The R-enantiomer of the nonsteroidal antiinflammatory drug etodolac binds retinoid X receptor and induces tumor-selective apoptosis.
ABSTRACT 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.
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ABSTRACT: Aspirin and other NSAIDs reduce the incidence of and mortality from colon cancer, but their mechanism of action remains unknown. We evaluated the effect of aspirin (ASA) and three other structurally unrelated NSAIDs (indomethacin, naproxen, and piroxicam) on cell proliferation, cell cycle phase distribution, and the development of apoptosis in HT-29 colon adenocarcinoma cellsin vitro.All of the NSAIDs examined reduced the proliferation and altered the morphology of these cells in a time- and concentration-dependent manner. In addition, they altered the cell cycle phase distribution of these cells. They increased the proportion of cells in the G0/G1phase and reduced the proportion in the S phase of the cell cycle. ASA and indomethacin also reduced the percentage of cells in the G2/M phase, whereas naproxen and piroxicam did not. Parallel to their effect on cell cycle, ASA and indomethacin also reduced the levels of p34cdc2and p33cdk2, two cyclin-dependent kinases that are important for cell cycle progression. Finally, all the NSAIDs analyzed, except ASA, induced apoptosis in these cells. There was a rough correlation between the relative potency of these compounds in inducing apoptosis and their effectiveness in retarding cell proliferation. Our findings indicate that NSAIDs can reduce the proliferation of HT-29 colon cancer cellsin vitro.In addition, they cause cell cycle quiescence and apoptosis, both of which could account for their anti-proliferative effect. These findings suggest possible mechanisms for the cancer preventive effects of these compounds in humans.Experimental Cell Research 02/1996; · 3.56 Impact Factor
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ABSTRACT: Ligand-induced structural alterations of retinoid X receptor (RXR) appear to facilitate receptor homodimerization, to enhance DNA binding of RXR homodimeric complexes, and to dictate the transcriptional activation properties of RXR complexes bound to response elements located in the promoter region of 9-cis-retinoic acid responsive genes. The technique of limited proteolysis was used to address 9-cis-retinoic acid-induced RXR conformational change and to identify receptor region(s) which undergo structural alteration upon ligand binding. Proteolytic digestion of 9-cis-retinoic acid-liganded, but not unliganded, RXR gave rise to a fragment of 31 kilodaltons (PF31), which contained a large portion of the RXR ligand binding domain. The potency with which 9-cis-retinoic acid induced formation of PF31 was nearly identical to that with which the ligand enhanced DNA binding of a RXR homodimeric complex to a response element composed of the directly repeated hexanucleotide, PuGGTCA, separated by 1 base pair. The amino-terminal limit of PF31 mapped to Ser229 of mouse RXR alpha, which lies roughly in the middle of the spacer region separating the DNA and ligand binding domains of the receptor. These results suggest that a fairly large region of the receptor protein undergoes a structural alteration upon ligand binding which may directly alter the multiple functions of the RXR ligand binding domain.Journal of Biological Chemistry 06/1994; 269(19):14175-81. · 4.65 Impact Factor
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ABSTRACT: Epidemiological studies and clinical observations suggest that nonsteroidal anti-inflammatory drugs and certain selective cyclooxygenase (COX)-2 inhibitors may reduce the relative risk of clinically evident prostate cancer. This prompted us to investigate the chemopreventive potential of celecoxib, a selective COX-2 inhibitor, against prostate carcinogenesis in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Similar to prostate cancer in humans, prostate malignancies in TRAMP mice progress from precursor intraepithelial lesions, to invasive carcinoma that metastasizes to lymph nodes, liver, lungs, and occasionally to bone. The basal enzyme activity and protein expression of COX-2 is significantly higher (>4-fold) in the dorsolateral prostate of TRAMP mice up to 24 weeks of age compared with their nontransgenic littermates. Eight-week-old TRAMP mice were randomly divided and fed either control diet (AIN 76A) or a custom prepared AIN 76A diet containing 1500-ppm celecoxib ad libitum for 24 weeks, a dosage that would compare with the normal recommended dose for the treatment of human disease. Studies from two independent experiments, each consisting of 10 mice on test, showed that the cumulative incidence of prostate cancer development at 32 weeks of age in animals fed with AIN 76A diet was 100% (20 of 20) as observed by tumor palpation, whereas 65% (13 of 20), 35% (7 of 20), and 20% (4 of 20) of the animals exhibited distant site metastases to lymph nodes, lungs, and liver. Celecoxib supplementation to TRAMP mice from 8-32 weeks of age exhibited significant reduction in tumor development (5 of 20) with no signs of metastasis. Celecoxib feeding resulted in a significant decrease in prostate (56%; P < 0.0003) and genitourinary weight (48%; P < 0.008). Sequential magnetic resonance imaging analysis of celecoxib-fed mice documented lower prostate volume compared with the AIN 76A-fed group. Histopathological examination of celecoxib-fed animals showed reduced proliferation, and down-modulation of COX-2 and prostaglandin E2 levels in the dorsolateral prostate and plasma, respectively. These results correlated with retention of antimetastasis markers, viz E-cadherin, and alpha- and beta-catenin, along with a significant decrease in vascular endothelial growth factor protein expression. Celecoxib supplementation also resulted in enhanced in vivo apoptosis in the prostate as monitored by several techniques including a recently perfected technique of 99mTc-labeled annexin V in live animals followed by phosphor imaging. One striking observation in an additional study was that celecoxib feeding to mice with established tumors (16 weeks of age) significantly improved their overall survival (P = 0.014), compared with AIN 76A-fed group. Our findings suggest that celecoxib may be useful in chemoprevention of prostate cancer.Cancer Research 06/2004; 64(9):3334-43. · 8.65 Impact Factor
The R-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
(RXR?) protein in the adenocarcinoma cells, but not in normal
prostatic epithelium. R-etodolac specifically bound recombinant
RXR?, 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
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
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).
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
were achieved after oral gavage in a xenograft prostate cancer
model to diminish the growth of the transplanted tumor (12).
of peroxisome proliferator-activated receptor ? (PPAR?) transac-
tivation (12). PPAR?, as well as other nuclear hormone receptors,
forms heterodimers with the retinoid X receptor ? (RXR?) (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-
everal epidemiological studies have shown that the use of
nonsteroidal antiinflammatory drugs (NSAIDs) is associated
independent effects of R-etodolac in malignant prostate cells might
be attributed to its binding and modulation of RXR? activities.
Here, we show that R-etodolac induced apoptosis of prostate
cancer cells, but not normal prostatic epithelial cells. The R-
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, suppression of RXR? expression
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 mCi?ml 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
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-
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-
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: firstname.lastname@example.org or xzhang@
© 2005 by The National Academy of Sciences of the USA
February 15, 2005 ?
vol. 102 ?
no. 7 ?
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 C57BL?6 mice were purchased from The Jackson
Laboratory and bred at UCSD. All animal protocols received prior
were measured on a group of seven 15-week-old C57BL?6 male
mice who were fed R-etodolac (1.25 mg?kg) chow for 2 weeks by a
bioanalytical LC?MS-based method developed by Maxxam Ana-
lytics (Mississauga, ON, Canada). Chiral HPLC (23) was used to
confirm the lack of R- to S-etodolac in vivo interconversion.
We started 46 male TRAMP mice at 9–12 weeks of age on chow
with R-etodolac 1.25 mg?kg 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
for the presence 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 ?g),
was incubated with radiolabeled ligand in the presence of different
The RXR? 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-
chow. The prostates were removed, and serial frozen sections were
assayed for terminal deoxyribonucleotidyl transferase (TdT;
TUNEL; Chemicon) or stained with anti-human RXR? (D20;
Santa Cruz Biotechnology), followed by staining with DAPI (50
?g?ml; Sigma) containing DNase-free RNase A (100 ?g?ml;
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. Expression vectors for RXR?, RA
receptor ? (RAR?), hemagglutinin (HA)-ubiquitin, and reporter
gene ?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
normalized for transfection efficiency on the basis of cotransfected
?-gal gene activity. Transfected cell lysates were separated by
SDS?PAGE and immunoblotted (see Supporting Materials and
RXR? Small Interfering RNA (siRNA) Transfections.ASMARTpoolof
Table 1. Incidence of primary tumors and metastases
Primary tumor incidence*
Animals with gross masses‡
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
30 weeks of age, or after development of a gross palpable mass, the mice were sacrificed, and the urogenital systems were removed and weighed. (b–f) 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.
Inhibition of prostate cancer progression in the TRAMP model. Male TRAMP mice were fed control chow or chow with R-etodolac (1.25 mg?kg). (a) At
www.pnas.org?cgi?doi?10.1073?pnas.0409721102Kolluri et al.
from Dharmacon. A 10-?l aliquot of 20 ?M siRNA per well was
transfected into cells in six-well plates by using Lipofectamine Plus
Antiproliferative and Proapoptotic Effects of R-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 ID50values 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 b–e). In the latter instance, the malignant
cells displayed shrunken and pyknotic nuclei, whereas the nuclei
from the adjacent normal prostatic epithelium appeared morpho-
Activity of R-Etodolac in the TRAMP Model.Preliminarydose-ranging
pharmacokinetic data showed that plasma concentrations of 370 ?
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-
tases 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).
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 a–l). Collectively, these data
indicated that R-etodolac retarded the progression and metastasis
of prostate cancer in the TRAMP system.
R-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
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) in R-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
Histological evaluation of R-etodolac-treated TRAMP prostate tis-
RXR? 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
?M or 1 mM R-etodolac for 24 h in medium containing 0.5% FBS. Apoptotic
were obtained in two separate experiments. The percentage of apoptosis
represents the percentage of annexin-V-positive drug-treated cells minus the
RXR? 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
determined after 44 h of treatment. Bars represent mean ? SD from three
RXR? is required for R-etodolac-induced apoptosis. (a) Inhibition of
Kolluri et al.PNAS ?
February 15, 2005 ?
vol. 102 ?
no. 7 ?
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.
RXR? Is Required for the Apoptotic Effect of R-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?-
RXR? expression in LNCaP cells (Fig. 3a). The apoptotic effect of
R-etodolac in RXR? siRNA-transfected cells was substantially
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.
bind RXR?, a ligand competition assay with [3H]9-cis-RA was
used. Both unlabeled 9-cis-RA and R-etodolac displaced [3H]9-
cis-RA bound to RXR? LBD (Fig. 4a), with an IC50value of
?200 ?M for R-etodolac. Furthermore, [3H]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
R-Etodolac Binding Induces Conformational Change in RXR?. Binding
of ligands to their receptors often induces changes in susceptibility
to proteolysis (13, 14). Digestion of the RXR? LBD with a low
concentration of trypsin (3 ?g?ml) yielded a proteolytic fragment
of ?20 kDa, whreeas higher concentrations of trypsin (10 or 30
the RXR? LBD with 9-cis-RA did not alter its sensitivity to trypsin
digestion, consistent with previous studies (29). However, incuba-
binding to RXR?. Human RXR? LBD was incubated with 1 nM [3H]9-cis-RA in
the presence of different concentrations of unlabeled 9-cis-RA (F) or R-
etodolac (?) at 4°C for 14 h. [3H]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 [3H]R-
etodolac were incubated with or without polyhistidine-tagged-RXR? LBD.
RXR?-bound [3H]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 [3H]R-etodolac in the presence of unlabeled 9-cis-RA
(10?6M) or R-etodolac (1 mM) as indicated. RXR?-bound [3H]R-etodolac was
separated and assayed as described above.
R-etodolac binds to RXR?. (a) R-etodolac competes with 9-cis-RA for
(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)2-
tk-CAT; 300 ng] and a ?-gal expression vector (50 ng). After subsequent
treatment with 9-cis-RA (10?8M) and the indicated concentrations of R-
etodolac for 24 h CAT activities were determined and normalized relative to
the ?-gal activity. (b) R-etodolac modulation of RXR??RAR heterodimer ac-
ng) and a ?-gal expression vector (50 ng), and they were then treated with
all-trans-RA (10?7M) 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?6M), R-etodolac (1 mM), or their combination
Inhibitory effect of R-etodolac on RXR??PPAR? heterodimer activity. ZR-75-1
cells were transfected with ?RARE-tk-CAT reporter plasmid (300 ng) and a
were determined. (e) R-etodolac inhibits RAR? protein expression induced by
without SR11237 (10-6M), ciglitazone (10 ?M), R-etodolac (1 mM) in the
indicated combinations. Cell lysates were immunoblotted and probed for
relative levels of RAR? and ?-actin.
R-etodolac inhibits 9-cis-RA-induced transcriptional activity of RXR?.
www.pnas.org?cgi?doi?10.1073?pnas.0409721102Kolluri et al.
tion of the RXR? LBD with R-etodolac before trypsin digestion (3
?g?ml) 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).
R-Etodolac Modulates RXR? Transcriptional Activity. To test whether
R-etodolac binding modulated RXR? transcriptional activity, a
reporter gene containing RXR? homodimer-responsive elements,
(TREpal)2-tk-CAT (26), was transfected with a RXR? expression
vector into CV-1 cells. Treatment of cells with 9-cis-RA strongly
induced reporter gene activity, whereas treatment with R-etodolac
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
RAR? promoter, which binds various RXR-containing het-
erodimers including RXR?RAR and RXR?PPAR? (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 RXR?RAR 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
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 RXR?PPAR?
heterodimer activity, ZR-75-1 cells were stimulated with the
PPAR? 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? expression by the two drugs in
combination was abolished by R-etodolac cotreatment (Fig. 5e).
Loss of RXR? Expression After R-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? was
performed on the prostate tissues 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
prostates of TRAMP mice fed with R-etodolac.
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
RXR? 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
The standard therapy for progressive prostate cancer is androgen
ablation. However, many patients become unresponsive and de-
velop metastatic disease (33). Thus, there is a compelling need for
the development of unconventional agents that can delay the
oral administration of the COX-inactive R-stereoisomer of the
common NSAID etodolac inhibited tumor expansion and metas-
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
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
enous RXR?. HEK 293T cells were transfected with a vector expressing HA-
ubiquitin and then treated with R-etodolac as described above and 1 ?M
anti-HA antibody and immunoblotted with an antibody to detect RXR?. (e)
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
R-etodolac induces RXR? degradation. (a) Diminished RXR? staining
Kolluri et al.PNAS ?
February 15, 2005 ?
vol. 102 ?
no. 7 ?
tasis in the TRAMP model. By analogy, R-etodolac could be a
prospective agent for the treatment of human prostate cancer.
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
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
PPAR? recombinant protein failed to demonstrate any direct
binding of R-etodolac to PPAR? (data not shown). Because
PPAR? activity depends on heterodimerization with RXR?, it is
is mediated by its binding to RXR?. Antagonists of RXR ho-
modimers are known to function as agonists of RXR?PPAR?
heterodimers (36, 37).
Exactly how RXR? mediates the apoptotic effects of R-etodolac
effect through its inhibition of RXR? transactivation (Fig. 5)
because many RXR? agonists potently inhibit the growth of
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 corresponding noncancerous surrounding
A recent population-based study of NSAID use and prostate
cancer revealed that the relative odds of prostate cancer among the
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 progression
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. Kim, and J. Town for technical assistance. We also
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 Program. Some of the histologic sections were performed by the
Cancer Center Histology Core Facility, which is supported in part by a
National Institutes of Health grant.
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www.pnas.org?cgi?doi?10.1073?pnas.0409721102Kolluri et al.