Tumorigenesis and Neoplastic Progression
Combined Low Doses of PPAR
and RXR Ligands
Trigger an Intrinsic Apoptotic Pathway in Human
Breast Cancer Cells
Daniela Bonofiglio,* Erika Cione,* Hongyan Qi,*
Attilio Pingitore,* Mariarita Perri,*
Stefania Catalano,* Donatella Vizza,*
Maria Luisa Panno,† Giuseppe Genchi,*
Suzanne A.W. Fuqua,‡ and Sebastiano Ando`†§¶
From the Departments of Pharmaco-Biology,* and Cellular
Biology,† the Centro Sanitario,§ and the Faculty of Pharmacy
Nutritional and Health Sciences,¶ University of Calabria,
Arcavacata di Rende (Cosenza), Italy; and the Lester and Sue
Smith Breast Center,‡ Department of Medicine, Baylor College of
Medicine, Houston, Texas
Ligand activation of peroxisome proliferator-acti-
vated receptor (PPAR)
and retinoid X receptor (RXR)
induces antitumor effects in cancer. We evaluated the
ability of combined treatment with nanomolar levels
of the PPAR
ligand rosiglitazone (BRL) and the RXR
ligand 9-cis-retinoic acid (9RA) to promote antiprolif-
erative effects in breast cancer cells. BRL and 9RA in
combination strongly inhibit of cell viability in
MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer
cells, whereas MCF-10 normal breast epithelial cells
are unaffected. In MCF-7 cells, combined treatment
with BRL and 9RA up-regulated mRNA and protein
levels of both the tumor suppressor p53 and its effec-
tor p21WAF1/Cip1. Functional experiments indicate
that the nuclear factor-B site in the p53 promoter is
required for the transcriptional response to BRL plus
9RA. We observed that the intrinsic apoptotic path-
way in MCF-7 cells displays an ordinated sequence of
events, including disruption of mitochondrial mem-
brane potential, release of cytochrome c , strong
caspase 9 activation, and, finally, DNA fragmentation.
An expression vector for p53 antisense abrogated the
biological effect of both ligands, which implicates
involvement of p53 in PPAR
/RXR-dependent activity
in all of the human breast malignant cell lines tested.
Taken together, our results suggest that multidrug
regimens including a combination of PPAR
and RXR
ligands may provide a therapeutic advantage in breast
cancer treatment. (Am J Pathol 2009, 175:1270–1280; DOI:
10.2353/ajpath.2009.081078)
Breast cancer is the leading cause of death among
women in the world. The principal effective endocrine
therapy for advanced treatment on this type of cancer is
anti-estrogens, but therapeutic choices are limited for
estrogen receptor (ER)-negative tumors, which are of-
ten aggressive. The development of cancer cells that are
resistant to chemotherapeutic agents is a major clinical
obstacle to the successful treatment of breast cancer,
providing a strong stimulus for exploring new ap-
proaches in vitro. Using ligands of nuclear hormone re-
ceptors to inhibit tumor growth and progression is a novel
strategy for cancer therapy. An example of this is the
treatment of acute promyelocytic leukemia using all-trans
retinoic acid, the specific ligand for retinoic acid recep-
tors.1–3 A further paradigm for the use of retinoids in
cancer therapy is for early lesions of head and neck
cancer4 and squamous cell carcinoma of the cervix.5
The retinoic acid receptor, retinoid X receptor (RXR),
and peroxisome proliferator receptor (PPAR)
, ligand-
activated transcription factors belonging to the nuclear
hormone receptor superfamily, are able to modulate
gene networks involved in controlling growth and cellular
differentiation.6 Particularly, heterodimerization of PPAR

with RXR by their own ligands greatly enhances DNA
binding to the direct-repeated consensus sequence
AGGTCA, which leads to transcriptional activation.7 Pre-
vious data show that PPAR
, poorly expressed in normal
breast epithelial cells,8 is present at higher levels in
Supported by AIRC, MURST, and Ex 60%.
Portions of this work were presented as an Abstract at Societa` Italiana
di Patologia XXIX National Congress in Rende, Italy, on September 10–
13, 2008.
Accepted for publication June 5, 2009.
Supplemental material for this article can be found on http://ajp.
amjpathol.org.
Address reprint requests to Prof. Sebastiano Ando`, Faculty of Phar-
macy Nutritional and Health Sciences, University of Calabria, 87036
Arcavacata - Rende (Cosenza), Italy. E-mail: sebastiano.ando@unical.it.
The American Journal of Pathology, Vol. 175, No. 3, September 2009
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2009.081078
1270

breast cancer cells,9 and its synthetic ligands, such as
thiazolidinediones, induce growth arrest and differen-
tiation in breast carcinoma cells in vitro and in animal
models.10–11 Recently, studies in human cultured
breast cancer cells show the thiazolidinedione rosigli-
tazone (BRL), promotes antiproliferative effects and
activates different molecular pathways leading to dis-
tinct apoptotic processes.12–14
Apoptosis, genetically controlled and programmed
death leading to cellular self-elimination, can be initiated
by two major routes: the intrinsic and extrinsic pathways.
The intrinsic pathway is triggered in response to a variety
of apoptotic stimuli that produce damage within the cell,
including anticancer agents, oxidative damage, and UV
irradiation, and is mediated through the mitochondria.
The extrinsic pathway is activated by extracellular li-
gands able to induce oligomerization of death receptors,
such as Fas, followed by the formation of the death-
inducing signaling complex, after which the caspases
cascade can be activated.
Previous data show that the combination of PPAR

ligand with either all-trans retinoic acid or 9-cis-retinoic
acid (9RA) can induce apoptosis in some breast cancer
cells.15 Furthermore, Elstner et al demonstrated that the
combination of these drugs at micromolar concentrations
reduced tumor mass without any toxic effects in mice.8
However, in humans PPAR
agonists at high doses exert
many side effects including weight gain due to increased
adiposity, edema, hemodilution, and plasma-volume ex-
pansion, which preclude their clinical application in pa-
tients with heart failure.16–18 The undesirable effects of
RXR-specific ligands on hypertriglyceridemia and sup-
pression of the thyroid hormone axis have been also
reported.19 Thus, in the present study we have eluci-
dated the molecular mechanism by which combined
treatment with BRL and 9RA at nanomolar doses triggers
apoptotic events in breast cancer cells, suggesting po-
tential therapeutic uses for these compounds.
Materials and Methods
Reagents
BRL49653 (BRL) was from Alexis (San Diego, CA), the
irreversible PPAR
-antagonist GW9662 (GW), and 9RA
were purchased from Sigma (Milan, Italy).
Plasmids
The p53 promoter-luciferase plasmids, kindly provided
by Dr. Stephen H. Safe (Texas A&M University, College
Station, TX), were generated from the human p53 gene
promoter as follows: p53-1 (containing the
1800 to 12
region), p53-6 (containing the
106 to  12 region),
p53-13 (containing the
106 to
40 region), and p53-14
(containing the
106 to
49 region).20 As an internal
transfection control, we cotransfected the plasmid pRL-
CMV (Promega Corp., Milan, Italy) that expresses Renilla
luciferase enzymatically distinguishable from firefly lucif-
erase by the strong cytomegalovirus enhancer promoter.
The pGL3 vector containing three copies of a peroxisome
proliferator response element sequence upstream of the
minimal thymidine kinase promoter ligated to a luciferase
reporter gene (3XPPRE-TK-pGL3) was a gift from Dr. R.
Evans (The Salk Institute, San Diego, CA). The p53 anti-
sense plasmid (AS/p53) was kindly provided from Dr.
Moshe Oren (Weizmann Institute of Science, Rehovot,
Israel).
Cell Cultures
Wild-type human breast cancer MCF-7 cells were grown
in Dulbecco’s modified Eagle’s medium-F12 plus glu-
tamax containing 5% newborn calf serum (Invitrogen,
Milan, Italy) and 1 mg/ml penicillin-streptomycin. MCF-7
tamoxifen resistant (MCF-7TR1) breast cancer cells were
generated in Dr. Fuqua’s laboratory similar to that de-
scribed by Herman21 maintaining cells in modified Ea-
gle’s medium with 10% fetal bovine serum (Invitrogen), 6
ng/ml insulin, penicillin (100 units/ml), streptomycin (100
g/ml), and adding 4-hydroxytamoxifen in tenfold in-
creasing concentrations every weeks (from 10
9 to 10
6
final). Cells were thereafter routinely maintained with 1
mol/L 4-hydroxytamoxifen. SKBR-3 breast cancer cells
were grown in Dulbecco’s modified Eagle’s medium with-
out red phenol, plus glutamax containing 10% fetal bo-
vine serum and 1 mg/ml penicillin-streptomycin. T-47D
breast cancer cells were grown in RPMI 1640 medium
with glutamax containing 10% fetal bovine serum, 1
mmol/L sodium pyruvate, 10 mmol/L HEPES, 2.5g/L glu-
cose, 0.2 U/ml insulin, and 1 mg/ml penicillin-streptomy-
cin. MCF-10 normal breast epithelial cells were grown in
Dulbecco’s modified Eagle’s medium-F12 plus glutamax
containing 5% horse serum (Sigma), 1 mg/ml penicillin-
streptomycin, 0.5 g/ml hydrocortisone, and 10 g/ml
insulin.
Cell Viability Assay
Cell viability was determined with the 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.22 Cells
(2  105 cells/ml) were grown in 6 well plates and ex-
posed to 100 nmol/L BRL, 50 nmol/L 9RA alone or in
combination in serum free medium (SFM) and in 5%
charcoal treated (CT)-fetal bovine serum; 100 l of MTT
(5 mg/ml) were added to each well, and the plates were
incubated for 4 hours at 37°C. Then, 1 ml 0.04 N HCl in
isopropanol was added to solubilize the cells. The absor-
bance was measured with the Ultrospec 2100 Pro-spec-
trophotometer (Amersham-Biosciences, Milan, Italy) at a
test wavelength of 570 nm.
Immunoblotting
Cells were grown in 10-cm dishes to 70% to 80% conflu-
ence and exposed to treatments in SFM as indicated.
Cells were then harvested in cold PBS and resuspended
in lysis buffer containing 20 mmol/L HEPES (pH 8), 0.1
mmol/L EGTA, 5 mmol/L MgCl2, 0.5 M/L NaCl, 20% glyc-
erol, 1% Triton, and inhibitors (0.1 mmol/L sodium or-
thovanadate, 1% phenylmethylsulfonylfluoride, and 20
PPAR
and RXR Ligands in Breast Cancer 1271
AJP September 2009, Vol. 175, No. 3