Raddeanin A suppresses breast cancer-associated osteolysis through inhibiting osteoclasts and breast cancer cells
Wang et al. Cell Death and Disease
Raddeanin A suppresses breast cancer- associated osteolysis through inhibiting osteoclasts and breast cancer cells
Qiang Wang 0
Jian Mo 0
Chenchen Zhao 1 3
Kangmao Huang 0
Mingxuan Feng 4
Wenxin He 0
Jiying Wang 0
Shuai Chen 0
Zi'ang Xie 0
Jianjun Ma 0
Shunwu Fan 0
0 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University , 3
1 Department of Orthopedic Surgery, the Second Affiliated Hospital, Medical College of Zhejiang University , 88
2 Jiefang Road, Hangzhou , Zhejiang Province 310016 , China
3 Department of Orthopedic Surgery, the Second Affiliated Hospital, Medical College of Zhejiang University , 88
4 Orthopaedic Department, Taizhou Central Hospital, Affiliate Hospital Of Taizhou University , Taizhou, Zhejiang Province 318000 , China
Bone metastasis is a severe complication of advanced breast cancer, resulting in osteolysis and increased mortality in patients. Raddeanin A (RA), isolated from traditional Chinese herbs, is an oleanane-type triterpenoid saponin with anticancer potential. In this study, we investigated the effects of RA in breast cancer-induced osteolysis and elucidated the possible mechanisms involved in this process. We first verified that RA could suppress osteoclast formation and bone resorption in vitro. Next, we confirmed that RA suppressed Ti-particle-induced osteolysis in a mouse calvarial model, possibly through inhibition of the SRC/AKT signaling pathway. A breast cancer-induced osteolysis mouse model further revealed the positive protective effects of RA by micro-computed tomography and histology. Finally, we demonstrated that RA inhibited invasion and AKT/mammalian target of rapamycin signaling and induced apoptosis in MDA-MB-231 cells. These results indicate that RA is an effective inhibitor of breast cancer-induced osteolysis.
Anemone raddeana Regel has been widely used to treat
cancer, rheumatism, and neuralgia1?3. This traditional
Chinese medicinal herb belongs to the Ranunculaceae
family and exhibits antitumor efficacy, anti-inflammatory
efficacy, and analgesic activity4. Raddeanin A (RA), an
oleanane-type triterpenoid saponin, has been shown to be
the main bioactive constituent of Anemone raddeana
Regel4?6. Recent studies have demonstrated that RA can
prevent proliferation, induce apoptosis, and inhibit
invasion in various human tumor cells, including gastric
cancer cells, hepatocellular carcinoma cells, and
nonsmall-cell lung carcinoma cells6?8. The mechanisms
through which RA exerts these effects may be attributed
to its ability to inhibit angiogenesis by preventing the
phosphorylation of vascular endothelial growth factor
receptor 2 and associated protein kinases, including
phospholipase C ?1, Janus kinase 2, focal adhesion kinase,
Src, and AKT9. Further research has indicated that RA
can also induce apoptosis and autophagy in SGC-7901
cells10. Therefore, RA may be a promising agent with
broad antitumor effects.
Breast cancer is the most common cancer in women
worldwide and is related to a high frequency of bone
metastasis. A previous report demonstrated that bone
metastasis occurs in 70% of patients who died from
prostate cancer or breast cancer11. The mechanism of
bone metastasis, sometimes referred to as the ?vicious
cycle,? is complex and involves interactions among
metastatic breast cancer cells, osteoblasts, and
osteoclasts12,13. It is believed that inflammatory cytokines and
parathyroid hormone-related protein secreted by breast
cancer cells can stimulate osteoblasts to produce receptor
activator of nuclear factor-?B (NF-?B) ligand (RANKL)
and further enhance osteoclast differentiation and bone
resorption12,14. Thus, a number of factors with potential
chemoattractive properties are released to stimulate
breast cancer cell proliferation and migration15.
Bisphosphonate and denosumab have been shown to slow down
the progression of breast cancer-induced osteolysis16,17.
However, due to adverse events, such as osteonecrosis of
the jaw, toothache, and hypocalcemia, and because
antiresorptive treatment is only palliative, novel therapies for
breast cancer-induced osteolysis should be considered.
The aim of this study was to assess the effects of RA on
osteoclasts, osteoblasts, and MDA-MB-231 breast cancer
cells. Subsequently, we evaluated the effects of RA in
mouse models of Ti-particle-induced calvarial osteolysis
and breast cancer-induced osteolysis. The related
molecular mechanisms were further determined.
RA inhibited RANKL-induced osteoclast formation in vitro
To explore the effect of RA on RANKL-induced
osteoclast differentiation, bone marrow-derived
macrophages (BMMs) were treated with 0, 0.2, 0.4, and 0.8 ?M
RA in the presence of macrophage-colony stimulating
factor (M-CSF) and RANKL. RANKL differentiated
BMMs into mature tartrate-resistant acid phosphatase
(TRAP)-positive multinucleated osteoclasts, but RA
produced an inhibitory effect on the formation of
TRAPpositive multinucleated osteoclasts in a
concentrationdependent manner (Fig. 1a, b). We further treated BMMs
with 0.4 ?M RA for 3, 5, and 7 days. As shown in Fig. 1c,
RA significantly suppressed osteoclast formation at day 7.
The number of dead osteoclasts was also calculated and
an increase of osteoclast apoptosis was observed with the
increasing of the RA doses (Supplementary 1A, B). The
results of cytotoxicity assays on BMMs revealed that slight
cytotoxic effect was observed for a dose of 0.391 ?M, and
no significant inhibitory effects for doses below 0.195 ?M
(Fig. 1e). Collectively, these evidences suggested that RA
prevented RANKL-induced osteoclast formation in vitro.
RA suppressed RANKL-induced osteoclast-related gene expression in vitro
To confirm the inhibitory potential of RA on
RANKLinduced osteoclast differentiation, we examined the
osteoclast-related genes, including TRAP (ACP5),
cathepsin k (CTSK), calcitonin receptor (CTR), V-ATPase-a3,
V-ATPase-d2, and the nuclear factor of activated T cells 1
(NFATc1). Compared to the control group treated by
RANKL, the expression of CTSK and NFATc1 was
dramatically suppressed with the addition of RA (Fig. 2a?f).
The protein expression level of CTSK and NFATc1 was
also decreased in the RA treatment group (Fig. 2g). These
data confirmed that RA inhibited the expressions of
RA inhibited osteoclastic bone resorption in a concentration-dependent manner
We performed pit formation assay to investigate the
function of RA on the osteoclastic bone resorption
activity. BMMs without RA treatment obviously resorbed
the bone surface (Fig. 3a), while the RA treatment group
showed fewer resorption pits and almost no resorption
pits were observed in the 0.8 ?M RA group. The average
resorption area in each group were 43, 18, 7, 1%,
respectively (Fig. 3b). These results suggested that RA
inhibited osteoclastic bone resorption in vitro.
RA did not inhibited osteoblast differentiation and osteoblastic-related genes expression in vitro
No inhibitory effect was observed on the survival of
MC3T3-E1 cells below doses of 0.781 ?M
(Supplementary 2A). To determine the role of RA on osteoblast
differentiation, we further cultured MC3T3-E1 cells and
analyzed alkaline phosphatase (ALP) activity at day 7. No
significant difference of ALP was detected between
control and the 0.2, 0.4, and 0.8 ?M RA treatment groups
(Supplementary 2B, D). We also evaluated osteoblastic
mineralization with Alizarin red staining at day 21, and
found that more total mineralized area was observed in
0.2 ?M RA compared to the control group
(Supplementary 2C, E). Though no significant difference between RA
treatment and control group was observed in the mRNA
expressions of osteoblastic-specific genes at day 7;
however, secreted protein acidic and rich in cysteine were
significantly increased after 14 days of treatment with RA
(Supplementary 2F). The above results suggested that RA,
at least, had no inhibitory effect on osteoblast
RA suppressed Ti-particle-induced osteolysis in vivo
Since RA could inhibit osteoclastic bone resorption
in vitro, we further explored its property on
Ti-particleinduced osteolysis in a mouse calvarial model.
Microcomputed tomography (CT) revealed that massive surface
erosion was seen in the vehicle group. On the contrary,
treatment with low or high concentration of RA
significantly reduced Ti-particle-induced osteolysis (Fig. 4a).
We then measured and calculated the ratio of bone
volume to total volume (BV/TV) as well as the percentage
of total porosity in the region of interest from
threedimensional (3D) reconstruction images. Compared with
the vehicle group, treatment with low or high
concentration of RA significantly increased the BV/TV
and decreased the percentage of porosity (Fig. 4b).
Meanwhile, TRAP staining indicated that the number of
multinucleated osteoclasts (arrows) lining along the
eroded bone surface was increased in the vehicle group, but
was significantly decreased after low or high dose of RA
treatment (Fig. 4c, d). CTSK immunohistochemical
staining showed similar trends (Fig. 4e, f). These results
illustrated that RA also inhibited osteoclasts formation
and function in vivo.
RA inhibited SRC/AKT signaling during osteoclastogenesis
We next focused on elucidating the potential
mechanism of RA in inhibiting osteoclasts formation and
function. RAW264.7 cells were cultured with RANKL for
different time to investigate mitogen-activated protein
kinase (MAPK), NF-?B, and SRC/AKT signaling
pathways. We found the RANKL-induced phosphorylation of
AKT was significantly inhibited by RA at 10 and 30 min
(Fig. 5a). This inhibitory effect can be partly rescued by
the AKT activator, SC79. Moreover, expression of SRC
increased since day 3 after stimulating by RANKL, but the
addition of RA significantly inhibited this trend at the
same time point (Fig. 5b). SC79 could reverse the
RArelated decrease of SRC. However, RA did not show any
suppressive effect on the RANKL-induced
phosphorylation of c-Jun N-terminal kinase (JNK), p38, extracellular
signalregulated kinase (ERK), as well as degradation of
I?B? (Fig. 5c). These results revealed that RA specifically
inhibited the SRC/AKT signaling pathways during
osteoclastogenesis without affecting MAPK or NF-?B
RA inhibits breast cancer-associated osteolysis in vivo
To determine whether RA suppressed breast
cancerassociated osteolysis, MDA-MB-231 cells were injected
in mice tibiae plateau and treated with
phosphatebuffered saline (PBS) or RA (100 ?g/kg) for 28 days.
Micro-CT and histology was performed to assess
osteolytic bone metastasis. Compared with the RA
treatment group, trabecular bone loss in the mice tibias
was more remarkable in the vehicle group (Fig. 6a).
Quantitative analysis revealed that the RA treatment
group had significantly higher BV/TV ratios and smaller
trabecular separation (Tb. Sp) compared to the vehicle
group (Fig. 6b). From histology, extensive trabecular
bone resorption and discrete cortical bone could be
observed in the vehicle group, while intact bone cortex
was remained in the RA treatment group (Fig. 6c). The
transferase-mediated dUTP nick end labeling (TUNEL)
assay further revealed that the degree of apoptosis was
significantly increased in the RA treatment group
compared to the vehicle group (Fig. 6d). All above data
suggested that RA could inhibit breast cancer-induced
RA inhibits growth and invasion of breast cancer cells
in vitro through promotion of apoptosis and inhibition and
We further explored the mechanism how RA regulated
the growth of breast cancer cells. Cell Counting Kit-8
(CCK-8) assays were performed on MDA-MB-231 cells
after a 48 and 96 h culture, and RA treatment significantly
decreased cell amount at doses higher than 6.25 ?M
(Fig. 7a). Next, we used ethynyl-2-deoxyuridine (EdU)
incorporation assays to determine the effect of RA. After
RA treatment for 24 h, the proliferation of MDA-MB-231
cells showed a significant decrease at both 6.25 and 12.5
?M doses (Fig. 7b, c). The flow cytometric analysis
revealed that RA could increase the percentages of
apoptotic cells (Fig. 7d, e). We used the transwell assay to
examine the effect of RA on cell invasion. Our results
revealed that RA significantly reduced the invasion of
MDA-MB-231 cell in a concentration-dependent manner
(Fig. 7f, g). We also used another breast cancer cell,
BCAP37, and generated similar results (Supplementary 3).
Further, MDA-MB-231 cells were cultured with RA for 0,
6, and 12 h to investigate AKT/mTOR signaling pathways.
Both phosphorylation of p-AKT and expression of mTOR
was significantly downregulated by treatment with RA (3
?M), indicating the inhibitory effect of RA on AKT/
mTOR signaling (Fig. 7h). These data suggested RA could
suppress the growth and invasion of breast cancer cells
in vitro with inhibition of AKT/mTOR signaling.
One of the major causes of cancer-associated death
among women is breast cancer, and bone is the major site
of metastasis in invasive breast cancer18. The mechanisms
underlying bone metastasis in breast cancer are still
unclear; however, the concept of the ?vicious cycle?
during bone breakdown and tumor invasion has been widely
accepted19. It is believed that pro-osteoclastic factors
released by tumor cells stimulate osteoclastogenesis,
whereas pro-tumorigenic growth factors secreted from
the bone matrix promote tumor expansion12?15.
Currently, no available treatment is sufficient to treat bone
metastasis and resulting osteolysis20. RA is one such
compound that is derived from anemone raddeana Regel
and has been demonstrated to suppress the growth of
gastric and colorectal tumors6,9. Our results revealed that
RA possessed inhibitory effects on breast
cancerassociated osteolysis through suppression of osteoclasts
and breast cancer cells. The possible mechanisms might
be that RA inhibited the SRC/AKT signaling pathway in
osteoclasts as well as AKT/mTOR signaling in breast
Our study provided evidence for the effects of RA on
RANKL-induced osteoclastogenesis. Different doses of
RA were used in our experiment, and the number of
TRAP-positive multinuclear osteoclasts was significantly
decreased after RA exposure. The levels of osteoclast
phenotypic markers, including CTSK and NFATc1, were
also downregulated following the addition of RA.
Furthermore, results of bone resorption assays indicated that
the area of bone resorption pits was significantly reduced
when treated with RA. The effects of RA on
Ti-particleinduced osteolysis were further explored with a murine
calvarial model. Micro-CT assessments demonstrated
that Ti-particle-induced osteolysis was obviously inhibited
in the RA treatment group compared with that in the
To elucidate the molecular mechanisms underlying the
above results, we first investigated the effects of RA on the
RANKL-initiated signaling pathway, because RANKL has
been shown to be a key regulator of osteoclast activation
by breast cancer cells12,14,21. RANKL-induced signaling
pathways include MAPK, NF-?B, and SRC/AKT
pathways, which play a pivotal role in osteoclast differentiation
and function22?24. A significant outcome of our study was
that the RANKL-related SRC expression in osteoclasts
was significantly downregulated after treatment with RA.
Previous studies have shown that SRC is essential for the
normal function of osteoclasts. Inhibition of SRC
suppresses osteoclastogenesis and the formation of
resorption pits25, which was consistent with our results.
Although the numbers of osteoclasts increased compared
with that in wild-type mice, Src?/? mice developed
osteopetrosis, suggesting the vital role of SRC in
osteoclast function rather than differentiation26. AKT is a
downstream target of SRC in response to RANKL27. TNF
receptor-associated factor 6 (TRAF6) is recruited upon
the activation of RANK by RANKL, which also leads to a
complex of c-Src and TRAF6 and ultimately the activation
of phosphoinositide 3-kinase (PI3K) and AKT28,29.
Specifically, the expression of Src251, which lacks the entire
kinase domain, inhibits AKT activity and osteoclast
survival in transgenic mice30. In our study, decreased AKT
phosphorylation was observed following the addition of
RA, consistent with the above reports. We also found that
the addition of AKT activator, SC79, can rescue the
(see figure on previous page)
Fig. 7 RA inhibits the proliferation and invasion of MDA-MB-231 cells through promotion of apoptosis and inhibition of AKT/mTOR
signaling pathways. a Cell viability of RA-treated MDA-MB-231 cells tested by CCK-8 assays at 48 and 96 h. b MDA-MB-231 cells were treated with
various concentrations of RA for 24 h and then evaluated with EdU incorporation assay (n = 3 per group). Magnifications: ?100. c The percentages of
EdU-positive cells for each field. d MDA-MB-231 cells were treated with various doses of RA for 48 h and then stained with Annexin V and propidium
iodide for flow cytometric analysis (n = 3 per group). e Apoptotic rate was defined as the percentage of dead and apoptotic cells (quandrants 2 and
3). f RA inhibited the invasion of MDA-MB-231 cells by Transwell invasion assay (n = 3 per group). Magnifications: ?200. g The number of invaded
cells of each field was counted. h MDA-MB-231 cells were treated with or without RA (3 ?M) for 0, 6, or 12 h, respectively. Western blotting for p-AKT
and mTOR was analyzed with the cell lysates (*p < 0.05, **p < 0.01)
inhibitory effect of RA on AKT phosphorylation and SRC
expression. Because MAPK and NF-?B signaling
pathways were not affected by RA, it is tempting to speculate
that RA may inhibit the formation and function of
osteoclasts through downregulation of the SRC/AKT
signaling pathway, which may explain why osteolysis was
reduced in the RA group.
We then investigated the effects of RA on osteolysis
using a breast cancer-associated osteolysis mouse model.
Our results revealed that RA reversed severe osteolysis
caused by MDA-MB-231 cells. Moreover, RA significantly
increased BV/TV ratios and decreased trabecular
separation compared with that in the vehicle group, which
was in accordance with the results of histological analysis.
In TUNEL assays, higher levels of apoptosis were detected
in the RA treatment group than in the vehicle group.
Based on the above results, we further explored the
direct effects of RA on MDA-MB-231 breast cancer cells.
The survival and invasion of MDA-MB-231 cells was
inhibited by RA, and RA also suppressed breast cancer
cell proliferation and invasion. Flow cytometric analysis
revealed that apoptosis rates in MDA-MB-231 cells
increased significantly when treated with RA, in
accordance with the results of TUNEL analysis. The
mechanism may involve the inhibitions of phosphorylation of
AKT and expression of mTOR. The PI3K/AKT/mTOR
pathway is believed to be the main signaling pathway
regulating cell proliferation, survival, metabolism, and
angiogenesis31?34. Hyperactivation of the PI3K/AKT/
mTOR pathway is frequently observed in breast cancer
and is often associated with resistance to both
antiERBB2-targeted and endocrine therapies35. Various PI3K/
AKT/mTOR inhibitors have been identified as promising
antitumor drugs in advanced breast cancer. Everolimus,
an inhibitor of mTOR, was found to increase
progressionfree survival among patients in a phase 3, randomized
trial36. Therefore, suppression of AKT activation and
mTOR expression mediated the inhibitory effects of RA
on breast cancer cell-associated osteolysis.
Another interesting finding in our study was that RA
tended to promote osteoblast differentiation and
osteoblastic-related genes expression in vitro. This is the
first study reporting the potential effects of RA on
osteoblast differentiation; however, further studies are still
required to determine this effect and underlying
In conclusion, RA exerted protective effects against
breast cancer-associated bone osteolysis by decreasing
osteoclast formation and resorption and by suppressing
tumor cell proliferation and invasion. Analysis of the
mechanisms involved in this process showed that RA
inhibited SRC/AKT signaling in osteoclasts and AKT/
mTOR signaling in MDA-MB-231 cells. Therefore, RA
may serve as a potential therapeutic agent for the
treatment of breast cancer-associated bone diseases in the
Materials and methods
Reagents and antibodies
RA was purchased from Meilunbio (Dalian, China). The
alpha modification of Eagle?s medium (?-MEM),
Dulbecco?s modified Eagle?s medium (DMEM), fetal bovine
serum (FBS), and penicillin/streptomycin were obtained
from Gibco-BRL (Gaithersburg, MD, USA). Recombinant
mouse M-CSF and mouse RANKL were obtained from
R&D (Minneapolis, MN, USA). SC79 was purchased from
Selleck Chemicals (Texas, TX, USA). Specific antibodies
targeting SRC, ERK, JNK, p38, I?B?, phospho-I?B?,
phospho-ERK, phospho-JNK, phospho-p38,
phosphoAKT, and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) were obtained from Cell Signalling Technology
(Cambridge, MA, USA). Antibodies against NFATc1 and
CTSK were purchased from Abcam (Cambridge, MA,
USA). CCK-8 was obtained from Dojindo Molecular
Technology (Kumamoto, Japan). The TRAP staining kit,
Triton X-100, and all other reagents were purchased from
Sigma Aldrich (St. Louis, MO, USA), unless otherwise
BMMs were isolated from femoral and tibial bone
marrow of 6-week-old female C57BL/6 mice, incubated in
?-MEM containing 10% FBS, 100 U/mL
penicillin/streptomycin, and 30 ng/mL M-CSF in a T75 flask in a 5% CO2
atmosphere at 37 ?C until it reached 90% density. Then
BMMs were moved to a 96-well plate at a density of 8 ?
103 cells per well and incubated for further differentiation.
RAW264.7 cells and MC3T3-E1 cells were obtained from
American Type Culture Collection. Human breast cancer
cell lines (MDA-MB-231 and BCAP37) were gifts from
Dr. Linbo Wang (Sir Run Run Shaw Hospital, Zhejiang
University). They were cultured in DMEM supplemented
with 10% FBS and antibiotics with the condition
mentioned above. Cell culture media were replaced every
Cell viability assay
BMMs (8 ? 103 cells per well) were seeded into a 96-well
plate, adherent cells were treated with various
concentrations of RA in ?-MEM containing 10% FBS, and 30
ng/mL M-CSF for 48, 72, or 96 h. MC3T3-E1 cells (5 ?
103 cells per well) were seeded into a 96-well plate with
DMEM containing 10% FBS and treated with indicated
concentrations of RA for 48 or 96 h. MDA-MB-231 cells
(5 ? 103 cells per well) were seeded into a 96-well plate
with DMEM containing 10% FBS and various
concentrations of RA for 48 or 96 h. The culture medium was
replaced every second days. The cytotoxic effect of RA on
BMMs, MC3T3-E1, or MDA-MB-231 cells were assessed
by a CCK-8 assay, 10 ?L of CCK-8 buffer was added to
each well, and plates were incubated for an additional 2 h.
The absorbance was measured at 450 nm (650 nm
reference) using an ELX800 microplate reader (Bio-Tek, USA).
Bone resorption assay
BMMs (2 ? 104 cells per well) were seeded on bovine
bone slices in 96-well plates for 24 h, and then stimulated
with 0, 0.2, 0.4, and 0.8 ?M RA in the presence of M-CSF
(30 ng/mL) and RANKL (50 ng/mL) for another 3 days.
Cells were then fixed with 2.5% glutaraldehyde. Bone
slices were visualized under a scanning electron
microscope (SEM, FEI Quanta 250; FEI, Hillsboro, OR, USA),
and the resorption areas were quantified with Image J
BMMs were seeded into a 96-well plate at a density of
8 ? 103 cells per well and treated with 0, 0.2, 0.4, and 0.8
?M RA in the presence of 30 ng/mL M-CSF and 50 ng/
mL RANKL. The culture medium was replaced every
second day until mature osteoclasts were formed. Then,
the cells were washed twice with PBS, fixed with 4%
paraformaldehyde for 30 min, and stained for TRAP.
TRAP-positive cells with five or more nuclei were
counted under the light microscopy. To assess the survival of
osteoclast, osteoclast ghosts were identified as dead
osteoclasts and the total number of each well was
ALP and Alizarin red staining
MC3T3-E1were cultured into a 12-well plate and
incubated with 0, 0.2, 0.4, or 0.8 ?M RA in osteogenic
medium (1 mM ?-glycerophosphate and 5 ?M L-ascorbic
acid 2-phosphate). At day 7, ALP staining was performed
and the area of positive cells was determined with Image J
software (NIH). For Alizarin red staining, at day 21, cells
were washed with PBS twice, fixed with 4%
paraformaldehyde for 30 min, and stained with Alizarin red
solution for 10 min at 4 ?C. The area of Alizarin red
S-stained mineralization nodules was also calculated with
Image J software (NIH).
EdU incorporation assay
Cell proliferation was evaluated with Click-iT EdU Cell
Proliferation Kit (KeyGEN, Nanjing, China) following the
manufacturer?s instructions. Breast cancer cells were
pretreated with 0, 6.25, and 12.5 ?M RA for 24 h. Then,
the cells were incubated with 25 ?M Edu for 2 h.
Subsequently, the cells were fixed for 20 min with 4%
paraformaldehyde. After permeabilization with 0.5% Triton
X-100, the cells were incubated with 1? Click-iT EdU
reaction cocktail for 30 min. Then, the cells were
subjected to 1? Hoechst 33342 solution for 30 min. The
cells were washed and observed under fluorescence
Flow cytometric analysis
Breast cancer cells were cultivated by the addition of 0,
6.25, 12.5, or 18.75 ?M RA with medium described above
for 24 h. Afterwards, the cells were washed twice with PBS
and then resuspended in binding buffer. The cells were
then stained with Annexin V and propidium iodide for 15
min at room temperature in the dark. Flow cytometric
analyses were carried out using a flow cytometer, and the
data were analyzed with the Cell Quest software, version
3.0 (BD Biosciences, Sunnyvale, CA, USA).
Transwell invasion assay
A 24-well invasion chamber system was used to
evaluate the effect of RA on invasion (Corning Inc., New
York, NY, USA). Cells were seeded in the upper chamber
at a density of 5 ? 104 cells in 200 ?l serum-free medium
by the addition of different concentrations of RA (0, 6.25,
12.5, and 25 ?M). The lower chamber was filled with 500
?l of 10% fetal bovine serum-containing medium. The
plates were incubated for 24 h at 37 ?C. Then, the cells
were fixed with methanol and stained with Trypan blue.
Cotton swabs were used to remove the non-migrating
cells on the upper side. The number of migrating cells
was calculated by counting one randomly selected field
of each well.
The fixed calvaria and tibiae were analyzed by micro-CT
scanner (Skyscan 1072; Skyscan, Aartselaar, Belgium).
The scanning protocol was set at an isometric resolution
of 9 mm, with X-ray energy settings of 80 kV and 800 ?A.
3D images were reconstructed using Cone Beam
Reconstruction software (SkyScan). BV, bone mineral density,
BV/TV, mean trabecular number, and mean trabecular
separation were recorded with resident reconstruction
After micro-CT analysis, the calvaria and tibiae were
decalcified in 10% EDTA for 3 weeks, followed by paraffin
embedding. Hematoxylin and eosin, TRAP, and CTSK
staining were performed, after which specimens were
examined and photographed under a high-quality
microscope. The number of TRAP-positive and
CTSKpositive multinucleated osteoclasts was counted.
Tumor tissues were decalcified in 10% EDTA for
3 weeks, and embedded in paraffin. TUNEL assay was
performed with an In Situ Cell Death Detection Kit
(Roche Applied Science, Indianapolis, IN, USA) according
to the manufacturer?s instructions.
Cells were lysed with RIPA buffer (Beyotime, Shanghai,
China), then the lysate was centrifuged at 12,000 rpm for
10 min, and the protein in the supernatants was collected
and quantified. Each protein lysate (30 ?g) was resolved
using sodium dodecyl sulfate?polyacrylamide gel
electrophoresis and transferred to a polyvinylidene difluoride
membrane (Millipore, Bedford, MA, USA). Following
transfer, membranes were blocked with 5% skim milk for
2 h and probed with primary antibodies at 4 ?C overnight
and incubated with appropriate secondary antibodies.
Antibody reactivity was detected by exposure in an
Odyssey V3.0 image scanning (Li-COR Inc., Lincoln, NE,
RNA isolation and real-time PCR analysis
BMMs were cultured in 6-well plates at a density of
2 ? 105 cells per well, treated with 30 ng/mL M-CSF, 50
ng/mL RANKL, and 0, 0.2, 0.4, and 0.8 ?M RA for
5 days. MC3T3-E1 cells were cultured in osteogenic
medium at the same density with above indicated
concentrations of RA for 7 or 14 days. Total RNA was
extracted using the RNeasy Mini Kit (Qiagen, Valencia,
CA, USA). RT-PCR was performed using SYBR Premix
Ex Tag Kit (TaKaRa, Biotechnology, Otsu, Japan) and
an ABI 7500 Sequencing Detection System (Applied
Biosystems, Foster City, CA, USA). The following
cycling conditions were used: denaturation at 95 ?C for
10 min, 40 cycles at 95 ?C for 10 s, and amplification at
60 ?C for 34 s. The quantity of each target was
normalized to GAPDH.
Ti-particle-induced calvarial osteolysis mice model
A mouse calvarial osteolysis model was established
using 8-week-old male C57BL/6 mice. After anesthesia,
30 mg of Ti particles were embedded under the
periosteum at middle suture of calvaria in the Ti, low and high
RA groups. In the sham group, the incision was closed
without further intervention. Mice in the low or high RA
groups were injected daily with 50 or 100 ?g/kg per day
RA, while mice in the sham or Ti group received PBS.
After 14 days, mice were sacrificed and the calvariae were
collected for micro-CT assessment and histological
Breast cancer-induced osteolysis model
The model of human breast cancer bone metastasis was
established through injection of the MDA-MB-231 cells
(1 ? 106/mL) into the tibiae plateau of 5-week-old BALB/c
nu/nu female mice. The mice were then randomly
assigned to two groups, treated with PBS (n = 6) or RA
(100 ?g/kg body weight in vehicle, n = 6) by
intraperitoneal injection every other day for 28 days and then
sacrificed. The tibiae were scanned with a micro-CT and
proceeded with histological or immunohistochemical
The SPSS 20.0 software was used to analyze the data
which were expressed as the mean ? SD. Groups were
compared using the Student?s t test. Results with values of
P < 0.05 were considered statistically significant.
All animal experiments were performed in accordance
with guidelines for animal treatment of Sir Run Run Shaw
Hospital. All experimental protocols in our study were
approved by the Ethics Committee of Sir Run Run Shaw
The study was sponsored by National Nature Science Fund of China
(81472064), Natural Science Fund of Zhejiang Province (Y17H060034), and
Foundation of Zhejiang Health Committee (2017PY018). No benefits in any
form have been or will be received from a commercial party related directly or
indirectly to the subject of this study.
Conflict of interest
The authors declare that they have no conflict of interest.
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