15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Osteolytic Breast Cancer Bone Metastasis and Estrogen Deficiency-Induced Bone Loss
15-Deoxy-12,14-Prostaglandin J2 Inhibits Osteolytic Breast Cancer Bone Metastasis and Estrogen Deficiency-Induced Bone Loss
Ki Rim Kim 0 1 2
Hyun Jeong Kim 0 1 2
Sun Kyoung Lee 0 1 2
Gwang Taek Ma 0 1 2
Kwang Kyun Park 0 1 2
Won Yoon Chung 0 1 2
0 1 Department of Dental Hygiene, Kyungpook National University , Sangju, 742-711, Korea , 2 Department of Oral Biology, Oral Cancer Research Institute, BK21 PLUS project, Yonsei University College of Dentistry , Seoul, 120-752, Korea , 3 Department of Applied Life Science, The Graduate School, Yonsei University , Seoul, 120-749 , Korea
1 Funding: This work was supported by a faculty research grant from Yonsei University College of Dentistry for 2009 (6-2009-0025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
2 Academic Editor: Dominique Heymann, Faculte de medecine de Nantes , FRANCE
Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPAR) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPAR-dependent and/or PPAR-independent pathways. We investigated the inhibitory activity of 15d-PGJ2 on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ2 dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15dPGJ2 suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ2 blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ2 exerted its effects on breast cancer and bone cells via PPAR-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ2 substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ2 prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ2 may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.
Competing Interests: The authors have declared
that no competing interests exist.
Breast cancer is inextricably linked to two bone diseases, bone metastasis and osteoporosis.
Metastatic breast cancer cells in the bone microenvironment disturb the balance between
osteoclasts and osteoblasts, which disrupts the bone remodeling cycle and results in bone
destruction . Therefore, a vicious cycle between tumor cells and the bone microenvironment
plays a critical role in breast cancer-mediated bone loss . Four essential contributors to
this vicious cycle are tumor cells, osteoblasts, osteoclasts, and resorbed bone matrix. Tumor
cells produce osteolytic factors, including parathyroid hormone-related protein (PTHrP) and
several interleukins . These factors stimulate the expression of receptor activator of nuclear
factor-kappaB (RANK) ligand (RANKL) and inhibit the production of osteoprotegerin (OPG),
which is a decoy receptor of RANKL, in osteoblastic/stromal cells. RANKL triggers osteoclast
differentiation via binding to RANK on osteoclast precursors . Bone resorption by mature
osteoclasts releases calcium and growth factors, such as transforming growth factor-beta
(TGF-) and insulin-like growth factor-1, from the bone matrix. These growth factors further
stimulate tumor growth and the secretion ofosteolytic factors from tumor cells, which causes
severe osteolytic lesions [3,6]. In addition to the direct harm of bone metastasis, cancer therapy
for early stage and/or estrogen receptor-positive breast cancer, including cytotoxic
chemotherapy, induces premature ovarian failure and hormone deprivation therapy, which ultimately
increases the risk of bone loss because of estrogen deficiency . Therefore, the maintenance and
restoration of bone health is particularly important to promote the efficacy of cancer treatment
and the quality of life in breast cancer patients.
15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) is one of the terminal products of the
cyclooxygenase-mediated arachidonic acid pathway, and it is an endogenous ligand of peroxisome
proliferator-activated receptor gamma (PPAR) . Its cyclopentenone structure forms a
covalent adduct with cysteine residues in protein targets, which contributes to its anti-inflammatory
activity at micromolar concentrations . Unlike pro-inflammatory prostaglandins, 15d-PGJ2
suppresses proliferation and induces apoptosis in different cancer cells . 15d-PGJ2
inhibited the invasive capacities of MDA-MB-231 human breast cancer cells via by upregulating
a tissue inhibitor of matrix metalloproteinase-1 and decreasing gelatinase activity in
conditioned media . However, 15d-PGJ2 increased the expression of matrix metalloproteinase
(MMP)-1 and vascular endothelial growth factor to induce angiogenesis in MCF-7 breast
cancer cells [18,19]. PPAR activation by rosiglitazone induced bone loss by reducing osteoblast
differentiation and activating osteoclast differentiation . However, a recent study showed
that rosiglitazone inhibited TNF--induced osteoclast differentiation and bone resorption
. Several studies also demonstrated the inhibitory effect of PPAR agonists, including
15dPGJ2, ciglitazone, and troglitazone, on osteoclast formation .
This study determined the inhibitory activity of 15d-PGJ2 on cancer-associated bone
diseases. We examined the effect of 15d-PGJ2 on the viability, migration, invasion, and secretion
of PTHrP in MDA-MB-231 metastatic human breast cancer cells, RANKL and OPG
expression in hFOB1.19 osteoblastic cells, RANKL-induced osteoclastogenesis in mouse bone
marrow macrophages, and bone resorption by mature osteoclasts. We further evaluated the effect
of 15d-PGJ2 on bone loss in mice that received an intracardiac inoculation of human metastatic
breast cancer cells and ovariectomized mice, which reflected estrogen deficiency.
15d-PGJ2 and the PPAR antagonist GW9662 were purchased from Cayman Chemicals (Ann
Arbor, MI), dissolved in dimethyl sulfoxide (DMSO), and diluted with culture media
immediately prior to use. Dulbeccos modified Eagles medium (DMEM), minimum essential
medium-alpha (-MEM), DMEM:nutrient mixture F-12 (DMEM/F-12) without phenol red,
Dulbeccos phosphate-buffered saline (PBS), Hanks balanced salt solution (HBSS), fetal bovine
serum (FBS), an antibiotic-antimycotic mixture (100 U/ml penicillin and 100 g/ml
streptomycin), 0.25% trypsin-EDTA, and Geneticin (G-418) were products of Gibco-BRL (Grand Island,
NY). Recombinant mouse soluble RANKL and murine macrophage-colony stimulating factor
(M-CSF) were obtained from Koma Biotech (Seoul, South Korea) and R&D Systems
(Minneapolis, MN), respectively. Zoledronic acid (di-sodium salt) was purchased from Enzo Life
Sciences (Farmingdale, NY) and D-luciferin potassium salt was obtained from Goldbio
Technology (St. Louis, MO). Histopaque-1083,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 17-estradiol (E2), and DMSO were obtained from Sigma-Aldrich
(St. Louis, MO). All reagents used in this study were of analytical grade.
MDA-MB-231 human mammary carcinoma cells (Korean Cell Line Bank, Seoul, South Korea)
were cultured in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic mixture
at 37C under a humidified atmosphere of 5% CO2. Human fetal osteoblastic hFOB1.19 cells
(American Type Culture Collection, Manassas, VA) were grown in DMEM/F-12 without
phenol red, containing 10% FBS, 1% antibiotic-antimycotic mixture, and 0.3 mg/ml G418 at 34C
in 5% CO2 in a humidified incubator. Mouse bone marrow macrophages (BMMs) were
isolated from the tibiae of male ICR mice using histopaque density gradient centrifugation as
described previously  and cultured in -MEM containing 10% FBS, 1% antibiotic-antimycotic
mixture, and 30 ng/ml M-CSF at 37C in a humidified atmosphere of 5% CO2.
Male ICR mice (3 weeks old, 20 3 g), female Balb/c nu/nu mice (5 weeks old, 20 3 g), and
female sham-operated and ovariectomized (OVX) ICR mice (89 weeks old, 28 2 g) were
obtained from Central Lab Animal (Seoul, South Korea). The mice were provided free access to a
standard chow diet (Orient, Seongnam, Korea) and tap water ad libitum and housed under
specific pathogen-free conditions with a 12-h light/dark cycle and a relative humidity of 50 5%
at 22 2C. The Institutional Animal Care and Use Committee of Department of Laboratory
Animal Resources, Yonsei Biomedical Research Institute, Yonsei University College of
Medicine approved all animal experiments.
Luciferase vector construction and transfection
The firefly luciferase gene from Photinus pyralis was amplified using polymerase chain reaction
(PCR)-based methods and a pTAL-Luc vector (Clontech Laboratories, Palo Alto, CA) followed
by subcloning into the pLenti6/V5 Directional TOPO cloning vector in the ViraPower
Lentiviral Expression System (Invitrogen, Carlsbad, CA) to generate lentiviral particles with
lentiviral vector-based luciferase. The pLenti6/V5-Luc plasmid was subjected to DNA sequencing
analysis to confirm successful construction. Lentivirus particles were produced using
cotransfection of the 293FT producer cell line with the pLenti6/V5-Luc plasmid and ViraPower
Packaging Mix. Cells were transduced using 2 x 107 lentiviral particles in the transduction enhancer
Polybrene at 10 g/ml to establish luciferase-transfected MDA-MB-231 stable cells
(MDA-MD-231/Luc+). Blasticidin (10 g/ml) was added to select stably transduced cells.
Blasticidin-resistant clones exhibited V5 epitope detection against an anti-V5 antibody on Western
blot analysis and revealed the maximum level of luciferase activity in a microplate
spectrofluorometer (Molecular Devices, Palo Alto, CA).
MDA-MB-231 cells (1 x 104 cells/well) were seeded into a 96-well plate with 10% FBS-DMEM.
The cells were incubated in serum-free media with various concentrations of 15d-PGJ2 for 24
or 72 h. hFOB1.19 human osteoblastic cells (1x 104 cells/well) were incubated in serum-free
media with the indicated concentrations of 15d-PGJ2 for 6 h or 24 h. BMMs (5 x 104 cells/well)
were cultured in media with the indicated concentrations of 15d-PGJ2 in the presence of 10%
FBS and 30 ng/ml M-CSF for 5 days. Cell viability was determined using the MTT assay as
described previously .
MDA-MB-231 cells were seeded in a 6-well plate and allowed to grow to 90% confluency. One
artificial wound per well was scratched into monolayers using the narrow end of a sterile
micropipette tip, and the wounded areas were photographed. Cells were incubated in serum-free
DMEM with mitomycin (5 g/ml) and various concentrations of 15d-PGJ2. The scratched
areas were photographed again 40 h later at the identical location of the initial image. The
width of the wounded cell monolayer was measured using ImageJ software, and the percentage
of wound closure was derived using the following formula: (1(current wound width/initial
wound width)) 100.
A cell invasion assay was performed using a Transwell chamber (Corning, Cambridge, MA)
that contained a polycarbonate membrane filter (6.5 mm diameter, 8 m pore size). The
bottom of the filter was coated with 0.1% (w/v) gelatin. Matrigel (BD Biosciences, San Jose, CA),
which is a mixture of basement membrane extracellular matrix proteins, was diluted with
DMEM to a final concentration of 1 mg/ml and applied to coat the membrane filter.
MDA-MB-231 cell suspensions (2 x 104 cells/100 l) with various concentrations of 15d-PGJ2
were added to the inserts of each coated Transwell. The lower chamber contained 600 l of
media with 1% FBS and 15d-PGJ2. Transwell chambers were incubated for 24 h at 37C. Cells
were fixed with 70% methanol, and the membranes were stained with hematoxylin.
Non-invading cells on the upper surface of the membrane were scraped with cotton swabs, and
invading cells that remained on the bottom surface were mounted on slides. Four random fields for
each membrane were captured, and cells in the captured fields were quantified under a Zeiss
AXio imager microscope (Carl Zeiss AG, Gttingen, Germany).
MDA-MB-231 cells were seeded at a density of 1 x 105 cells into a 96-well plate and incubated
to adhere overnight. Cells were treated with the indicated concentrations of 15d-PGJ2, TGF-,
and/or GW9662 for 24 h. The plate was centrifuged, and media were collected. PTHrP levels in
the collected culture media were quantified using a human PTHrP enzyme-linked
immunosorbent assay (ELISA) kit (USCN Life Science, Wuhan, China) .
MDA-MB-231 cells were seeded at 1 x 106 cells in 100 mm cell culture dishes and incubated
with the indicated concentrations of TGF-, 15d-PGJ2, and/or GW9662 for 24 h. Cells were
lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA) containing 1 mM
phenylmethylsulfonyl fluoride and protease inhibitor cocktail tablets (Roche Diagnostics) or the
nuclear/cytosol fractionation kit (BioVision, Mountain View, CA). Samples were centrifuged, and
proteins in the supernatants were quantitated using BCA protein assay reagents (Pierce
Biotechnology, Rockford, IL). Proteins were separated using 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes
(Millipore, Danvers, MA). Membranes were blocked for 1 h in 5% skim milk and incubated
with rabbit anti-Smad2, rabbit anti-phosphorylated Smad2 (Cell Signaling Technology), rabbit
anti- -actin (Sigma-Aldrich), and mouse anti-Lamin B (Invitrogen) at 4C overnight, followed
by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room
temperature. Proteins were visualized using an enhanced chemiluminescence kit (GE
Healthcare, Buckinghamshire, UK).
The medium was replaced with serum-free DMEM/F-12 when seeded MDA-MB-231 cells
reached 7080% confluency in 10% FBS-DMEM. Conditioned media (CM), including
MDA-MB-231 cell-secreted osteolytic factors, were collected after a 24-h incubation.
Osteoblastic hFOB1.19 cells (1 x 106 cells/100 mm culture dish) were treated with the indicated
concentrations of 15d-PGJ2 and/or GW9662 in DMEM with 75% CM or PTHrP (100 nM) for 6 h.
Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA). First-strand cDNA
from 1 g total RNA was synthesized using the PrimeScript RT reagent kit (TaKaRa, Dalian,
China). Real-time quantitative RT-PCR was performed using the 7300 Real-Time PCR System
(Applied Biosystems, Foster City, CA) and the SYBR Premix Ex Taq (TaKaRa) in a 96-well
optical reaction plate according to the manufacturers instructions. The following PCR conditions
were used: initial denaturation at 95C for 30 s, followed by 40 cycles of denaturation at 95C
for 5 s and annealing at 60C for 31 s. Cycle threshold (Ct) values were established. Relative
gene expressions of RANKL and OPG to the reference gene GAPDH were determined using
the 2Ct method. The following primer sequences were used: RANKL, forward 5-ATGGTG
GATGGCTCATGGTTAG-3 and reverse 5-GAGCAAAAGGCTGAGCTTCAAG-3; OPG,
forward 50-CCAGTGACCAGATCCTGAAGCT-30 and reverse 50-GGTGTCTTGGT
CGCCATTTT-30; and GAPDH, forward 50-AGTCCTTCCACGATACCAAAGT-30 and
Osteoclast formation assay
BMMs (5 x 104 cells/well) were seeded into a 96-well plate and cultured in -MEM with 10%
FBS, 30 ng/ml M-CSF and the indicated concentrations of 15d-PGJ2 in the absence or presence
of 100 ng/ml RANKL. Cells were cultured for 5 days, and fresh media containing the
appropriate chemicals was replaced every other day. Cells were fixed using 3.7% (v/v) formaldehyde
and stained for tartrate-resistant acid phosphatase (TRAP) activity for 10 min at 37C using
the Acid Phosphatase Leukocyte kit (Sigma-Aldrich) as described previously [6,26].
TRAPpositive multinucleated cells (MNCs) with more than three nuclei were quantified as
differentiated osteoclasts using an Olympus IX70 inverted microscope (Olympus Optical, Tokyo, Japan)
BMMs (5 x 104 cells/well) were plated onto BD BioCoat Osteologic MultiTest Slides with
mineralized calcium phosphate thin films (BD Biosciences) and cultured in -MEM containing
10% FBS, 30 ng/ml M-CSF, and 100 ng/ml RANKL for 4 days as described previously .
Differentiated osteoclasts were treated with 15d-PGJ2 at the indicated concentrations for an
additional 10 days. Media were replaced every other day. Media were collected to measure
cathepsin K and MMP activity, and cells were treated with 4% sodium hypochlorite. Slides
were washed twice using distilled water, and resorbed pits were observed under a light
microscope (100x magnification).
Cathepsin K activity assay and gelatin zymography
The activity of cathepsin K and MMPs in the collected media was measured using a SensiZyme
Cathepsin K Activity Assay Kit (Sigma-Aldrich) and gelatin zymography as described
previously . Cathepsin K activity was calculated using a standard curve and the gelatinolytic
activities of MMPs were detected as clear bands against a dark blue background.
Animal model of breast cancer bone metastasis
MDA-MB-231/Luc+ cells (1 x 106 cells/0.1 ml in HBSS) were injected into the left cardiac
ventricle of female Balb/c nu/nu mice as previously described [27,28]. Animals were divided into
four groups of 10 mice on the following day and subcutaneously administered vehicle (PBS
containing 2% DMSO) alone, 0.5 or 2 mg/kg 15d-PGJ2, or 0.1 mg/kg zoledronic acid as a
positive control, three times per week for six weeks. Metastatic progression in nude mice was
visualized using bioluminescence imaging three and five weeks after intracardiac injections. Mice
were anesthetized for imaging using a Xenogen XGI-8 Gas Anesthesia System and injected
intraperitoneally with 150 mg/kg of D-luciferin potassium salt in PBS. The luciferase activity
was visualized using an intensified CCD video camera connected to the in vivo IVIS Imaging
System 200 Series (Caliper Life Sciences, Hopkinton, MA). Bioluminescence from mice was
expressed as total photon flux measured in photons/sec/cm2 per steradian (sr) using Xenogen
Living Image software. Blood was collected by intracardiac puncture at the end of the
experiment for the serum PTHrP assay. The femora, tibiae, and mandibles of nude mice were also
collected for CT analysis.
A murine model of ovariectomy-induced bone loss
OVX mice were divided into four groups of 10 mice, and subcutaneously administered vehicle
(PBS containing 2% DMSO), 0.5 or 2 mg/kg 15d-PGJ2, or 10 g/kg E2 as a positive control,
three times per week for 10 weeks. Sham-operated mice were treated with vehicle alone. Body
weights were measured weekly using an electronic scale. Blood samples were collected by
cardiac puncture at the end of the experimental period, and the femora were dissected.
The femora, tibiae, and mandibles of nude mice and the femora of sham-operated and OVX
mice were examined using a SkyScan 1076 CT scanner (SkyScan, Aartselaar, Belgium) with
100 kV, 140 A current, rotation step 0.6, and camera pixel size of 35 m as described
previously [6,26]. Three-dimensional (3D) images were reconstructed based on the CT images
using SkyScan NRecon software and analyzed using SkyScan's computed tomography analyzer
software (CTAn). The following parameters were analyzed in the proximal tibiae of nude mice
and the distal femora of sham-operated and OVX mice for quantitative analyses of bone
histomorphometry: percent bone volume (BV/TV, %), trabecular thickness (Tb.Th, mm),
trabecular number (Tb.N, mm-1), trabecular separation (Tb.Sp, mm), and structure model index
(SMI). Values for bone mineral density (BMD) were measured in the femora of sham-operated
and OVX mice.
Determination of biochemical bone parameters
The collected blood samples were allowed to clot for 2 h at room temperature and centrifuged
at 2,000 g for 20 min to obtain sera as described previously [6,26]. Serum PTHrP levels from
10 nude mice were quantified in triplicate using a specific PTHrP ELISA kit. Calcium and
alkaline phosphatase (ALP) levels in the sera of sham-operated and OVX mice were determined
using QuantiChrome calcium and ALP assay kits (BioAssay Systems, Hayward, CA),
respectively. TRAP and C-terminal telopeptides of type I collagen (CTX) levels were measured using
mouse TRAP and RatLaps enzyme immunoassay (EIA) kits (Immunodiagnostic Systems,
Fountain Hills, AZ), respectively. Serum levels of osteocalcin were detected using a mouse
osteocalcin EIA kit (Biomedical Technologies, Stoughton, MA), and tumor necrosis
factoralpha (TNF-) and interleukin-1 beta (IL-1) levels were quantified using respective
commercially available ELISA kits (R&D Systems). The serum levels of these factors in 10
sham-operated and OVX mice were measured in duplicate.
Goldner's trichrome staining
Mouse femora were fixed in a 10% buffered formalin solution for 48 h, decalcified with a 10%
EDTA solution, and embedded in paraffin. Five-mthick serial sagittal sections were stained
using Goldner's trichrome according to the protocol specified by Electron Microscopy Sciences
Data are expressed as the means standard error (SEM) and analyzed using one-way ANOVA
and Students t-test. A value of P< 0.05 was considered statistically significant.
The viability of MDA-MB-231 cells treated with the indicated concentrations of 15d-PGJ2 for
24 or 72 h was reduced in a dose-dependent manner (Fig 1A). Cells exposed to 3, 5, 10, and
30 M concentrations of 15d-PGJ2 were viable to 91%, 65%, 54%, and 38%, respectively, after
24 h of treatment and 65%, 33%, 22%, and 8%, respectively, after 72 h of treatment. The scratch
wound healing assay showed that a 40 h treatment with 15d-PGJ2 at noncytotoxic
concentrations inhibited the migratory ability of MDA-MB-231 cells by 32% at 0.5 M, 46% at 1 M,
and 62% at 3 M (Fig 1B). 15d-PGJ2 also decreased cell invasion in a dose-dependent manner.
The number of invaded cells was inhibited by 25%, 53%, and 73% following treatment for 24 h
with 15d-PGJ2 at concentrations of 0.5, 1, and 3 M, respectively (Fig 1C).
We investigated the effect of 15d-PGJ2 on the secretion of PTHrP, which plays a central role in
the osteolytic bone metastasis of MDA-MB-231 cells . PTHrP levels were decreased by
Fig 1. Effect of 15d-PGJ2 on the viability, migration, and invasion of MDA-MB-231 cells. (A) Cells were
incubated in serum-free media containing various concentrations of 15d-PGJ2 for 24 or 72 h. Cell viability
was determined using the MTT assay. (B) Cells were grown to confluency in monolayers, scratched using a
micropipette tip, and treated with the indicated concentrations of 15d-PGJ2 for 40 h. Scratched areas on
cultured MDA-MB-231 cells were observed under a light microscope immediately and 40 h after scratching
(40x magnification). Relative migrating distances of cells into scratched areas were measured using ImageJ
software. Data are expressed as percentages of cell migrating distances at 40 h compared with 0 h. (C) Cells
were stimulated with a 1% FBS attractant and treated with 15d-PGJ2 at the indicated concentrations for 24 h.
Cells that traversed across the Matrigel matrix were stained with hematoxylin, and representative images
were visualized using light microscopy (200x magnification). The numbers of invaded cells were counted in
four random fields per membrane filter. Data are expressed as means SEM. *P<0.05, **P<0.001 vs.
normalize the reduced PTHrP levels following 15d-PGJ2 treatment in MDA-MB-231 cells
reof GW9662 (Fig 2C). These results demonstrate that 15d-PGJ2 inhibits PTHrP production
osteoblastic cells (Fig 3A). Real-time PCR analysis indicated that RANKL mRNA levels
increased considerably and OPG mRNA levels decreased noticeably in osteoblastic hFOB1.19
Fig 2. Effect of 15d-PGJ2 on PTHrP production in MDA-MB-231 cells. The cells were treated with (A)
various concentrations of 15d-PGJ2 or (B) TGF-, 15d-PGJ2 and/or GW9662 for 24 h. PTHrP levels were
measured in the cultured media of MDA-MB-231 cells using a commercial human PTHrP ELISA kit. Data are
expressed as the means SEM. *P<0.05, **P<0.01 vs. untreated cells. #P<0.01 vs. TGF- -treated cells.
(C) The level of Smad2 in total lysates and nuclear and cytoplasmic fractions was determined using western
blotting in MDA-MB-231 cells stimulated by TGF- , 15d-PGJ2 and/or GW9662 PPAR antagonist for 24 h.
The cropped blots are representative of experiments that were repeated three times.
cells stimulated with 75% CM from MDA-MB-231 breast cancer cells or PTHrP (100 nM) for
6 h, which elevated the RANKL/OPG ratio. However, treatment with 15d-PGJ2
dose-dependently suppressed the CM- and PTHrP-induced increase in the RANKL/OPG ratio by blocking
increases in RANKL mRNA levels and decreases in OPG mRNA levels in hFOB1.19 cells
exposed to CM and PTHrP, respectively (Fig 3B). GW9662 treatment did not rescue RANKL and
OPG mRNA levels that were altered by 15d-PGJ2 treatment in CM and PTHrP-treated
hFOB1.19 cells. These results indicate that 15d-PGJ2 inhibits the RANKL/OPG ratio in
hFOB1.19 cells stimulated with MDA-MB-231 cell-derived osteolytic factors, particularly
Treatment with 15d-PGJ2 for 5 days inhibited cell viability by 19% at a concentration of 3 M,
36% at 5 M, and 53% at 10 M in BMMs. BMMs were differentiated to TRAP-positive
multinucleated cells as osteoclasts in the presence of M-CSF and RANKL for 5 days, but treatment
with 15d-PGJ2 inhibited RANKL-induced osteoclast formation in a dose-related manner.
Treatment with 0.5, 1, and 3 M 15d-PGJ2 reduced the number of differentiated osteoclasts by
32%, 55%, and 93%, respectively. GW9662 treatment did not attenuate the inhibitory effect of
15d-PGJ2 on RANKL-induced osteoclast formation (Fig 4A). The formation of resorption pits
determined the activity of mature osteoclasts. Osteoclast differentiation was induced on
calcium phosphate-coated plates, and cells were incubated with 15d-PGJ2 in the presence of M-CSF
and RANKL for 10 days. Treatment with 15d-PGJ2 dose-dependently suppressed the
Fig 3. Effect of 15d-PGJ2 on RANKL and OPG mRNA expression in hFOB1.19 human osteoblastic
cells. (A) hFOB1.19 cells were treated with the indicated concentrations of 15d-PGJ2 in DMEM/F12 for 6 h or
24 h. Cell viability was determined using the MTT assay. (B) hFOB1.19 cells were treated with the indicated
concentrations of 15d-PGJ2 in DMEM/F12 containing 75% CM of MDA-MB-231 cells or PTHrP (100 ng) for 6 h.
mRNA levels of RANKL and OPG were analyzed using real time-PCR. Graphs are expressed as the ratio of the
densitometric intensity of RANKL to OPG after normalization to GAPDH. Data represent the means SEM.
*P<0.05, **P<0.001 vs. untreated cells, #P<0.05, ##P<0.001 vs. CM- or PTHrP-treated cells.
formation of resorbed areas (Fig 4B). RANKL treatment significantly increased the activity of
cathepsin K in cultured media, but 15d-PGJ2 treatment suppressed the activity almost to
control levels (Fig 4C). Gelatin zymography indicated that the levels of pro- and active forms of
MMP-2 and MMP-9 were considerably enhanced in cultured media of RANKL-induced
mature osteoclasts, but 15d-PGJ2 treatment decreased the levels of these MMPs in a
dose-dependent manner (Fig 4D).
MDA-MB-231/Luc+ cells were inoculated into the left ventricles of nude mice to induce bone
metastasis of breast cancer cells, and 15d-PGJ2 or zoledronic acid was subcutaneously injected
for six weeks. Bioluminescence imaging showed that subcutaneous administration of 15d-PGJ2
or zoledronic acid for three and five weeks inhibited the metastatic progression of
MDA-MB231/Luc+ cells (Fig 5A). Osteolytic lesions of all mice were analyzed using CT six weeks after
cancer cell injections. Radiographic images of MDA-MB-231/Luc+ cell-injected mice indicated
that osteolytic lesions were substantially developed in mandibles, distal femora, and proximal
tibiae. However, 15d-PGJ2 or zoledronic acid administration inhibited cancer cell-induced
osteolytic lesions in a dose-dependent manner (Fig 5B). 3D-images also revealed that
MDA-MB-231/Luc+ cells induced severe destruction in the inner part of the mandible, but
treatment with 15d-PGJ2 or zoledronic acid noticeably reduced this damage (Fig 5C). Bone
morphometric analyses demonstrated that the injection of MDA-MB-231/Luc+ cells decreased
BV/TV, Tb.Th, and Tb.N and increased Tb.Sp and SMI, but treatment with 15d-PGJ2 or
zoledronic acid induced a recovery of these bone morphometric parameters. Furthermore, the
Fig 4. Effect of 15d-PGJ2 on RANKL-induced osteoclast differentiation and activation. (A) BMMs
isolated from ICR mice were treated with M-CSF (30 ng/ml), RANKL (100 ng/ml), 15d-PGJ2, and/or GW9662
for 5 days. TRAP staining was performed to detect osteoclast formation. TRAP-positive multinucleated cells
( 3 nuclei) as differentiated osteoclasts were observed (100x magnification) and counted under an inverted
microscope. (B) The differentiated BMMs were treated with 15d-PGJ2 in the presence of M-CSF (30 ng/ml)
and RANKL (100 ng/ml) for an additional 10 days. The formed resorption pits were visualized using light
microscopy (100x magnification). (C) The level of cathepsin K in the cultured media was measured using a
commercially available ELISA kit. (D) The activities of MMPs were determined using gelatin zymograpy as
clear bands against a blue background that corresponded to active MMP-2/9 (62/92 kDa) and pro-MMP-2/9
(72/105 kDa). The cropped gel image is representative of experiments that were repeated three times. Data
are expressed as the means SEM. *P<0.001 vs. RANKL-untreated cells. #P<0.05, ##P<0.001 vs.
levels of PTHrP were significantly increased in the sera of mice that received an intracardiac
injection of MDA-MB-231/Luc+ cells, but treatment with 15d-PGJ2 or zoledronic acid inhibited
the increase in PTHrP levels (Fig 5D).
15d-PGJ2 inhibited ovariectomy-induced bone loss
Ovariectomy-induced osteoporosis is commonly used as an animal model of bone loss due to
decreased, but Tb.Sp and SMI increased, in OVX mice compared to the sham group. However,
these bone morphometric parameters significantly recovered to control levels in 15d-PGJ2- or
E2-treated OVX mice (Fig 6A). 3D-images and Goldners trichrome staining of distal femoral
metaphyses revealed that the subcutaneous administration of 15d-PGJ2 or E2 in OVX mice
dose-dependently suppressed severe trabecular bone loss in OVX mice (Fig 6B). In addition,
treatment with 15d-PGJ2 or E2 blocked elevations in body weight in OVX mice. We measured
ALP activity and osteocalcin level as markers of bone formation and calcium, TRAP, and CTX
Fig 5. Effect of 15d-PGJ2 on osteolytic bone metastasis in nude mice that received intracardiac
injections of MDA-MB-231 cells. MDA-MB-231/Luc+ cells were inoculated into the left ventricles of female
nude mice. 15d-PGJ2 or zoledronic acid (ZA) was subcutaneously injected 3 times per week for 6 weeks at
the indicated doses (n = 10). (A) Metastatic progression was detected by measuring bioluminescence in the
same mice at 3 and 5 weeks after the injection of cancer cells. The formed metastases were quantified by
measuring total photon flux per second. (B) Radiographic images of mandibles, distal femora, and proximal
tibiae were scanned using CT 6 weeks after the injection of cancer cells. Arrowheads indicate osteolytic
lesions. (C) The mandibles of mice were analyzed using 3D-images. (D) Bone morphometric parameters,
including BV/TV, Tb.N, Tb.Th, Tb.Sp, and SMI, were measured using CT analysis of the proximal tibiae
from mice. (E) Serum PTHrP levels were assayed using a commercially available ELISA kit. Data are
expressed as the means SEM. #P<0.05, ##P<0.01 vs. control group.*P<0.05, **P<0.01 vs.
vehicletreated group inoculated with cancer cells.
were used as the markers of bone resorption. The serum levels of these bone turnover markers
were elevated significantly in OVX mice, but these levels noticeably decreased in 15d-PGJ2- or
E2-treated OVX mice (Fig 6C). Treatment with 15d-PGJ2 or E2 also significantly reduced
TNF- and IL-1 levels in the sera of OVX mice (Fig 6D).
The present study determined the inhibitory activity of 15d-PGJ2 on breast cancer-associated
bone diseases. We first investigated whether 15d-PGJ2 blocked breast cancer bone metastasis
and the resulting bone loss. We found that 15d-PGJ2 attenuated cell migration and the invasion
of MDA-MB-231 human breast cancer cells at noncytotoxic concentrations. Treatment with
15d-PGJ2 reduced the secreted levels of PTHrP through a PPAR-independent pathway in
MDA-MB-231 cells regardless of TGF- stimulation. Cancer cell-derived PTHrP attracted
attention as a key mediator of the triggering and intensification of the vicious cycle of osteolytic
bone metastasis in the bone microenvironment . Among the bone matrix-derived growth
factors, TGF- regulates PTHrP production via Smad-dependent or Smad-independent
pathways . Our data indicated that 15d-PGJ2 inhibited Smad2 phosphorylation regardless
of the PPAR pathway, and consequently, reduced levels of pSmad2 were detected in the
nucleus and cytoplasm. These results suggest that treatment with 15d-PGJ2 suppresses the metastatic
progression of MDA-MB-231 cells and decreases PTHrP production through a
PPAR-independent but Smad2-dependent pathway in MDA-MB-231 cells that primarily metastasized
The majority of patients with breast cancer bone metastasis exhibit osteolytic lesions that
are accompanied by severe bone destruction. Osteolysis is caused by mature osteoclasts derived
from hematopoietic mononuclear precursors not a direct effect of cancer cells on bone .
Almost all of the other mediators that induce osteoclast differentiation and activation transduce
and amplify signals through RANKL, which is produced by osteoblastic stromal cells .
PTHrP also enhances osteoclastogenesis and the activity of mature osteoclasts via an
up-regulation of RANKL and down-regulation of its decoy receptor OPG in osteoblasts .
Noncytotoxic concentrations of 15d-PGJ2 significantly inhibited an increase in RANKL mRNA
expression and a decrease in OPG mRNA expression in hFOB1.19 human osteoblastic cells
stimulated with an MDA-MB-231 cell-derived conditioned medium or PTHrP in our study.
Osteoclasts that are differentiated by RANKL produce acidic conditions to dissolve calcium
hydroxyapatite and secrete various proteolytic enzymes, such as cathepsin K and MMPs, to
degrade organic components in the bone matrix, which releases bone-stored growth factors
[37,38]. Therefore, the inhibition of osteoclast-mediated bone resorption can ultimately
contribute to the prevention of additional tumor growth and cancer cell-mediated osteolysis.
15dPGJ2 reduced RANKL-induced osteoclast differentiation and inhibited the formation of
osteoclast-mediated resorption pits by suppressing the proteolytic activities of cathepsin K and
MMP-2/9. Treatment with GW9662 did not affect the anti-osteoclastogenic activity of
15dPGJ2. These results indicate that 15d-PGJ2 blocks breast cancer-mediated bone destruction by
reducing the RANKL/OPG ratio in osteoblasts that are exposed to MDA-MB-231 cell-derived
osteolytic factors and inhibiting the formation and function of osteoclasts in RANKL-treated
osteoclast precursors in a PPAR-independent manner.
We further estimated the inhibitory activity of 15d-PGJ2 on osteolytic bone metastasis in
nude mice inoculated with MDA-MB-231/Luc+ cells into the left cardiac ventricle and
Fig 6. Effect of 15d-PGJ2 on ovariectomy-induced bone loss. OVX mice were subcutaneously injected
with vehicle, 15d-PGJ2, or E2 (10 g/kg) for 10 weeks (n = 10). Sham-operated mice received vehicle alone
(n = 10). (A) Bone morphometric parameters, including BMD, BV/TV, Tb.Th, Tb.N, Th.Sp, and SMI, were
measured using CT analysis of the femora from mice. (B) 3D images of distal femora of mice were obtained
from the reconstruction of CT data (upper). Sagittal sections of distal femora from mice were stained with
Goldner's trichrome. Bone trabeculae appear green, and bone marrow appears red. Stained sections were
photographed using a light microscope (100x magnification). (C) Body weights of all mice were measured,
and blood sera were collected from all mice for analyses of biochemical parameters. Serum levels of calcium,
ALP, osteocalcin, TRAP, and CTX were evaluated using the respective kits as described in Materials and
Methods. (D) Serum levels of TNF- and IL-1 were determined using specific ELISA kits. Data are
expressed as the means SEM. #P< 0.05, ##P< 0.01 vs. sham group. *P<0.05, **P<0.01 vs. OVX group.
subcutaneously injected with 15d-PGJ2 or zoledronic acid for six weeks. Metastatic progression
was delayed and osteolytic lesions in mandibles, femora, and tibiae were decreased in
15dPGJ2-treated mice, which was supported by bioluminescence imaging, radiographic and 3D
images, and bone morphometric parameters. Moreover, the reduced serum PTHrP levels in
MDA-MB-231cell-injected mice treated with15d-PGJ2 may be linked with the in vitro
inhibitory effect of 15d-PGJ2 on PTHrP production. Zoledronic acid used as a positive control is an
anti-bone resorptive agent administered to cancer patients with bone metastases at the clinical
dose of 4 mg via intravenous injection every 34 weeks . Although treatment with clinical
doses of bisphosphonates (a daily dose 3 g/kg or a weekly dose of 20 g/kg) has been reported
to inhibit skeletal tumor growth in murine models , zoledronic acid at high doses, 0.1 or
0.125 mg/kg, was administered to exhibit its in vivo anti-tumor effect in recent studies .
In this study, zoledronic acid at 0.1 mg/kg also reduced bone metastasis and bone loss in
mandibles, femora, and tibiae in mice with intracardiac injection of MDA-MB-23l cells. 15d-PGJ2
at 2 mg/kg showed a similar effect with zoledronic acid at 0.1 mg/kg. These results demonstrate
that administration of 15d-PGJ2 inhibits the bone metastasis of breast cancer and the resulting
Currently, the main clinical drugs for the treatment of cancer-associated skeletal lesions are
inhibitors of osteoclastic bone resorption, including bisphosphonates and denosumab as a
monoclonal antibody against RANKL . These agents are beneficial for the prevention and
treatment of osteoporosis in postmenopausal women . In addition, bisphosphonates and
denosumab decrease estrogen deficiency-related bone loss due to aromatase inhibitor therapy
and cytotoxic chemotherapy in cancer patients . We determined whether 15d-PGJ2, which
exhibits potent anti-osteoclastic and anti-bone resorptive activity, prevented bone loss in OVX
mice as a standard model for the pharmaceutical evaluation of estrogen deficiency-induced
osteoporosis. Subcutaneously administered 15d-PGJ2 delayed weight gain and damage to femoral
trabecular bone in OVX mice, as evidenced by bone morphometric parameters, reconstructed
3D images, histological analyses, and biochemical parameters. The serum levels of the
pro-inflammatory cytokines TNF- and IL-1, which are key mediators of bone loss following
estrogen withdrawal via the promotion of osteoclastic bone resorption , were also reduced in
15d-PGJ2-treated OVX mice. These results indicate that 15d-PGJ2 prevents bone loss during
estrogen deficiency-inducing cancer treatment.
In summary, 15d-PGJ2 inhibited the proliferation, migration, and invasion of
MDA-MB231 cells and the production of a major osteolytic factor, PTHrP. 15d-PGJ2 also suppressed the
RANKL/OPG ratio in osteoblastic cells exposed to breast cancer cell-derived osteolytic factors,
the RANKL-induced differentiation of osteoclast precursors, and the formation of resorbed
pits by decreasing the activities of cathepsin K and MMPs. Furthermore, subcutaneous
injections of 15d-PGJ2 reduced the metastasis of breast cancer cells into bone and the generation of
osteolytic lesions in mice. Treatment with 15d-PGJ2 inhibited estrogen deficiency-induced
bone loss. Therefore, 15d-PGJ2 and 15d-PGJ2-inducing agents may be potent candidates for
the prevention and treatment of bone loss caused by breast cancer bone metastasis and
The authors thank Chae-Eun Lee in the Oral Science Research Institute for technical assistance
for CT and Won-Gyeong Ahn at the Chuncheon Center of the Korea Basic Science Institute
for technical assistance in real-time IVIS Imaging System 200.
Conceived and designed the experiments: KKP WYC. Performed the experiments: KRK.
Analyzed the data: KRK KKP HJK SKL WYC. Contributed reagents/materials/analysis tools: KRK
HJK SKL GTM. Wrote the paper: KRK WYC.
1. Suva LJ , Washam C , Nicholas RW , Griffin RJ . Bone metastasis: mechanisms and therapeutic opportunities . Nat Rev Endocrinol . 2011 ; 7 : 208 - 218 . doi: 10.1038/nrendo.2010.227 PMID: 21200394
2. Coleman RE . Clinical features of metastatic bone disease and risk of skeletal morbidity . Clin Cancer Res . 2006 ; 12 : 6243s - 6249s . PMID: 17062708
3. Kozlow W , Guise TA . Breast cancer metastasis to bone: mechanisms of osteolysis and implications for therapy . J Mammary Gland Biol Neoplasia . 2005 ; 10 : 169 - 180 . PMID: 16025223
4. Esposito M , Kang Y. Targeting tumor-stromal interactions in bone metastasis . Pharmacol Ther . 2014 ; 141 : 222 - 233 . doi: 10.1016/j.pharmthera. 2013 . 10.006 PMID: 24140083
5. Azim HA , Kamal NS , Azim HA Jr. Bone metastasis in breast cancer: the story of RANK-ligand . J Egypt Natl Canc Inst . 2012 ; 24 : 107 - 114 . doi: 10.1016/j.jnci. 2012 . 06.002 PMID: 22929916
6. Lee SK , Park KK , Park JH , Lim SS , Chung WY . The inhibitory effect of roasted licorice extract on human metastatic breast cancer cell-induced bone destruction . Phytother Res . 2013 ; 27 : 1776 - 1783 . doi: 10.1002/ptr.4930 PMID: 23401151
7. Brufsky AM . Cancer treatment-induced bone loss: pathophysiology and clinical perspectives . Oncologist . 2008 ; 13 : 187 - 195 . doi: 10.1634/theoncologist. 2007-0152 PMID: 18305064
8. Diez-Dacal B , Perez-Sala D. Anti-inflammatory prostanoids: focus on the interactions between electrophile signaling and resolution of inflammation . Scientific World Journal . 2010 ; 10 : 655 - 675 . doi: 10. 1100/tsw.2010.69 PMID: 20419278
9. Surh YJ , Na HK , Park JM , Lee HN , Kim W , Yoon IS , et al. 15-deoxy-12,14-prostaglandin J2, an electrophilic lipid mediator of anti-inflammatory and pro-resolving signaling . Biochem Pharmacol . 2011 ; 82 : 1335 - 1351 . doi: 10.1016/j.bcp. 2011 . 07.100 PMID: 21843512
10. Ishihara S , Rumi MA , Okuyama T , Kinoshita Y. Effect of prostaglandins on the regulation of tumor growth . Curr Med Chem Anticancer Agents . 2004 ; 4 : 379 - 387 . PMID: 15281909
11. Shen ZN , Nishida K , Doi H , Oohashi T , Hirohata S , Ozaki T , et al. Suppression of chondrosarcoma cells by 15-deoxy-Delta 12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21 . Biochem Biophys Res Commun . 2005 ; 328 : 375 - 382 . PMID: 15694358
12. Ciucci A , Gianferretti P , Piva R , Guyot T , Snape TJ , Roberts SM , et al. Induction of apoptosis in estrogen receptor-negative breast cancer cells by natural and synthetic cyclopentenones: role of the IkappaB kinase/nuclear factor-kappaB pathway . Mol Pharmacol . 2006 ; 70 : 1812 - 1821 . PMID: 16908599
13. Ray DM , Akbiyik F , Phipps RP . The peroxisome proliferator-activated receptor gamma (PPARgamma) ligands 15-deoxy-12,14-prostaglandin J2 and ciglitazone induce human B lymphocyte and B cell lymphoma apoptosis by PPARgamma-independent mechanisms . J Immunol . 2006 ; 177 : 5068 - 5076 . PMID: 17015690
14. Qiao L , Dai Y , Gu Q , Chan KW , Zou B , Ma J , et al. Down-regulation of X-linked inhibitor of apoptosis synergistically enhanced peroxisome proliferator-activated receptor gamma ligand-induced growth inhibition in colon cancer . Mol Cancer Ther . 2008 ; 7 : 2203 - 2211 . doi: 10.1158/ 1535 - 7163 . MCT-08-0326 PMID: 18645029
15. Kaikkonen S , Paakinaho V , Sutinen P , Levonen AL , Palvimo JJ . Prostaglandin 15d-PGJ(2) inhibits androgen receptor signaling in prostate cancer cells . Mol Endocrinol . 2013 ; 27 : 212 - 223 . doi: 10.1210/ me. 2012-1313 PMID: 23192983
16. Wang JJ , Mak OT . Induction of apoptosis by 15d-PGJ2 via ROS formation: an alternative pathway without PPAR activation in non-small cell lung carcinoma A549 cells . Prostaglandins Other Lipid Mediat . 2011 ; 94 : 104 - 111 . doi: 10.1016/j.prostaglandins. 2011 . 01.004 PMID: 21396480
17. Liu H , Zang C , Fenner MH , Possinger K , Elstner E. PPARgamma ligands and ATRA inhibit the invasion of human breast cancer cells in vitro . Breast Cancer Res Treat . 2003 ; 79 : 63 - 74 . PMID: 12779083
18. Kim EH , Na HK , Surh YJ . Upregulation of VEGF by 15-deoxy-12,14-prostaglandin J2 via heme oxygenase-1 and ERK1/2 signaling in MCF-7 cells . Ann N Y Acad Sci . 2006 ; 1090 : 375 - 384 . PMID: 17384282
19. Kim DH , Kim JH , Kim EH , Na HK , Cha YN , Chung JH , et al. 15 - Deoxy-12,14-prostaglandin J2 upregulates the expression of heme oxygenase-1 and subsequently matrix metalloproteinase-1 in human breast cancer cells: possible roles of iron and ROS. Carcinogenesis . 2009 ; 30 : 645 - 654 . doi: 10.1093/ carcin/bgp012 PMID: 19136476
20. Wei W , Wan Y. Thiazolidinediones on PPAR: The roles in bone remodeling . PPAR Res . 2011 ; 2011 : 867180. doi: 10.1155/2011/867180 PMID: 22135675
21. Yang CR , Lai CC . Thiazolidinediones inhibit TNF--mediated osteoclast differentiation of RAW264.7 macrophages and mouse bone marrow cells through downregulation of NFATc1 . Shock . 2010 ; 33 : 662 - 667 . doi: 10.1097/SHK.0b013e3181cc0738 PMID: 19953004
22. Hounoki H , Sugiyama E , Mohamed SG , Shinoda K , Taki H , Abdel-Aziz HO , et al. Activation of peroxisome proliferator-activated receptor gamma inhibits TNF-alpha-mediated osteoclast differentiation in human peripheral monocytes in part via suppression of monocyte chemoattractant protein-1 expression . Bone. 2008 ; 42 : 765 - 774 . doi: 10.1016/j.bone. 2007 . 11.016 PMID: 18242157
23. Okazaki R , Toriumi M , Fukumoto S , Miyamoto M , Fujita T , Tanaka K , et al. Thiazolidinediones inhibit osteoclast-like cell formation and bone resorption in vitro . Endocrinology . 1999 ; 140 : 5060 - 5065 . PMID: 10537132
24. Mbalaviele G , Abu-Amer Y , Meng A , Jaiswal R , Beck S , Pittenger MF , et al. Activation of peroxisome proliferator-activated receptor- pathway inhibits osteoclast differentiation . J Biol Chem . 2000 ; 275 : 14388 - 14393 . PMID: 10799521
25. Park SY , Kim HJ , Kim KR , Lee SK , Lee CK , Park KK , et al. Betulinic acid, a bioactive pentacyclic triterpenoid, inhibits skeletal-related events induced by breast cancer bone metastases and treatment . Toxicol Appl Pharmacol . 2014 ; 275 : 152 - 162 . doi: 10.1016/j.taap. 2014 . 01.009 PMID: 24463094
26. Jun AY , Kim HJ , Park KK , Son KH , Lee DH , Woo MH , et al. Extract of Magnoliae Flos inhibits ovariectomy induced osteoporosis by blocking osteoclastogenesis and reducing osteoclast-mediated bone resorption . Fitoterapia . 2012 ; 83 : 1523 - 1531 . doi: 10.1016/j.fitote. 2012 . 08.020 PMID: 22981503
27. Park SI , Kim SJ , McCauley LK , Gallick GE . Pre-clinical mouse models of human prostate cancer and their utility in drug discovery . Curr Protoc Pharmacol . 2010 ; Chapter 14: Unit 14 .15.
28. Campbell JP , Merkel AR , Masood-Campbell SK , Elefteriou F , Sterling JA . Models of bone metastasis . J Vis Exp . 2012 ; 67 : e4260. doi: 10.3791/4260 PMID: 22972196
29. Guise TA , Yin JJ , Thomas RJ , Dallas M , Cui Y , Gillespie MT . Parathyroid hormone-related protein (PTHrP)-(1-139) isoform is efficiently secreted in vitro and enhances breast cancer metastasis to bone in vivo . Bone . 2002 ; 30 : 670 - 676 . PMID: 11996903
30. Inada M , Matsumoto C , Miyaura C. Animal models for bone and joint disease. Ovariectomized and orchidectomized animals . Clin Calcium . 2011 ; 21 : 164 - 170 . doi: CliCa1102164170 PMID: 21289412
31. Pratap J , Wixted JJ , Gaur T , Zaidi SK , Dobson J , Gokul KD , et al. Runx2 transcriptional activation of Indian Hedgehog and a downstream bone metastatic pathway in breast cancer cells . Cancer Res . 2008 ; 68 : 7795 - 7802 . doi: 10.1158/ 0008 - 5472 . CAN-08-1078 PMID: 18829534
32. Safina A , Sotomayor P , Limoge M , Morrison C , Bakin AV . TAK1-TAB2 signaling contributes to bone destruction by breast carcinoma cells . Mol Cancer Res . 2011 ; 9 : 1042 - 1053 . doi: 10.1158/ 1541 - 7786 . MCR-10-0196 PMID: 21700681
33. Lindemann RK , Ballschmieter P , Nordheim A , Dittmer J. Transforming growth factor beta regulates parathyroid hormone-related protein expression in MDA-MB-231 breast cancer cells through a novel Smad/Ets synergism . J Biol Chem . 2001 ; 276 : 46661 - 46670 . PMID: 11590145
34. Kakonen SM , Selander KS , Chirgwin JM , Yin JJ , Burns S , Rankin WA , et al. Transforming growth factor-beta stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways . J Biol Chem . 2002 ; 277 : 24571 - 24578 . PMID: 11964407
35. Mundy GR . Metastasis to bone: Causes, consequences and therapeutic opportunities . Nat Rev Cancer . 2002 ; 2 : 584 - 593 . PMID: 12154351
36. Karaplis AC , Goltzman D. PTH and PTHrP effects on the skeleton . Rev Endocr Metab Disord . 2000 ; 1 : 331 - 341 . PMID: 11706747
37. Andersen TL , del Carmen Ovejero M , Kirkegaard T , Lenhard T , Foged NT , Delaiss JM . A scrutiny of matrix metalloproteinases in osteoclasts: evidence for heterogeneity and for the presence of MMPs synthesized by other cells . Bone . 2004 ; 35 : 1107 - 1119 . PMID: 15542036
38. Costa AG , Cusano NE , Silva BC , Cremers S , Bilezikian JP . Cathepsin K: its skeletal actions and role as a therapeutic target in osteoporosis . Nat Rev Rheumatol . 2011 ; 7 : 447 - 456 . doi: 10.1038/nrrheum. 2011.77 PMID: 21670768
39. Daubin F , Le Gall C , Gasser J , Green J , Clzardin P. Antitumor effects of clinical dosing regimens of bisphosphonates in experimental breast cancer bone metastasis . J Natl Cancer Inst . 2007 ; 99 : 322 - 330 . PMID: 17312309
40. Thudi NK , Martin CK , Nadella MV , Fernandez SA , Werbeck JL , Pinzone JJ , et al. Zoledronic acid decreased osteolysis but not bone metastasis in a nude mouse model of canine prostate cancer with mixed bone lesions . Prostate . 2008 ; 68 : 1116 - 1125 . doi: 10.1002/pros.20776 PMID: 18461562
41. Jeong J , Lee KS , Choi YK , Oh YJ , Lee HD . Preventive effects of zoledronic acid on bone metastasis in mice injected with human breast cancer cells . J Korean Med Sci . 2011 ; 26 : 1569 - 1575 . doi: 10.3346/ jkms.2011. 26.12.1569 PMID: 22147993
42. Luo KW , Ko CH , Yue GG , Lee MY , Siu WS , Lee JK , et al. Anti-tumor and anti-osteolysis effects of the metronomic use of zoledronic acid in primary and metastatic breast cancer mouse models . Cancer Lett . 2013 ; 339 : 1569 - 1575 .
43. . Bundred N. Antiresorptive therapies in oncology and their effects on cancer progression . Cancer Treat Rev . 2012 ; 38 : 776 - 786 . doi: 10.1016/j.ctrv. 2012 . 02.002 PMID: 22370427
44. Guise TA , Brufsky A , Coleman RE . Understanding and optimizing bone health in breast cancer . Curr Med Res Opin . 2010 ; 26 Suppl. 3 : 3 - 20 . doi: 10.1185/03007995.2010.533162 PMID: 21050131
45. Mundy GR . Osteoporosis and inflammation . Nutr Rev . 2007 ; 65 : S147 - 151 . PMID: 18240539