andrographolide prevents human breast cancer-induced osteoclastic bone loss via attenuated rankl...

PRECLINICAL STUDY

Andrographolide prevents human breast cancer-inducedosteoclastic bone loss via attenuated RANKL signaling

Zanjing Zhai • Xinhua Qu • Wei Yan •

Haowei Li • Guangwang Liu • Xuqiang Liu •

Tingting Tang • An Qin • Kerong Dai

Received: 22 July 2013 / Accepted: 16 January 2014 / Published online: 31 January 2014

� Springer Science+Business Media New York 2014

Abstract Bone metastasis is a common and serious

complication in advanced cancers such as breast cancer,

prostate cancer, and multiple myeloma. Agents that prevent

bone loss could be used to develop an alternative therapy

for bone metastasis. RANKL, a member of the tumor

necrosis factor superfamily, has been shown to play a

significant role in cancer-associated bone loss. In this

study, we examined the efficacy of the natural compound

andrographolide (AP), a diterpenoid lactone isolated from

the traditional Chinese and Indian medicinal plant And-

rographis paniculata, in reducing breast cancer-induced

osteolysis. AP prevented human breast cancer-induced

bone loss by suppressing RANKL-mediated and human

breast cancer cell-induced osteoclast differentiation.

Molecular analysis revealed that AP prevented osteoclast

function by inhibiting RANKL-induced NF-jB and ERK

signaling pathway in lower dose (20 lM), as well as

inducing apoptosis at higher dose (40 lM). Thus, AP is a

potent inhibitor of breast cancer-induced bone metastasis.

Keywords Andrographolide � RANKL � Osteoclast �Breast cancer � Bone metastasis

Introduction

Bone metastasis is a common and serious complication of

advanced cancers such as breast cancer, prostate cancer, and

multiple myeloma. Up to 70 % of patients with advanced

cancer develop bone metastasis, followed by sequential

skeletal complications such as bone pain, fractures, hyper-

calcemia, and spinal cord compression, all of which pro-

foundly affect a patient’s quality of life [1, 2]. In the

development of metastatic bone disease, many interactions

occur between tumor and bone cells. Bone metastases usu-

ally occur as osteolytic, osteoblastic/osteosclerotic, or mixed

lesions. The development of osteolytic lesions is due to a

significant increase in osteoclast number and reduced

osteoblastic activity [2]. For example, breast cancer tumor

cells produce various factors that induce osteoclastogenesis,

including interleukins (IL)-1, -6, and -11, which stimulate

production of receptor activator of nuclear factor-kappaB

(RANK) ligand (RANKL, an essential osteoclast stimulator)

[3–5]. Breast cancer tumor cells are also capable of secreting

RANKL and inducing osteoclastogenesis [6–9]. Once

secreted, RANKL binds the RANK receptor on the surface of

osteoclast precursor cells, activating a cascade of signaling

pathways [10, 11] and inducing the formation of functional

osteoclasts that are capable of bone destruction.

Therefore, selective inhibition of osteoclast formation or

RANKL signaling may have therapeutic potential for

Zanjing Zhai, Xinhua Qu and Wei Yan have contributed equally to

this study.

Z. Zhai � X. Qu � H. Li � X. Liu � T. Tang � A. Qin (&) �K. Dai (&)

Shanghai Key Laboratory of Orthopaedic Implants, Department

of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong

University School of Medicine, Shanghai, The People’s

Republic of China

e-mail: [email protected]

K. Dai

e-mail: [email protected]

W. Yan

Wendeng Zhenggu Hospital of Shandong Province, Wendeng,

Shandong, The People’s Republic of China

G. Liu

Department of Orthopaedic Surgery, The Central Hospital of

Xuzhou, Affiliated Hospital of Medical Collage of Southeast

University, Xuzhou, Jiangsu, The People’s Republic of China

123

Breast Cancer Res Treat (2014) 144:33–45

DOI 10.1007/s10549-014-2844-7

cancer-induced bone loss and related complications such as

pathological fractures and hypercalcemia. Indeed, in breast

cancer patients with bone metastasis, several bisphospho-

nates and denosumab (the first fully human monoclonal

antibody to RANKL, which has been approved by the US

Food and Drug Administration in patients with breast or

prostate cancer receiving hormone ablation therapy) have

demonstrated clinical efficacy. Both the drugs prevent

skeletal-related events (SREs) in patients with solid tumors

and bone metastases [12–15]; however, these drugs have

some disadvantages. For example, bisphosphonates have

yielded conflicting results in several studies on antitumor

efficacy in breast cancer [16]. Therefore, there remains

considerable scientific and public interest in investigating

alternative agents and treatments for osteolytic bone

metastasis.

Andrographolide (AP), a diterpenoid lactone isolated

from Andrographis paniculata, has been widely used as a

traditional Chinese and Indian medicine for the treatment

of various diseases [17–23] due to its effectiveness and

favorable safety profile. Recently, AP has attracted sub-

stantial research emphasis for its anticancer [24, 25], anti-

inflammation [26–28], hepatoprotective [29, 30], and anti-

infection [31] activities. In this study, we investigated

whether AP influences osteoclast differentiation induced by

RANKL or breast cancer tumor cells. We also studied the

effects of AP in a mouse xenograft breast cancer tumor

model to evaluate the prevention of breast cancer-induced

bone metastasis and osteolysis [32–35].

Materials and methods

Reagents and antibodies

AP was purchased from Sigma Aldrich (USA). Alpha-MEM,

fetal bovine serum (FBS), and penicillin were purchased

from Gibco BRL (Gaithersburg, MD, USA). Soluble mouse

recombinant M-CSF and RANKL were purchased from

R&D Systems (USA). Tartrate-resistant acid phosphatase

(TRAP) staining solution was from Sigma Aldrich (USA).

Primary antibodies for b-actin, phospho-IjBa, and IjBa

were purchased from Cell Signaling Technology (USA).

Primary antibodies for Bax, Bcl-2, caspase-3, and cleaved

caspase-3 were purchased from Affinity (USA).

Cell lines

RAW 264.7 and MDA-MB-231 cells were obtained from

American Type Culture Collection. RAW 264.7 cells were

cultured in DMEM/F12 supplemented with 10 % FBS and

antibiotics. This cell line is a well-established osteopro-

genitor cell system that expresses RANK and differentiates

into functional TRAP-positive osteoclasts when cultured

with soluble RANKL [36]. MDA-MB-231 cells were cul-

tured in DMEM with 10 % FBS.

In vitro osteoclastogenesis assay

RAW 264.7 cells were cultured in 24-well dishes at

5 9 103 cells/well and allowed to adhere overnight. The

medium was replaced and the cells were treated with

50 nmol/L RANKL for 5 days. All cell lines were stained

with TRAP using a leukocyte acid phosphatase kit. For co-

culture experiments with tumor cells, RAW 264.7 cells

were seeded at 5 9 103 cells/well and allowed to adhere

overnight. The following day, MDA-MB-231 and MC3T3-

E1 cells at 1 9 103 of each cells/well were added to the

RAW 264.7 cells, treated with AP, and co-cultured for

7 days before TRAP staining. TRAP? multinucleated cells

with [5 nuclei were counted as osteoclasts.

Cytotoxicity assay

The proliferation effect of AP on RAW 264.7 cells was

determined with the Cell Counting Kit-8 (CCK-8, Dojindo

Molecular Technology, Japan). Cells were plated in 96-well

plates at 3 9 103 cells/well in triplicate. After 24 h, the cells

were treated with increasing concentrations of AP (0, 5, 10,

20, and 40 lM) for 48 h or other indicated time. Then, 10 lL

CCK-8 was added to each well and the plates were incubated

at 37 �C for 2 h. Optical density (OD) was measured with an

ELX800 absorbance microplate reader (Bio-Tek, USA) at

450 nm (650 nm reference). Cell viability was calculated

relative to the control ([experimental group OD-blank OD]/

[control group OD-blank OD]).

Apoptosis assay

The apoptosis effect of AP on RAW 264.7 was determined

with the Vybrant� Apoptosis Assay Kit #2 (Invitrogen,

USA). Cells were treated with increasing concentrations of

AP (0, 5, 10, 20, and 40 lM) for 48 h. Then cells were

washed twice with cold PBS and pelleted; the supernatants

were discarded and the cells resuspended in 19 annexin-

binding buffer. Early apoptosis was detected by staining

with Alexa Fluor� 488 annexin V and propidium iodide

using the Vybrant� Apoptosis Assay Kit #2 (Invitrogen,

USA). FACS was performed using a FACScan flow

cytometer (Becton-Dickinson, Sunnyvale, CA, USA). Data

were acquired using CELL Quest software.

Clonogenic assay

RAW 264.7 cells were seeded in triplicate in 48-well plates

at 3 9 103 cells/well and cultured for 4 days in the

34 Breast Cancer Res Treat (2014) 144:33–45

123

presence of increasing concentrations of AP. After 4 days,

the cells were fixed and stained with DAPI (Sigma). Col-

onies with C50 were counted.

NF-jB Luciferase reporter gene activity assay

To examine the NF-jB activation in RAW cells, RAW 264.7

cells were stably transfected with a luciferase reporter gene

as previously described [37, 38]. The 3kB-Luc-SV40 re-

porter, which contains three NF-jB sites from the interferon

gene upstream of the luciferase coding region, has been

described previously [39, 40]. For stable transfection, the

3kB-Luc-SV40 reporter construct (20 lg) and pcDNA3.1

(2 lg) vectors were transfected into RAW 264.7 cells. The

transfected cells were selected with 400 lg/mL of G418

(Gibco BRL, Life Technologies, Melbourne, Australia). The

resulting stable cell line, named as P3K-RAW cell, was used

to investigate NF-jB activation by RANKL and AP. Briefly,

P3K-RAW cells were seeded in 48-well plates and main-

tained in culture media for 24 h. The cells were pretreated

with AP for 1 h, followed by RANKL (50 ng/mL) for 8 h.

Firefly luciferase expression was measured using the Pro-

mega Luciferase Assay System according to the manufac-

turer’s instructions (Promega, Sydney, Australia).

Western blotting

Cells were lysed in Ripa Lysis Buffer containing 50 mM

Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1 % Triton

X-100, 1 mM sodium fluoride, 1 mM sodium vanadate,

and 1 % deoxycholate and protease inhibitor cocktail. The

lysate was centrifuged at 12,000 rpm for 10 min and pro-

tein in the supernatants was collected and quantified. Each

protein lysate (30 lg) was resolved by 8–10 % sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–

PAGE) and transferred to a polyvinylidene difluoride

membrane (Millipore, Bedford, MA, USA). Nonspecific

interactions were blocked with 5 % skim milk for 2 h. The

membranes were probed with primary antibodies overnight

at 4 �C. Membranes were incubated with the appropriate

horseradish peroxidase-conjugated secondary antibodies

and reactivity was detected by exposure in an odyssey

infrared imaging system (LI-COR).

Caspase-3 activity assay

Cells were treated with different concentrations of AP (0, 10,

20, or 40 lM) for 48 h. Caspase-3 activity was determined

using the Caspase Colorimetric Assay Kit (KeyGen Biotech

Co., Ltd. Nanjing, China) according to the manufacturer’s

instruction. Briefly, cell lysate (100 lg total protein) was

added to a reaction mixture, which contained colorimetric

substrate peptides specific to caspase-3. The reaction was

incubated at 37 �C for 4 h. A spectrophotometer was used to

measure the absorbance at 405 nm, and the caspase activity

was expressed as the ratio OD inducer/OD control.

In vivo osteolytic bone metastasis

Cultured and resuspended human breast cancer cell line

MDA-MB-231 in PBS solution arrived at a final concen-

tration of 1 9 106/mL. BALB/c nu/nu mice (5–6 weeks old;

female; Harlan) were inoculated with MDA-MB-231 cells

(l 9 106/mL) directly into the tibiae plateau via a percuta-

neous approach. The mice were randomly assigned to 2

groups, treated with vehicle (0.9 % sodium chloride, n = 8)

or AP (30 mg/kg body weight in vehicle, n = 8) by intra-

peritoneal injection every other day for 28 days, and then

sacrificed. Radiographs (Faxitron Radiographic inspection

unit; Kodak) were obtained at baseline and just prior to

sacrifice. The tibiae of all animals were scanned with a high-

resolution micro-CT (Skyscan 1072; Skyscan, Aartse-laar,

Belgium). Bone histomorphometric analyses were per-

formed with the micro-CT data using the software described

previously [41]. The calculations of bone mineral density

(BMD), as well as the microstructural indices of trabecular

bone density (BV/TV), bone surface/volume ratio (BS/BV),

structure model index (SMI), connectivity density

(Conn.Dn), Euler number (Eu.N), trabecular thickness

(Tb.Th), trabecular number (Tb.N), and trabecular space

(Tb.Sp) were measured to assess the bone microstructure of

the tibiae. Tissues were removed and fixed in 4 % parafor-

maldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 1 day

at 4 �C and decalcified in 12 % EDTA. Decalcified bones

were paraffin-embedded and sectioned. For histologic

examination, sections were stained with hematoxylin and

eosin (H&E), and another section was stained with TRAP to

identify osteoclasts on the bone surface.

Statistical analysis

All values are presented as the mean ± standard deviation

(SD) of the values obtained from three or more experiments.

Statistical significance was determined by Student’s t test. A

value of *P \ 0.05 or **P \ 0.01 was considered significant.

Results

AP inhibits RANKL-induced osteoclastogenesis

in RAW 264.7 cells

We initially examined the effect of AP on osteoclast dif-

ferentiation induced by RANKL from osteoclast precursor

murine monocyte RAW 246.7 cells. Cells were treated

with 5, 10, or 20 lM AP in the presence of RANKL and

Breast Cancer Res Treat (2014) 144:33–45 35

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allowed to differentiate into osteoclasts. As shown in

Fig. 1a, the control group formed numerous TRAP-positive

multinucleated osteoclasts. In contrast, osteoclast forma-

tion is inhibited after AP treatment, demonstrated by the

dose-dependent decrease in the number of osteoclasts

(Fig. 1b). In addition, TRAP? osteoclasts start to form after

3 days’ RANKL stimulation. More mature osteoclasts were

formed and fused over the following 2 days (Fig. 1c).

However, in the AP treatment group, osteoclast differen-

tiation was inhibited throughout the process (Fig. 1c, d).

These data indicate that AP suppresses osteoclast forma-

tion in a dose-dependent manner.

AP inhibited early-stage osteoclastogenesis

To determine at which stage AP inhibits osteoclastogene-

sis, AP (20 lM) was added to culture medium at day 0, 1,

2, 3, or 4 of osteoclast differentiation. AP provided maxi-

mum inhibition of osteoclastogenesis when added early

with RANKL treatment (Fig. 1e, f). Exposure of precursor

cells to AP at later stages (after 3 days) provided less

effective suppression (Fig. 1f, fifth column). These data

suggest that AP blocks early osteoclast differentiation.

AP inhibits osteoclastogenesis induced by tumor cells

Bone loss is one of the most common complications in

patients with breast cancer [42]. We investigated whether

AP blocks tumor cell-induced osteoclastogenesis in RAW

264.7 cells. As shown in Fig. 2a, co-culture of RAW 264.7

cells with human breast cancer MDA-MB-231 cells

induced osteoclast differentiation and AP suppressed this

effect in a dose-dependent manner (Fig. 2b). Thus, AP

suppresses tumor-induced osteoclastogenesis.

In order to exclude the possibility of AP cytotoxicity, we

performed cell viability assays (Fig. 2c, d). AP did not

induce apoptosis in RAW 264.7 cells at doses up to 20 lM.

In addition, the CCK-8 proliferation assay showed that AP

was not cytotoxic in RAW 264.7 cells at doses up to 20 lM

(Fig. 2e). RAW 264.7 colony formation was also

Fig. 1 AP inhibits RANKL-

induced early

osteoclastogenesis in RAW

264.7 cells. a RAW 264.7 cells

(5 9 103 cells) were incubated

with RANKL (50 nmol/L) in

the presence of various

concentrations of AP for 5 days

and TRAP-stained to examine

osteoclast formation. TRAP-

positive cells were

photographed (original

magnification 9100). b TRAP-

positive multinucleated

osteoclasts were counted.

c RAW 264.7 cells (5 9 103

cells) were incubated with

RANKL (50 nmol/L) in the

presence of 20 lM AP for 3, 4,

or 5 days and then TRAP-

stained to examine osteoclast

formation. TRAP-positive cells

were photographed (original

magnification 9100). d TRAP-

positive multinucleated

osteoclasts were counted.

e RAW 264.7 cells (5 9 103

cells) were incubated with

RANKL (50 nmol/L) and AP

(20 lM) was added on day 0, 1,

2, 3, or 4. At the end of 5 days,

cells were stained for TRAP

expression. f TRAP-positive

multinucleated osteoclasts were

counted. AP-untreated,

RANKL-exposed cells served as

a control (CTRL)


123

unaffected by increasing AP concentrations (Fig. 2f).

These data suggest dose-dependent AP (B20 lM) sup-

pression of osteoclast formation without cytotoxic effects

in RAW 264.7.

AP suppresses osteolysis in MDA-MB-231 breast

cancer tumor-bearing mice

To determine whether AP suppresses osteolytic bone

metastasis and osteolysis, we injected nude mice tibia with

human breast cancer MDA-MB-231 cells, which are triple

negative (negative for estrogen receptor, progesterone

receptor, and human epidermal growth factor receptor 2),

and treated them with AP (30 mg/kg) or vehicle control for

28 days. To identify osteolytic bone metastasis, we per-

formed microradiography, micro-CT, and histology. As

shown in Figure 3a, osteolytic bone metastasis and

destruction of cortices (arrows) were observed in MDA-

MB-231 tumor-bearing control mice (left, upper). In con-

trast, there were fewer osteolytic lesions and the cortices

remained intact in the AP (30 mg/kg)-treated group (right,

upper). Micro-CT confirmed that the vehicle-treated tumor-

bearing mice induced extensive cancellous/trabecular bone

loss in the mouse tibias (Fig. 3a). Quantitative analysis of

bone parameters verified that MDA-MB-231 tumor in

vehicle control-induced osteolysis exhibited a significant

reduction in BMD, BV/TV, Tb.Th, Tb.N, and Conn.Dn,

and increased BS/BV, Tb.Sp, and SMI (Fig. 3b). In con-

trast, AP (30 mg/kg) reduced the extent of bone loss

induced by MDA-MB-231 tumor (Fig. 3a, b), indicating

AP protects against breast cancer-induced osteolysis.

Histology suggested the protective effect of AP on

MDA-MB-231 tumor-induced bone loss. As shown in

Fig. 4a, vehicle-treated tumor-bearing mice exhibited

serious osteolysis, resulting in discrete cortical bone and

severe trabecular bone resorption, especially alongside the

tumor sites. Tumors traversed the bone cortex and caused

invasion outside the bone marrow cavity, some even

destroyed the metaphysis and gave rise to tumor growth in

the articular cavity of the knee. AP treatment maintained

the intact bone cortex and complete metaphysis, and tumor

tissues remained in the bone marrow cavity. Meanwhile,

Fig. 2 AP blocks

osteoclastogenesis induced by

MDA-MB-231 tumor cells. In

addition, AP was noncytotoxic

in RAW 264.7 cells. a RAW

264.7 cells co-cultured with

MDA-MB-231 cells, treated

with AP (20 lM), and co-

cultured for 7 days before

TRAP staining. b TRAP?

multinucleated osteoclasts.

c AP-treated RAW 264.7 cells

were incubated for 48 h;

apoptosis was assessed by flow

cytometry. d The calculated

apoptosis ratio. e Cell viability

was measured in AP-stimulated

RAW 264.7 treated with CCK-

8. f AP-treated RAW 264.7 cells

fixed and stained with DAPI.

Colonies with C50 cells were

counted


123

Fig. 3 AP reduces osteolysis and preserves trabecular/cancellous

bone in human MDA-MB-231 breast cancer-bearing mice. a Repre-

sentative radiographs of mice treated with vehicle (left, upper) or AP

(right, upper). Arrows indicate osteolytic bone lesions caused by

injection of MDA-MB-231 cells; treatment with AP reduced these

osteolytic bone lesions. Three-dimensional computer reconstructions

of residual bone by micro-CT revealed extensive cancellous/trabec-

ular bone loss in vehicle-treated tumor-bearing mice (left, lower) and

inhibition of bone loss in a tumor-bearing mouse treated with AP

(right, lower). Each computer rendering is from the mouse with the

median BV/TV in each group. b Quantitative analysis of bone

parameters verified that MDA-MB-231 tumor in vehicle control

induced osteolysis with a significant reduction in BMD, BV/TV,

Tb.Th, Tb.N, and Conn.Dn, and increased BS/BV, Tb.Sp, and SMI.

AP rescued these bone parameters


123

numerous TRAP? osteoclasts were observed along the

junction zone between the tumor and bone tissues, and

there were fewer TRAP? cells in AP (30 mg/kg)-treated

mice (Fig. 4c). Quantitative analysis of TRAP? osteoclasts

confirmed this observation (Fig. 4d). We conclude that AP

inhibits human breast cancer MDA-MB-231 cell-induced

osteolytic lesions by inhibiting osteoclast activity.

AP represses RANKL-induced NF-jB signaling

by attenuating IjBa phosphorylation and degradation

through inhibition of IKK activity

Next, we focused on the underlying molecular mechanisms

of AP suppression of breast cancer-induced osteolysis. The

NF-jB pathway is essential for osteoclast differentiation

and function [10, 11, 43]. Therefore, we investigated

whether AP modulates RANKL-induced NF-jB activation

in monocytic RAW 264.7 cells using the NF-jB luciferase

reporter gene activity assay as previously reported by our

group [37, 38]. As shown in Fig. 5a, transcription of NF-

jB steeply increased in the presence of RANKL; however,

the addition of AP dose-dependently inhibited NF-jB

activation.

In unstimulated cells, NF-jB is retained in the cyto-

plasm as a complex with the inhibitory IjB protein. Upon

stimulation of RANKL, phosphorylation and subsequent

degradation of IjBa liberate NF-jB proteins, which can

enter the nucleus and bind to DNA target sites [44] to

stimulate osteoclast function. The previous study reported

that phospho-IjBa could be observed in 5 min after

RANKL stimulation. Consistently, as shown in Figure 5b,

our western blotting assay showed that the highest

expression of phospho-IjBa appeared at 5 min, which was

decreased at 30 min. In contrast, the AP-treated group

attenuated this trend, phospho-IjBa was not observed even

at 5 min. Similarly, due to the phosphorylation and sub-

sequent degradation of IjBa, the total amount of IjBadecreased with the stimulation of RANKL, especially at

5 min, while no obvious decrease in the expression of IjBawas observed in the AP-treated group.

Because IKK phosphorylates IjBa [45], we determined

whether AP alters the activity or levels of IKK. In vitro, cells

Fig. 4 AP inhibits MDA-MB-231 breast cancer-induced bone

destruction and tumor metastasis. a Decalcified bones stained with

H&E. Vehicle-treated tumor-bearing mice exhibited serious osteol-

ysis, with discrete cortical bone. The tumor traversed the bone cortex

and caused invasive metastasis outside the bone marrow cavity; some

even destroyed the metaphysis and gave rise to tumor growth in the

articular cavity of the knee (left). AP treatment retained the intact

bone cortex and complete metaphysis, and tumor tissues were limited

to the bone marrow cavity. b Osteolytic area/total bone was

calculated. c TRAP-stained decalcified bones. TRAP? osteoclasts

were observed alongside the junction zone between the tumor and

bone tissues; fewer TRAP? cells were observed in the AP (30 mg/

kg)-treated mice. d Quantitative analysis of the TRAP? osteoclasts

number confirmed this observation


123

treated with RANKL showed a sharp rise in IKK activity as

indicated by phosphorylation of GST-IjBa within 5 min. In

contrast, cells pretreated with AP could not phosphorylate

GST-IjBa upon RANKL treatment (Fig. 5c, upper panel).

To determine whether the loss of IKK activity was due to the

loss of IKK protein expression, we measured IKK subunits

IKK-a and IKK-b levels by western blotting. AP treatment

did not alter the expression of IKK-a or IKK-b (Fig. 5c

middle and lower panels). These results suggest that AP

might repress RANKL-induced NF-jB signaling by atten-

uating IjBa phosphorylation and degradation.

AP inhibits RANKL-induced ERK1/2 phosphorylation

RANKL induced activation of MAPK pathways (including

ERK1/2, p38, and JNK) are also critical for osteoclast

Fig. 5 AP represses RANKL-induced NF-jB signaling by attenuat-

ing IjBa phosphorylation and degradation by inhibiting IKK activity

and RANKL-induced ERK1/2 phosphorylation. Moreover, AP pro-

motes RAW 264.7 apoptosis at higher concentration though up-

regulating caspase-3 activity. a AP-treated P3K-RAW cells were

incubated with RANKL (50 ng/mL). Luciferase activity for NF-jB

was measured and normalized to control. b AP-treated RAW 264.7

cells were incubated with RANKL (50 nmol/L). Western blotting

with anti-IjBa, phospho-IjBa, and actin. c AP-pretreated RAW

264.7 cells were incubated with RANKL (50 nmol/L). Whole-cell

extracts were immunoprecipitated using antibody against IKKa and

analyzed by an immune complex kinase assay using recombinant

GST-IjBa as described in Materials and Methods. Whole-cell

extracts were western blotted with anti-IKK a and anti-IKK bantibodies. d AP-pretreated RAW 264.7 cells were incubated with

RANKL (50 nmol/L). Cytoplasmic extracts were western blotted with

anti-p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, and JNK antibodies.

e Cell viability of AP-stimulated RAW 264.7 cells treated with CCK-

8 was measured over time at various concentrations of AP. f AP-

treated RAW 264.7 cells were incubated for 48 h; apoptosis was

assessed by flow cytometry. g The apoptosis ratio was calculated at

various concentrations of AP. h Bax or Bcl-2 mRNA expression in

AP-stimulated RAW 264.7 cells over 48 h was measured. i AP-

treated RAW 264.7 cells were incubated for 48 h, the protein level of

Bcl-2 and BAX was analyzed by Western blot. j AP-treated RAW

264.7 cells were incubated for 48 h, AP-induced caspase-3 cleavage

was detected by Western blot. k RAW 264.7 cells were treated with

different concentrations of AP (0–40 lM) for 48 h. Spectrophotom-

etry was used to determine the activities of caspase-3


123

differentiation and function [10, 11, 43]. ERK induces

c-Fos which is implicated in osteoclastogenesis [46].

Inhibition of ERK suppresses osteoclast formation [47, 48],

while repression of JNK retards RANKL-induced osteo-

clastogenesis [49]. p38 is important in the early stage of

osteoclast generation because it regulates the micro-

phthalmia-associated transcription factor [50]. Further-

more, inhibition of MAPK by specific inhibitors resulted in

strong suppression of RANKL-induced osteoclast forma-

tion from precursor cells [51–53], suggesting the MAPK

signaling pathways play a critical role in osteoclast

formation.

Therefore, we also investigated whether AP modulates

RANKL-induced MAPK activation in monocytic RAW

264.7 cells. ERK1/2 phosphorylation peaked within 20 min

of RANKL stimulation (Fig. 5d, left); however, pretreat-

ment with AP significantly inhibited ERK1/2 phosphory-

lation (Fig. 5d, right). In contrast, AP treatment had no

obvious effect on JNK or p38 phosphorylation (Fig. 5d).

These data suggest that AP also inhibits ERK/MAPK sig-

naling during osteoclastogenesis.

AP promotes RAW 264.7 apoptosis at higher

concentration though up-regulating caspase-3 activity

The previous researches had demonstrated that there was

closed relationship between NF-jB signaling and apoptosis

[54–56]. However, different studies gained contradictory

observation, where NF-jB seemed to exhibit either antia-

poptosis or proapoptosis effect in different scenario

[57–60]. Since AP inhibited NF-jB activity, we performed

further experiments to investigate the effect of AP on

osteoclast apoptosis. Especially, we examined the effect of

AP on RAW 264.7 viability over time (1, 3, 5, and 7 days)

and at different concentrations (0, 10, 20, and 40 lM). As

shown in Fig. 5e, low concentrations of AP (B20 lM)

were not cytotoxic to cell viability at any time point. In

contrast, 40 lM AP severely reduced cell viability at all

the time points. In addition, while AP at 20 lM is sufficient

to suppress osteoclast differentiation but had little effect on

cell apoptosis, AP at 40 lM significantly induced cellular

apoptosis. As seen in Fig. 5f, the percentage of apoptotic

cells was statistically similar in the control group and the

groups treated with a low dose of AP (B20 lM) after 48 h.

However, treatment with AP at 40 lM resulted in a sta-

tistically significant increase in the number of apoptotic

cells (Fig. 5g).

In order to elucidate the potential mechanisms of AP-

induced apoptosis, we examined the functional role of the

Bcl-2 family members, including both antiapoptotic Bcl-2

and proapoptotic Bax, which are particularly important

apoptosis-regulatory proteins [61]. Our experiment further

demonstrated that AP dose-dependently stimulated Bax

gene expression (Fig. 5h). It is interesting to note that AP

treatment induced Bcl-2 expression. However, higher dose

of AP actually suppressed Bcl-2 expression (Fig. 5h). We

also investigated their protein expression level and found

that AP had similar effects on Bax and Bcl-2 expression

level. In particular, the protein level of Bcl-2 is high at

20 lM but decreased at 40 lM (Fig. 5i). It is thus believed

that in AP modulated Bcl-2 and Bax expression in

osteoclasts.

Caspase-3, the principal member of the caspase cascade,

is a terminal effector of apoptosis. Once cleaved and

activated, caspase-3 executes the cell death program [62–

64]. To determine the involvement of caspase-3 cascade in

AP induced apoptosis, the cleavage of caspase-3 from

proform into its active form (cleaved caspase-3) was

detected by western blot after AP treatments. As shown in

Fig. 5j, AP induced caspase-3 cleavage, especially at

40 lM. Consistent with this observation, caspase-3 activity

assay performed by Caspase Colorimetric Assay Kit (Ke-

yGen Biotech Co., Ltd. Nanjing, China) showed that

around four-fold increases of caspase-3 activity were

observed after treatment with 40 lM AP for 48 h, while

lower concentrations groups (B20 lM) had no obvious

increase versus control. To sum up, in our present study,

apoptosis was not detected when cells were treated with

low concentrations of AP (B20 lM). However, higher

concentration of AP (40 lM) resulted in a sharp increase in

cell apoptosis.

Discussion

Cancer is one of the most serious problems affecting public

health in developed and developing countries [65]. Breast

cancer is the most common cancer and remains the second

leading cause of cancer death in women [66]. While bone

metastasis is the major cause of morbidity in patients with

advanced breast cancer, it rarely leads directly to disease-

related death; however, severe complications frequently

occur in bone metastatic patients, such as chronic pain,

hypercalcemia, SREs, incontinence, and paralysis, all of

which dramatically affect the patients’ quality of life

[67–69].

There is no effective treatment for breast cancer

metastasis to the bone. Treatment of osteolytic diseases

mainly rely on selective estrogen receptor modulators

(SERM), calcitonin, estrogen replacement therapy, bis-

phosphonates, synthetic parathyroid hormone, and novel

antibodies such as denosumab. Unfortunately, these ther-

apies are associated with side effects such as estrogen-

related diseases, GI problems, thromboembolism, and

endocrine disorders [70–75]. Bisphosphonates, potent

inhibitors of osteoclast formation and activity, are the


123

current standard for treatment of cancer-induced osteolytic

diseases; however, several studies have shown that bis-

phosphonates increase the risk of severe osteonecrosis of

the jaw in cancer patients [76], with devastating conse-

quences for affected patients [77]. It is important, therefore,

to develop safer antiresorptive strategies for treating bone

lesions in patients with metastatic cancer.

RANKL is a member of the TNF superfamily, which is

the dominant mediator of osteoclast differentiation,

resorption, function, and survival [10, 11, 78], and is tightly

associated with cancer-induced osteolytic lesions. Numer-

ous active cytokines are released in metastatic breast can-

cer, increasing RANKL expression and leading to

excessive osteoclast activity and osteolysis [4], which in

turn causes release of growth factors and calcium from the

bone matrix, producing a ‘‘vicious cycle’’ of bone break-

down and tumor proliferation [2]. Thus, RANKL inhibition

prevents the development and progression of tumor-

induced bone lesions and reduces skeletal tumor burden in

clinical settings [79–81]. This suggests the RANKL path-

way is central to the pathology of bone destruction in bone

metastasis, and blocking RANKL signaling is a promising

therapeutic target.

This study provides insight into the mechanisms of AP

action, starting from its effects on in vitro inhibition of

RANKL- and tumor cell-induced osteoclastogenesis and

in vivo remission of MDA-MB-231 cancer cell-induced

bone destruction. AP suppressed osteoclast formation

stimulated by RANKL or MDA-MB-231 cancer cells at

nonlethal concentrations, especially when the preosteo-

clasts were treated early with AP. Based on these in vitro

results, we hypothesized that AP may attenuate osteoclast

activity and thus retard cancer-induced bone loss in tumor-

bearing mice. Indeed, micro-CT detection showed signifi-

cant bone loss in vehicle-treated vs. AP-treated tumor-

bearing mice, as demonstrated by trabecular and cortical

bone parameters, including decreased BMD, BV/TV,

Tb.Th, Tb.N, and Conn.Dn, and increased BS/BV, Tb.Sp,

and SMI. These results were confirmed by histology.

Vehicle-treated tumor-bearing mice had discrete bone

cortex, accompanied by tumor growth outside the bone

marrow cavity. Interestingly, tumor-induced bone resorp-

tion in vehicle-treated mice was consistent with numerous

activated osteoclasts, especially alongside the junction

zone between the tumor and bone tissues. In the AP-treated

group, bone mass improved, there was limited tumor

growth within the bone marrow cavity, and limited osteo-

clasts alongside the junction zone. Our in vitro and in vivo

results and the ‘‘vicious cycle’’ theory of the tight rela-

tionship between tumor growth, osteoclast activity, and

bone lesions [2] suggest that AP inhibits human breast

cancer MDA-MB-231 cell-induced osteolytic lesions by

inhibiting osteoclast activity.

RANKL-induced NF-jB and MAPK (ERK1/2, p38,

JNK) signaling pathways are the dominant mediators of

osteoclastogenesis [10, 11, 43, 82]. The previous

researchers found that knockout of NF-jB subunits resul-

ted in disastrous effects in osteoclastogenesis and led to

serious osteopetrosis [43, 83, 84]. IKK b, a component of

the NF-jB signaling pathway, is also a critical mediator of

osteoclast survival and is required for inflammation-

induced bone loss; IKKb-ablation or specific inhibition

results in a lack of osteoclastogenesis and unresponsiveness

of IKK b-deficient mice to inflammation [85, 86]. The

MAPKs (ERK, JNK, and p38) are activated by RANKL

stimulation and are associated with osteoclastogenesis

[10, 87]. ERK induces c-Fos for osteoclastogenesis [46];

inhibition of ERK reduces osteoclast formation [47, 48],

while dominant-negative JNK prevents RANKL-induced

osteoclastogenesis [49]. In comparison, p38 is important in

the early stage of osteoclastogenesis because it regulates

the microphthalmia-associated transcription factor [50].

Furthermore, inhibition of MAPKs by specific inhibitors

caused strong suppression of RANKL-induced osteoclast

formation from precursor cells [51–53], suggesting the

MAPK signaling pathways have a role in osteoclast for-

mation. Our results showed that AP inhibited phosphory-

lation and degradation of NF-jB inhibitory subunit IjBa,

resulting in reduced levels of NF-jB transactivation. AP

also attenuated ERK phosphorylation, while it had no

obvious effect on JNK and p38 phosphorylation. Together,

these data suggested the effects of AP on RANKL/Breast

cancer-induced osteoclast formation may, at least partially,

be due to the inhibition of both the NF-jB and ERK/

MAPK signaling pathways.

Moreover, NF-jB is known to be involved in cell apop-

tosis. Here, we found that AP at higher dose can suppress NF-

jB activation and subsequently induce cell apoptosis by

enhancing Bax expression and reducing Bcl-2 expression,

leading to the activation of caspase-3-induced apoptosis.

However, it is still interesting to notice that this effect is

obvious when AP at used higher than 40 lM. Thus, further

studies are required to understand the molecular mechanisms

of AP induced Bcl-2/Bax imbalance.

Breast cancer stimulates RANKL signaling by produc-

ing RANKL in the tumor microenvironment [6, 88]. We

have demonstrated that AP attenuates RANKL or breast

cancer-induced osteoclastogenesis in vitro and inhibits

bone destruction and extended metastasis in tumor-bearing

mice, likely due to AP suppression of NF-jB or ERK/

MAPK signaling. Our results suggest AP may be useful for

treatment of cancer-induced bone lesions. In comparison to

bisphosphonates or denosumab, AP is inexpensive and has

few side effects. Our research provides a foundation for

further studies of AP and breast cancer-associated bone

loss.


123

Acknowledgments This work was supported by the Program for

Innovative Research Team of Shanghai Municipal Education Com-

mission (Phase I), a grant from the Innovative Research from

Shanghai Municipal Education Commission (13YZ031), a grant for

scientific research from the National Natural Science Foundation for

the Youth of China (No. 81201364), grant from the scientific research

foundation for returned overseas Chinese scholars from the state

human resource ministry, the Key National Basic Research Program

of China (Grant No. 2012CB619101), a scientific research grant for

youth of Shanghai (Grant No. ZZjdyx 2097), a scientific research

grant from 985 project–stem cell and regenerative medicine centre, a

scientific research grant from Zhejiang National Science Foundation

(Grant No.Y2110653), and the Major Basic Research of Science and

Technology Commission of Shanghai Municipality (Grant No.

11DJ1400303).

Conflict of interest The authors declare that they have no conflict

of interest.

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