apoptosis Assays. For 2 weeks, we fed 6-
to 7-month-old TRAMP mice R-etodolac supplemented or control
chow. The prostates were removed, and serial frozen sections were
assayed for terminal deox yribonucleotidyl transferase (TdT;
TUNEL; Chemicon) or stained with anti-human RXR
Santa Cruz Biotechnology), followed by staining with DAPI (50
gml; Sigma) containing DNase-free RNase A (100
Boehringer Mannheim) to visualize the nuclei and examined by
fluorescence microscopy (18, 19, 24). Single-cell apoptosis was
detected in vitro by removing adherent cells from the plate with 5
mM EDTA, incubating them with annexin-V–phycoerythrin (BD
PharMingen) and analyzing by flow cytometry.
Transient-Transfection Assay. Expre ssion vectors for RXR
), hemagglutinin (HA)-ubiquitin, and reporter
RARE-tk-chloramphenicol transferase (CAT) were pre-
pared and transfected as described (25, 26). We mixed 300 ng of
reporter plasmid, 50 ng of
-gal expression vector (pCH 110;
Pharmacia), and vector expressing RXR
with carrier DNA
(pBluescript) to give 1.0
g of total DNA per well. CAT activity was
normalized for transfection efficiency on the basis of cotransfected
-gal gene activity. Transfected cell lysates were separated by
SDSPAGE and immunoblotted (see Supporting Materials and
Small Interfering RNA (siRNA) Transfections. A SMARTpool of
siRNAs specific for RXR
and GFP control siRNA were puchased
Table 1. Incidence of primary tumors and metastases
Measurement Control Treated
Primary tumor incidence* 2424 (100%) 1617 (94%)
Metastasis incidence
1424 (58%) 517 (29%)
Animals with gross masses
624 (25%) 217 (12%)
Percentages of mice are given in parentheses.
*No. of mice with histologic evidence of carcinoma (grade 4).
No. of mice found to have histologic evidence of metastasis to lymph node,
lung, or liver tissues. P 0.05 by Fischer’s exact test.
No. of mice found to have a gross urogenital mass at postmortem
Fig. 1. Inhibition of prostate cancer progression in the TRAMP model. Male TRAMP mice were fed control chow or chow with R-etodolac (1.25 mgkg). (a)At
30 weeks of age, or after development of a gross palpable mass, the mice were sacrificed, and the urogenital systems were removed and weighed. (bf ) The
prostate lobes (b) were separated from the other organs and weighed separately; anterior (c), ventral (d), lateral (e), and posterior (f). The prostates were not
dissectible in mice that had gross tumor masses (two in the treatment group and six in the control group). The weights of the control urogenital tracts, lateral,
and dorsal prostates were significantly higher than those of the treated group, as determined by the Mann–Whitney test.
www.pnas.orgcgidoi10.1073pnas.0409721102 Kolluri et al.
Page 2
from Dharmacon. A 10-
l aliquot of 20
M siRNA per well was
transfected into cells in six-well plates by using Lipofectamine Plus
(Invitrogen) (17).
Antiproliferative and Proapoptotic Effects of
-Etodolac. R-etodolac
dose dependently inhibited the proliferation of LNCaP prostate
cancer cells and PrEC normal human prostatic epithelial cells (see
Fig. 7, which is published as supporting information on the PNAS
web site). At 72 h, the ID
values were 150
M for LNCaP,
compared with 400
M for PrEC (Fig. 7a). Concentrations of
R-etodolac of 500
M induced apoptosis in primary prostate
cancer explants (Fig. 7 be). In the latter instance, the malignant
cells displayed shrunken and pyknotic nuclei, whereas the nuclei
from the adjacent normal prostatic epithelium appeared morpho-
logically normal.
Activity of
-Etodolac in the TRAMP Model. Preliminary dose-ranging
pharmacokinetic data showed that plasma concentrations of 370
M could be achieved by supplementing a standard mouse chow
diet with 1.25 mgkg R-etodolac for 2 weeks. Chiral HPLC revealed
no detectable conversion of R-etodolac to the S-stereoisomer.
Therefore, in vivo experiments in the TRAMP mouse model were
undertaken under these conditions. Male TRAMP mice develop
histological intraepithelial neoplasia of the prostate by 8–12 weeks
of age that progresses to adenocarcinoma with distant site metas-
tase s by 24–28 weeks of age (27, 28). Control chow or diets
supplemented with R-etodolac were initiated at 9–12 weeks. By 30
weeks, nearly all of the prostates in the R-etodolac treated and
untreated groups had macroscopic evidence of tumor (Table 1).
However, both the average tumor mass (Fig. 1) and the frequencies
of metastases (Table 1) were significantly lower in the R-etodolac-
treated animals. Histological evaluation of the excised tissues
confirmed the anti-metastatic effects of the drug and did not show
evidence of drug toxicity (Fig. 2 al). Collectively, these data
indicated that R-etodolac retarded the progression and metastasis
of prostate cancer in the TRAMP system.
-Etodolac Selectively Induces Apoptosis in Cancerous Prostates. To
determine whether R-etodolac treatment resulted in apoptosis in
vivo, 6- to 7-month-old male TRAMP and nontransgenic litter-
mates were fed with R-etodolac or control chow for 2 weeks and
Fig. 2. Histological evaluation of R-etodolac-treated TRAMP prostate tis-
sues. Examples of ventral (a), dorsal (c), and lateral (e) lobes of the prostate in
an untreated 30-week-old TRAMP mouse are shown. For comparison, histol-
ogy sections of the ventral (b), dorsal (d), and lateral ( f) prostate from an
R-etodolac treated TRAMP mouse at 30 weeks of age are shown. Metastases
in the lymph-nodes (g), lungs (i), and liver (k) of an untreated TRAMP mouse
at 30 weeks of age are shown also. Examples of lymph nodes (h), lung (j), and
liver (l)inR-etodolac treated TRAMP mice at 30 weeks of age are shown also.
(m) Induction of tumor cell apoptosis by R-etodolac. For 2 weeks, we fed 6- to
7-month-old TRAMP mice with R-etodolac-supplemented chow or control
chow, and they were then sacrificed. The prostate lobes were removed and
frozen in OCT, sectioned, and subjected to TUNEL staining (red) and DAPI (to
visualize nuclei) (200). Extensive apoptosis (TUNEL-positive) was detected in
the prostates of R-etodolac-fed TRAMP mice compared with control chow-fed
Fig. 3. RXR
is required for R-etodolac-induced apoptosis. (a) Inhibition of
expression by RXR
siRNA. LNCaP cells were untransfected or trans-
fected with a pool of RXR
siRNAs or control GFP siRNA for 48 h. Cell lysates
were analyzed by immunoblotting with antibodies against RXR
(b) RXR
siRNA suppresses the apoptotic effect of R-etodolac. LNCaP cells were
transfected with RXR
or control GFP siRNA for 48 h and then treated with 500
Mor1mMR-etodolac for 24 h in medium containing 0.5% FBS. Apoptotic
cells were quantified by annexin-V binding and flow cytometry. Similar results
were obtained in two separate experiments. The percentage of apoptosis
represents the percentage of annexin-V-positive drug-treated cells minus the
percentage of annexin-V-positive untreated transfected cells. (c) Expression of
confers apoptotic sensitivity of R-etodolac in CV-1 cells. CV-1 cells were
transfected with GFP or GFP-RXR
in six-well plates. After overnight transfec-
tion, cells were treated with either 0.1% DMSO (vehicle) or 1 mM R-etodolac
for 28 h, harvested, and fixed, and nuclei were stained by DAPI. Nuclear
morphology of the GFP-positive cells was visualized by fluorescence micros-
copy and cells showing nuclear condensation and fragmentation were scored
as apoptotic cells. Shown are averages means from two independent
evaluations of at least 200 transfected cells. (d) RXR
F9 cells are resistant
to the apoptotic effect of R-etodolac. F9 cells or F9 cells without RXR (F9
cellsRXR) were treated with DMSO or R-etodolac as indicated. Apoptosis was
determined after 44 h of treatment. Bars represent mean SD from three
independent experiments.
Kolluri et al. PNAS
February 15, 2005
vol. 102
no. 7
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sacrificed, and the prostates were examined for apoptosis. Extensive
apoptosis in the prostates of drug-treated TRAMP mice was seen
on TUNEL staining of frozen sections, whereas no apoptosis was
seen in mice fed with control chow (Fig. 2m). No apoptosis was
detectable in prostates of nontransgenic mice fed with R-etodolac.
Is Required for the Apoptotic Effect of
-Etodolac. A previous
study demonstrated that PPAR
was associated with the apoptosis
induced by R-etodolac (12). PPAR
heterodimerizes with RXR
and thus, we examined its possible role. Transfection of RXR
specific siRNA, but not control siRNA, almost completely inhibited
expre ssion in LNCaP cells (Fig. 3a). The apoptotic effect of
R-etodolac in RXR
siRNA-transfected cells was substantially
diminished (Fig. 3b). CV-1 cells lack detectable levels of RXR
26), and they were relatively resistant to R-etodolac-induced apo-
ptosis. However, transfection of CV-1 cells with RXR
, but not a
control vector, reversed this drug-resistant phenotype (Fig. 3c).
Similarly RXR
F9 embryonal cells did not undergo apoptosis
with drug treatment compared with the extensive apoptosis in
wild-type F9 cells (Fig. 3d). Thus, RXR
is required for the
apoptotic effect of R-etodolac.
-Etodolac Binds to RXR
. To investigate whether R-etodolac c ould
bind RXR
, a ligand competition assay with [
H]9-cis-RA was
used. Both unlabeled 9-cis-RA and R-etodolac displaced [
cis-RA bound to RXR
LBD (Fig. 4a), with an IC
value of
M for R-etodolac. Furthermore, [
H]R-etodolac directly
bound the RXR
LBD, and this binding was competitively
inhibited by both unlabeled R-etodolac and 9-cis-RA (Fig. 4 b
and c).
-Etodolac Binding Induces Conformational Change in RXR
. Binding
of ligands to their receptors often induces changes in susceptibility
to proteolysis (13, 14). Dige stion of the RXR
LBD with a low
concentration of trypsin (3
gml) yielded a proteolytic fragment
of 20 kDa, whreeas higher concentrations of trypsin (10 or 30
gml) completely digested the LBD (see Fig. 8, which is published
as supporting information on the PNAS web site). Preincubation of
the RXR
LBD with 9-cis-RA did not alter its sensitivity to trypsin
digestion, consistent with previous studies (29). However, incuba-
Fig. 4. R-etodolac binds to RXR
.(a) R-etodolac competes with 9-cis-RA for
binding to RXR
. Human RXR
LBD was incubated with 1 nM [
H]9-cis-RA in
the presence of different concentrations of unlabeled 9-cis-RA (
etodolac (
) at 4°C for 14 h. [
H]9-cis-RA bound to RXR
was measured after
capturing the RXR
LBD by nickel-coated beads. The data represent the
average of total bound cpm SEM. One of five experiments is shown. (b)
R-etodolac binds directly to RXR
. The indicated concentrations of [
etodolac were incubated with or without polyhistidine-tagged-RXR
-bound [
H]R-etodolac was separated with nickel-coated beads and
measured. One of three independent experiments is shown. (c)9-cis-RA
competes with R-etodolac for binding of RXR
. Purified RXR
LBD (1
g) was
incubated with 1 mM [
H]R-etodolac in the presence of unlabeled 9-cis-RA
M) or R-etodolac (1 mM) as indicated. RXR
-bound [
H]R-etodolac was
separated and assayed as described above.
Fig. 5. R-etodolac inhibits 9-cis-RA-induced transcriptional activity of RXR
(a) Inhibition of RXR
homodimer activity by R-etodolac. CV-1 cells were
cotransfected with or without an expression vector for RXR
(25 ng), a CAT
reporter vector containing RXR homodimer responsive elements [(TREpal)
tk-CAT; 300 ng] and a
-gal expression vector (50 ng). After subsequent
treatment with 9-cis-RA (10
M) and the indicated concentrations of R-
etodolac for 24 h CAT activities were determined and normalized relative to
-gal activity. (b) R-etodolac modulation of RXR
RAR heterodimer ac-
tivity. ZR-75-1 cells were transfected with
RARE-tk-CAT reporter plasmid (300
ng) and a
-gal expression vector (50 ng), and they were then treated with
all-trans-RA (10
M) and the indicated concentrations of R-etodolac. CAT
activities were determined as described above. (c) Inhibition of trans-RA-
induced RAR
protein expression by R-etodolac. ZR-75-1 cells were treated
with or without trans-RA (10
M), R-etodolac (1 mM), or their combination
for 24 h. RAR
protein expression was determined by Western blot analysis. (d)
Inhibitory effect of R-etodolac on RXR
heterodimer activity. ZR-75-1
cells were transfected with
RARE-tk-CAT reporter plasmid (300 ng) and a
-gal expression vector (50 ng), treated with RXR ligand SR11237 (10
M) and
ligand ciglitazone (10
M) as well as R-etodolac and then CAT activities
were determined. (e) R-etodolac inhibits RAR
protein expression induced by
the combination of RXR and PPAR
ligands. ZR-75-1 cells were treated with or
without SR11237 (10
M), ciglitazone (10
M), R-etodolac (1 mM) in the
indicated combinations. Cell lysates were immunoblotted and probed for
relative levels of RAR
www.pnas.orgcgidoi10.1073pnas.0409721102 Kolluri et al.
Page 4
tion of the RXR
LBD with R-etodolac before trypsin digestion (3
gml) resulted in a different digestion pattern, with two new
proteolytic fragments of 18 kDa, in contrast to the lack of
protection detected with PPAR
(Fig. 8).
-Etodolac Modulates RXR
Transcriptional Activity. To test whether
R-etodolac binding modulated RXR
transcriptional activity, a
reporter gene containing RXR
homodimer-responsive elements,
-tk-CAT (26), was transfected with a RXR
expre ssion
vector into CV-1 cells. Treatment of cells with 9-cis-RA strongly
induced reporter gene activity, whereas treatment with R-etodolac
did not (Fig. 5a). However, when transfected cells were treated with
9-cis-RA, the addition of R-etodolac dose dependently reduced the
transcriptional activity of RXR
We investigated whether R-etodolac inhibited transactivation of
endogenous RXR
. An RA-response element (
RARE) in the
promoter, which binds various RXR-containing het-
erodimers including RXRRAR and RXRPPAR
(26, 30, 31),
was transfected into ZR-75–1 breast cancer cells. Reporter activity
was induced by all-trans-RA, presumably because of binding of
endogenous RXRRAR heterodimer. Cotreatment with R-
etodolac suppressed all-trans-RA-induced reporter activity in a
R-etodolac concentration-dependent manner, suggesting that R-
etodolac inhibited transcriptional activity of endogenous RXR
RAR heterodimers (Fig. 5b). The transcriptional effect of R-
etodolac on RXR
was confirmed by analyzing its effect on the
expre ssion of RAR
. Treatment of ZR-75-1 cells with all-trans-RA
strongly induced the endogenous expression of RAR
(Fig. 5c),
which was completely inhibited by R-etodolac, consistent with the
inhibitory effect of R-etodolac on the
RARE reporter gene (Fig.
5b). To determine the effect of R-etodolac on RXRPPAR
heterodimer activity, ZR-75-1 cells were stimulated with the
ligand ciglitazone and the RXR
ligand SR11237 in the
presence or absence of R-etodolac (Fig. 5 d and e) (30). The
induction of endogenous RAR
expre ssion by the two drugs in
combination was abolished by R-etodolac cotreatment (Fig. 5e).
Loss of RXR
Expression After
-Etodolac Treatment. Subcellular
localization of RXR
plays a role in the regulation of apoptosis
(17). To analyze whether R-etodolac treatment altered RXR
subcellular localization, in vivo immunostaining of RXR
performed on the prostate tissue s from male TRAMP and non-
transgenic littermates fed with R-etodolac or control chow. RXR
was predominantly localized in the nucleus in the prostates of the
nontransgenic mice fed with or without R-etodolac chow (Fig. 6a).
TRAMP mice fed with control chow displayed similar RXR
nuclear staining. However, RXR
staining was greatly reduced in
prostate s of TRAMP mice fed with R-etodolac.
protein levels in LNCaP cells were also markedly reduced
after treatment with R-etodolac (Fig. 6b). R-etodolac-induced
degradation of RXR
levels was completely prevented by the
proteosome inhibitor MG132 (Fig. 6b). Proteins are often ubiqui-
tinated before degradation by proteasomes (32). Thus, we deter-
mined whether R-etodolac induced ubiquitination of RXR
. Myc-
tagged RXR
was cotransfected into HEK 293T cells with or
without an expression vector for HA-tagged ubiquitin, followed by
treatment with R-etodolac in the presence of MG132. Immuno-
precipitation with anti-myc antibody, followed by immunoblotting
with an anti-HA antibody, revealed that RXR
was extensively
ubiquitinated after R-etodolac treatment (Fig. 6c) but not after
treatment with the synthetic RXR
ligands SR11345 and SR11246
(Fig. 6d). Instead, these ligands abrogated R-etodolac-induced
ubiquitination (Fig. 6e), probably because of their compe-
tition for binding to RXR
. Collectively, these results demonstrate
that R-etodolac binds RXR
and induces its degradation in a
proteasome-dependent manner.
The standard therapy for progre ssive prostate cancer is androgen
ablation. However, many patients become unre sponsive and de-
velop metastatic disease (33). Thus, there is a compelling need for
the development of unconventional agents that can delay the
progression of prostate cancer. In this article, we report that chronic
oral administration of the COX-inactive R-stereoisomer of the
common NSAID etodolac inhibited tumor expansion and metas-
Fig. 6. R-etodolac induces RXR
degradation. (a) Diminished RXR
in prostate of R-etodolac-fed TRAMP mice. For 2 weeks, we fed 6- to 7-month-
old TRAMP mice R-etodolac-supplemented chow or control chow. The pros-
tate tissues were immunostained for human RXR
(red), and the nuclei were
stained with DAPI (200). (b) R-etodolac induces RXR
degradation. LNCaP
cells were untreated, treated with 1 mM R-etodolac for 18 h, treated with
R-etodolac with pretreatment for 1 h with 20
M MG132, or treated with
MG132 alone. Cell lysates were immunoblotted with antibodies to human
-tubulin. (c) R-etodolac induces ubiquitination of RXR
. Expression
vectors for myc-tagged RXR
or RAR
, and ubiquitin (HA-tagged) were trans-
fected into HEK 293T cells. After 24 h, the cells were treated with R-etodolac
(1 mM), lysed after 24 h, and immunoprecipitated with an anti-myc antibody.
The precipitated proteins were immunoblotted with an anti-HA antibody to
detect ubiquitinated protein. (d) R-etodolac induces ubiquitination of endog-
enous RXR
. HEK 293T cells were transfected with a vector expressing HA-
ubiquitin and then treated with R-etodolac as described above and 1
synthetic retinoids as indicated. The lysates were immunoprecipitated with an
anti-HA antibody and immunoblotted with an antibody to detect RXR
Synthetic RXR
ligands inhibit R-etodolac-induced ubiquitination of RXR
Expression vectors for RXR
(myc-tagged) and ubiquitin (HA-tagged) were
transfected into HEK 293T cells. Cells were treated with 1
M synthetic
retinoids, SR11237, SR11345, or SR11246, as indicated. Cell lysates were im-
munoprecipitated with an anti-myc antibody and the precipitated proteins
were immunoblotted with an anti-HA antibody to detect ubiquitinated
Kolluri et al. PNAS
February 15, 2005
vol. 102
no. 7
Page 5
tasis in the TRAMP model. By analogy, R-etodolac could be a
prospective agent for the treatment of human prostate cancer.
In the TRAMP model, treatment with the COX-2 selective agent
celecoxib or the R-enantiomer of the NSAID flurbiprofen resulted
in a significantly lower primary-tumor incidence and a reduced
incidence of metastases (34, 35). However, both of these drugs may
have exerted their effect by active COX inhibition because 15% of
the R-flurbiprofen was converted to the active COX inhibitor
S-flurbiprofen by 2–4 h after administration. In contrast, the
stereoisomers of the conformationally rigid etodolac molecule,
unlike all other approved racemic NSAIDs, cannot undergo chiral
transformation under physiologic conditions. Indeed, S-etodolac
was undetectable in the plasmas of the mice given diets supple-
mented with the R-stereoisomer. Hence, the cytostatic and anti-
metastatic effects of R-etodolac in the TRAMP model must be
attributed to the drug or to a metabolite.
The results presented here reveal an unexpected function of
as a mediator of the apoptotic effect of R-etodolac. A recent
study (12) demonstrated that inhibition of prostate tumor growth
by R-etodolac was associated with initial enhancement of PPAR
transcriptional activity, followed by degradation of the receptor
(12). However, ligand competition and direct binding assays using
recombinant protein failed to demonstrate any direct
binding of R-etodolac to PPAR
(data not shown). Because
activity depends on heterodimerization with RXR
possible that modulation of PPAR
and degradation by R-etodolac
is mediated by its binding to RXR
. Antagonists of RXR ho-
modimers are known to function as agonists of RXRPPAR
heterodimers (36, 37).
Exactly how RXR
mediates the apoptotic effects of R-etodolac
remains unknown. It is unlikely that R-etodolac exerts its anticancer
effect through its inhibition of RXR
transactivation (Fig. 5)
because many RXR
agonists potently inhibit the growth of
prostate cancer cells. However, the binding of RXR
by R-etodolac
could affect the function and stability of several nuclear receptors
that dimerize with RXR
, including PPAR
and Nur77.
Our results demonstrate that R-etodolac induced apoptosis of
prostate cancer, but not normal epithelium (Fig. 2 and 7). The
contrasting effects might be attributable to differences in RXR
posttranslational processing in cancer and normal cells (38). In this
regard, it was reported recently that RXR
was phosphorylated by
MAP kinase in surgically resected hepatocellular carcinoma
samples but not in the corre sponding noncancerous surrounding
tissues (39).
A recent population-based study of NSAID use and prostate
cancer revealed that the relative odds of prostate cancer among the
drug users was 0.2 (95% confidence interval 0.1–0.5) in men during
the eighth decade of life but only 0.9 in men during the sixth decade,
compared with similarly aged men who did not use NSAIDs (3).
The stronger effect among older men raised the possibility that
NSAIDs may prevent the progression of prostate cancer from
latent to clinical disease, rather than reduce the frequency of
primary lesions. The experimental results with R-etodolac in the
TRAMP model of prostate cancer display many parallels with the
human population data. Thus, it is possible that R-etodolac could
represent a potential approach toward preventing the progre ssion
of hormone refractory prostate cancer, especially in the very elderly
patients who are not candidates for cytotoxic therapy.
We thank M. Rosenbach, B. Crain, S. Wu, K. Pekny, P. Charos, J. Wei,
R. Tawatao, Y. Zhao, J. Stebbins, F. Lin, N. Bruey-Sedano, A. A.
Bhattacharya, J. K im, and J. Town for technical assistance. We also
thank N. Noon for secretarial support. This work was supported by grants
from CaPCURE (Association for the Cure of Cancer of the Prostate),
the National Institutes of Health, the U.S. Army Medical Research and
Materiel Command, the University of California Biotechnology Strate-
gic Targets for Alliances in Research Project (BioSTAR), the Susan B.
Komen Foundation, and the California Tobacco-Related Disease Re-
search Prog ram. Some of the histologic sections were performed by the
Cancer Center Histology Core Facilit y, which is supported in part by a
National Institutes of Health grant.
1. Norrish, A. E., Jackson, R. T. & McRae, C. U. (1998) Int. J. Cancer 77, 511–515.
2. Nelson, J. E. & Harris, R. E. (2000) Oncol. Rep. 7, 169–170.
3. Roberts, R. O., Jacobson, D. J., Girman, C. J., Rhodes, T., Lieber, M. M. &
Jacobsen, S. J. (2002) Mayo Clin. Proc. 77, 219–225.
4. Allison, M. C., Howatson, A. G., Torrance, C. J., Lee, F. D. & Russell, R. I.
(1992) N. Engl. J. Med. 327, 749–754.
5. Grosch, S., Tegeder, I., Niederberger, E., Brautigam, L. & Geisslinger, G. (2001)
FASEB. J. 15, 2742–2744.
6. Raz, A. (2002) Biochem. Pharmacol. 63, 343–347.
7. Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D. & DuBois, R. N. (1998)
Cancer Res. 58, 362–366.
8. Shiff, S. J., Koutsos, M. I., Qiao, L. & Rigas, B. (1996) Exp. Cell Res. 222,
9. Sawaoka, H., Kawano, S., Tsuji, S., Tsujii, M., Gunawan, E. S., Takei, Y.,
Nagano, K. & Hori, M. (1998) Am. J. Physiol. 274, G1061–G1067.
10. Demerson, C. A., Humber, L. G., Abraham, N. A., Schilling, G., Martel, R. R.
& Pace-Asciak, C. (1983) J. Med. Chem. 26, 1778–1780.
11. Brocks, D. R. & Jamali, F. (1994) Clin. Pharmacokinet. 26, 259–274.
12. Hedvat, M., Jain, A., Carson, D. A., Leoni, L. M., Huang, G., Holden, S., Lu,
D., Corr, M., Fox, W. & Agus, D. B. (2004) Cancer Cell 5, 565–574.
13. Kastner, P., Mark, M. & Chambon, P. (1995) Cell 83, 859869.
14. Mangelsdorf, D. J. & Evans, R. M. (1995) Cell 83, 841–850.
15. Huang, J., Powell, W. C., Khodavirdi, A. C., Wu, J., Makita, T., Cardiff, R. D.,
Cohen, M. B., Sucov, H. M. & Roy-Burman, P. (2002) Cancer Res. 62,
16. Liu, B., Lee, H. Y., Weinzimer, S. A., Powell, D. R., Clifford, J. L., Kurie, J. M.
& Cohen, P. (2000) J. Biol. Chem. 275, 33607–33613.
17. Cao, X., Liu, W., Lin, F., Li, H., Kolluri, S. K., Lin, B., Han, Y.-H., Dawson, M. I.
& Zhang, X.-k. (2004) Mol . Cell . Biol . 24, 9705–9725.
18. Li, H., Kolluri, S. K., Gu, J., Dawson, M. I., Cao, X., Hobbs, P. D., Lin, B., Chen,
G., Lu, J., Lin, F., et al. (2000) Science 289, 1159–1164.
19. Lin, B., Kolluri, S. K., Lin, F., Liu, W., Han, Y. H., Cao, X., Dawson, M. I., Reed,
J. C. & Zhang, X. K. (2004) Cell 116, 527–540.
20. Lu, D., Zhao, Y., Tawatao, R., Cottam, H. B., Sen, M., Leoni, L. M., Kipps, T. J.,
Corr, M. & Carson, D. A. (2004) Proc. Natl. Acad. Sci. USA 101, 3118–3123.
21. Dawson, M. I. & Zhang, X. K. (2002) Cur r. Med. Chem. 9, 623–637.
22. Clifford, J., Chiba, H., Sobieszczuk, D., Metzger, D. & Chambon, P. (1996)
EMBO J. 15, 4142–4155.
23. Jamali, F., Mehvar, R., Lemko, C. & Eradiri, O. (1988) J. Pharm. Sci. 77,
24. Kolluri, S. K., Bruey-Sedano, N., Cao, X., Lin, B., Lin, F., Han, Y. H., Dawson,
M. I. & Zhang, X. K. (2003) Mol. Cell. Biol. 23, 8651–8667.
25. Zhang, X. K., Lehmann, J., Hoffmann, B., Dawson, M. I., Cameron, J.,
Graupner, G., Hermann, T., Tran, P. & Pfahl, M. (1992) Nature 358, 587–591.
26. Zhang, X. K., Hoffmann, B., Tran, P. B., Graupner, G. & Pfahl, M. (1992) Nature
355, 441–446.
27. Greenberg, N. M., DeMayo, F., Finegold, M. J., Medina, D., Tilley, W. D.,
Aspinall, J. O., Cunha, G. R., Donjacour, A. A., Matusik, R. J. & Rosen, J. M.
(1995) Proc. Natl. Acad. Sci. USA 92, 3439–3443.
28. Gingrich, J. R., Barrios, R. J., Morton, R. A., Boyce, B. F., DeMayo, F. J.,
Finegold, M. J., Angelopoulou, R., Rosen, J. M. & Greenberg, N. M. (1996)
Cancer Res. 56, 40964102.
29. Leid, M. (1994) J. Biol. Chem. 269, 14175–14181.
30. James, S. Y., Lin, F., Kolluri, S. K., Dawson, M. I. & Zhang, X. K. (2003) Cancer
Res. 63, 3531–3538.
31. Wu, Q., Li, Y., Liu, R., Agadir, A., Lee, M. O., Liu, Y. & Zhang, X. (1997) EMBO
J. 16, 1656–1669.
32. Nawaz, Z. & O’Malley, B. W. (2004) Mol. Endocrinol . 18, 493– 499.
33. Culine, S. & Droz, J. P. (2000) Ann. Oncol. 11, 1523–1530.
34. Gupta, S., Adhami, V. M., Subbarayan, M., MacLennan, G. T., Lewin, J. S.,
Hafeli, U. O., Fu, P. & Mukhtar, H. (2004) Cancer Res. 64, 3334–3343.
35. Wechter, W. J., Leipold, D. D., Murray, E. D., Jr., Quiggle, D., McCracken, J. D.,
Barrios, R. S. & Greenberg, N. M. (2000) Cancer Res. 60, 2203–2208.
36. Lala, D. S., Mukherjee, R., Schulman, I. G., Koch, S. S., Dardashti, L. J., Nadzan,
A. M., Croston, G. E., Evans, R. M. & Heyman, R. A. (1996) Nature 383,
37. Cesario, R. M., K lausing, K., Razzaghi, H., Crombie, D., Rungta, D., Heyman,
R. A. & Lala, D. S. (2001) Mol. Endocrinol. 15, 1360–1369.
38. Matsushima-Nishiwaki, R., Shidoji, Y., Nishiwaki, S., Yamada, T., Moriwaki, H.
& Muto, Y. (1996) Mol . Cell . Endocr inol. 121, 179–190.
39. Matsushima-Nishiwaki, R., Okuno, M., Adachi, S., Sano, T., Ak ita, K., Mori-
waki, H., Friedman, S. L. & Kojima, S. (2001) Cancer Res. 61, 7675–7682.
www.pnas.orgcgidoi10.1073pnas.0409721102 Kolluri et al.
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    • "Dietary omega-3 fatty acids, such as DHA, exert their beneficial effects primarily through their anti-inflammatory effects. DHA induces growth inhibition and apoptosis by inhibiting NF-κB activity [156] and suppressing cytokine production in macrophages [157], whereas R-etodolac which is known to bind to RXRα [158] decreases constitutive and RANKL-stimulated NF-κB activation in macrophages and suppresses TNFα-induced IκB-kinase (IKK) phosphorylation and subsequent NF-κB activation in human multiple myeloma cells [159]. LGD1069 down-regulates COX-2 expression in breast cancer cells [160] and inhibits angiogenesis and metastasis in solid tumors [161]. "
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    Full-text · Article · Oct 2015 · Acta Biochimica et Biophysica Sinica
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    • "Etodolac, an FDA-approved NSAID, has excellent postmarketing safety data with gastrointestinal disturbances being the most frequently reported side effects141516. In addition to inhibiting COX-2 [17] , etodolac has COXindependent activities including inhibiting retinoid X receptor (RXRα) leading to apoptosis in cancer cells with high expression levels of the PPARγ/RXRα nuclear receptor complex [18]. Etodolac negatively regulates PPARγ function which then downregulates cyclin D1 leading to tumor growth inhibition [19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Observational data show that nonsteroidal anti-inflammatory drug (NSAID) use is associated with a lower rate of breast cancer. We evaluated the effect of etodolac, an FDA-approved NSAID reported to inhibit cyclooxygenase (COX) enzymes and the retinoid X receptor alpha (RXR), on rationally identified potential biomarkers in breast cancer. Patients with resectable breast cancer planned for initial management with surgical resection were enrolled and took 400 mg of etodolac twice daily prior to surgery. Protein and gene expression levels for genes related to COX-2 and RXRα were evaluated in tumor samples from before and after etodolac exposure. Thirty subjects received etodolac and 17 subjects were assayed as contemporaneous or opportunistic controls. After etodolac exposure mean cyclin D1 protein levels, assayed by immunohistochemistry, decreased (P = 0.03). Notably, pre- versus post cyclin D1 gene expression change went from positive to negative with greater duration of etodolac exposure (r = -0.64, P = 0.01). Additionally, etodolac exposure was associated with a significant increase in COX-2 gene expression levels (fold change: 3.25 [95% CI: 1.9, 5.55]) and a trend toward increased β-catenin expression (fold change: 2.03 [95% CI: 0.93, 4.47]). In resectable breast cancer relatively brief exposure to the NSAID etodolac was associated with reduced cyclin D1 protein levels. Effect was also observed on cyclin D1 gene expression with decreasing levels with longer durations of drug exposure. Increased COX-2 gene expression was seen, possibly due to compensatory feedback. These data highlight the utility of even small clinical trials with access to biospecimens for pharmacodynamic studies. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
    Full-text · Article · Aug 2015 · Cancer Medicine
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    • "Recent studies have revealed that some NSAIDs having anticancer effects on reducing cell growth and metastasis, and inducing apoptosis, require levels of dosage much higher than those required for Cox-inhibition. Therefore, NO-donating (R,S)-profens or (R)-profens such as (R)-etodolac and (R)-flurbiprofen lacking Cox inhibitory activity and in vivo metabolic interconversion to their (S)-antipodes were tested, demonstrating chemopreventive properties against a variety of cancers in several cell lines and animal tumor models [18,19]. Moreover, certain NSAIDs such as (R,S)-flurbiprofen were shown to decrease the production of amyloid-(1–42) peptide (A 42 ) associated with the Alzheimer's disease (AD), with the mechanism of action of A 42 -lowering activity not related to Cox-inhibition or other non-Cox targets of NSAIDs [20,21]. "
    [Show abstract] [Hide abstract] ABSTRACT: A lipase-catalyzed alcoholysis of (R,S)-flurbiprofenyl azolide in anhydrous methyl tert-butyl ether (MTBE) has been developed for the preparation of (R)-flurbiprofenyl ester, (S)-flurbiprofenyl azolide and hence (S)-flurbiprofen. On the basis of enzyme enantioselectivity and activity, the best reaction condition of using (R,S)-flurbiprofenyl 4-bromopyrazolide and 2,3-dibromo-1-propanol as the substrates for Candida antartica lipase B (CALB) at 45 oC was selected, and led to excellent enantioselectivity (VR/VS = 200.3) with two order-of-magnitudes higher specific initial activity for the fast-reacting enantiomer in comparison with those for other lipases. A thermodynamic analysis indicated that both -ΔΔH and -ΔΔS gave equal contributions to -ΔΔG = 14.03 kJ/mol, and hence the excellent enantioselectivity, at the best reaction condition. The kinetic constants estimated from a thorough kinetic analysis were successfully employed for modeling the time-course conversions of both enantiomers. The optically pure (R)-flurbiprofenyl 2,3-dibromo-1-propyl ester obtained via reactive extraction after the alcoholysis was then employed for the synthesis of optically pure (R)-flurbiprofenyl 2,3-bisnitrooxypropyl ester prodrug.
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